DGMS2010 abstracts com€¦ · Reviews, kurzen Trends oder von Originalarbeiten eingereicht werden, ... V6-V10 Seite:42-46 V25-V28 Seite:63-68 InstrumentelleEntwicklungen: V16-V20

��

��

�� !�"��#��$%��

&��

Impressum

Herausgeber:

Dr. Jürgen Schmidt, Leibniz-Institut für Pflanzenbiochemie Halle

Prof. Dr. Andrea Sinz, Institut für Pharmazie, Martin-Luther-Universität Halle-Wittenberg

Redaktion

Jürgen Schmidt

Andrea Sinz

Carsten Kuhl

Christian Schmelzer

Dieses Werk ist urheberrechtlich geschützt. Die dadurch begründeten Rechte, insbesondere die der Über-setzung, des Nachdrucks, der Entnahme von Abbildungen, der Wiedergabe auf photomechanischem oderähnlichem Wege und der Speicherung in Datenverarbeitungsanlagen bleiben – auch bei nur auszugswei-ser Verwendung – vorbehalten.

Die Autoren übernehmen die Verantwortung für den Inhalt des Abstracts.

Inhaltsverzeichnis

I Grußwort 3

II Allgemeine Informationen 5

III Danksagung 8

IV Programm 14

V Plenarvorträge 18

VI Abstracts der Vortragsbeiträge 36

VII Abstracts der Posterbeiträge 90

VIIIKeyword-Index 266

IX Teilnehmerverzeichnis 273

1

Lokales Organisations- und Programmkomitee

Andrea Sinz

Jürgen Schmidt

Christian Schmelzer

Christian Ihling

Andrej Frolov

Mathias Müller

Angelika Schierhorn

Sylvia Pieplow

Carsten Kuhl

2

Liebe DGMS-Mitglieder, liebe Tagungsteilnehmer und Gäste,

ImNamen derMartin-Luther-Universität Halle-Wittenberg, des Leibniz-Institutes für Pflanzenbiochemie,

des Organisationskomitees und der Deutschen Gesellschaft für Massenspektrometrie begrüßen wir Sie

sehr herzlich zur 43. Jahrestagung der DGMS.

Ein herzliches Willkommen in unserer 1200-jährigen Saalestadt Halle!

Auch in diesem Jahr erfreut sich dieses alljährliche, fachübergreifende Treffen von Wissenschaftlern aus

ganz Deutschland und dem Ausland ungebrochen einer sehr guten Resonanz. Die Teilnehmer aus Uni-

versitäten, Instituten und der Industrie werden in den nächsten Tagen einen Eindruck von der rasanten

Entwicklung und neuen Trends der Massenspektrometrie bekommen. Wir sind sicher, dass unsere Ta-

gung in Halle ein attraktives Vortragsprogramm bietet, das die Möglichkeiten der Massenspektrometrie

in den verschiedenen Arbeitsgebieten eindrucksvoll unterstreicht.

Die Tagung beginnt - einer mittlerweile guten Tradition folgend - mit dem „Wolfgang-Paul-Vortrag“, der

in diesem Jahr von Peter Roepstorff (Odense), einem Pionier der Protein-Massenspektrometrie, gehalten

wird. Die Themen der Plenarvorträge reichen von neuen Entwicklungen bei den Ionisierungsmethoden

bis hin zu Anwendungen der Massenspektrometrie in Toxikologie und Biochemie. Ein Gastvortrag über

archäologische Funde in Mitteldeutschland wird den Teilnehmern der DGMS-Tagung 2010 unsere Re-

gion von einer anderen Seite zeigen. Vier Workshops und etwa 170 Poster werden das Programm der

Tagung facettenreich illustrieren. Darüberhinaus lädt eine umfangreiche Firmenausstellung mit 25 teil-

nehmenden Firmen mit ihren Präsentationen neuer Geräteentwicklungen unsere Tagungsteilnehmer ein.

Halle ist 2010 nach 16 Jahren zum zweiten Mal Gastgeber dieser Tagung. In diesen 16 Jahren hat sich

vieles getan: Halles wissenschaftliche Einrichtungen, allen voran die Martin-Luther-Universität Halle-

Wittenberg, haben trotz mancher Herausforderungen eine überaus positive Entwicklung genommen.

Halle stellt mittlerweile in Deutschland einen wichtigen Standort der Protein- und Pflanzenforschung

dar – zwei Gebiete, bei denen die Massenspektrometrie eine zentrale Rolle einnimmt.

Die 500 Jahre alte Moritzburg, in deren GotischemGewölbe am 9.März 2010 unsere Abendveranstaltung

stattfinden wird, hat durch den modernen Erweiterungsbau im Nord- und Westflügel der Burg weiter an

Attraktivität gewonnen. Für die Teilnehmer der Tagung bietet sich die Möglichkeit, am Dienstag an Führ-

ungen durch die Dauerausstellungen, einschließlich der Sammlung Hermann Gerlinger, teilzunehmen.

Wir wünschen allen Tagungsteilnehmern eine anregende und informative Tagung sowie angenehme Tage

in Halle an der Saale.

Jürgen Schmidt Andrea Sinz

3

Wolfgang-Paul Lecture Award

Peter Roepstorff

Education: 1965 Diplôme d’études supérieures (physiological chemistry), Faculté des Sciences, Uni-versité d’Aix-Marseille; 1966 graduated in chemical engineering from the Danish Technical University;1974-1975 Assistant Professor at the Dept. of Molecular Biology, Odense University; 1988 Docent at theDepartment of Molecular Biology, Odense University; 1990-1995 Research Professor under the Ministryof Education, placed at the Dept. of Molecular Biology, Odense University; 1995-2005 Professor in Pro-tein Chemistry, Dept. of Molecular Biology, University of Southern Denmark; 2005- Professor emeritus(part time professor), Dept Biochemistry and Molecular Biology, University of Southern Denmark.Many administrative appointments in Denmark, Sweden, Finland, France and Netherlands from 1976-2008.

Awards:Kaj Hansens Foundation, "Limprisen"(1968)."Villum Kann Rasmussens Årslegat for TechnicalResearch"(1989). Kaj Linderstøm-Lang Gold Medal (2002). Novo Nordisk Award 2004. HUPO Distin-guished Achievement Award in Proteomic Sciences (2007). Honorary member of the British Society forProteome Research (2008). Honorary member of the Spanish Proteome Society (2009). EUPA SeniorScientist Award in Proteomics Sciences (2009). Thomson Medal (2009).

Academy memberships:Member of the Royal Danish Academy of Sciences and Letters (1990). Mem-ber of the Danish Academy of Technical Sciences (1991).

Academic memberships:Member of the American Society for Mass Spectrometry; the Protein Society;the American Association for the Advancement of Science; the American Association for Biochemistryand Molecular Biology.

Honorary doctor in philosophy at the Faculty of Science, Uppsala University, Sweden (2001) and intechnical sciences at the Danish Technical University (2009).

Member of the Editorial boards and organizing committees of many scientific journals as Biomedicaland Environmental Mass Spectrometry (1982-1994), Mass Spectrometry Reviews (1994-present), Jour-nal of Mass Spectrometry (1994- present) and Molecular Biotechnology (1996-present), Journal of theAmerican Society for Mass Spectrometry (1999-2005), Proteomics (2000-present), Molecular and Cel-lular Proteomics (2001).More than 430 scientific papers, 500 conference contributions and guest lectures

4

Allgemeine Informationen

Tagungsort

Die 43. Jahrestagung der Deutschen Gesellschaft für Massenspektrometrie findet im Audimax und imMelanchthonianum der Martin-Luther-Universität Halle-Wittenberg am Universitätsplatz statt. Die Vor-träge der Tagung finden Audimax statt. Postersitzungen, Lunchseminare und Firmenausstellungen wer-den im nur wenige Meter entfernten Melanchthonianum abgehalten (s. Lageplan).

Vom Hauptbahnhof Halle fahren verschiedene Straßenbahnlinien ins Zentrum (Marktplatz, Fahrzeit ca.8-10 min). Am günstigsten kann man das Audimax mit der Linie 7 erreichen (Haltestelle Neues Theater).PKW-Nutzer beachten bitte die begrenzten Parkmöglichkeiten in der Innenstadt. Während der Tagungwird die Nutzung des öffentlichen Nahverkehrs empfohlen.

Die Tagung wird am Sonntag, den 7. März, 18.00 Uhr, offiziell eröffnet. Im Anschluss an den traditio-nellen “Wolfgang-Paul-Vortrag” findet gegen 19.30 Uhr im Stadthaus ein Empfang statt.

Tagungsbüro

Das Tagungsbüro befindet sich im Foyer des Audimax und ist zu folgenden Zeiten geöffnet:

Sonntag, 7. März 2010: 12.00 – 17.30 UhrMontag, 8. März 2010: 8.00 – 18.00 UhrDienstag, 9. März 2010: 8.00 – 18.00 UhrMittwoch, 10. März 2010: 8.00 – 14.00 Uhr

Kleiderablage

Im Audimax befindet sich eine Garderobe (unbeaufsichtigt). Bitte beachten Sie, dass wir für abgelegteKleidungsstücke keine haftung übernehmen können. Wertgegenstände sollten keinesfalls in Ihrer Garde-robe zurückgelassen werden.

Tagungsprogramm

Das komplette Programm der DGMS-Tagung mit allen eingereichten Abstracts erhalten Sie zu-

sätzlich zum Konferenzband auch in elektronischer Form auf USB-Stick.

Vorträge

Die Plenarvorträge finden im Großen Hörsaal des Audimax statt. Die in Themenbereiche gegliedertenparallelen Sitzungen der Kurzvorträge (15 min + 5 min Diskussion) werden im Audimax (Großer Hörsaalund Hörsaal XXII) stattfinden. Es stehen jeweils ein Beamer und ein Overheadprojektor zur Verfügung.Präsentationen sollten spätestens am Vortag den zuständigen Mitarbeitern im Technikraum übergebenwerden und können dort auf Lauffähigkeit überprüft werden. Aus technischen Gründen ist die Nutzungeigener Notebooks für die Präsentationen nicht möglich.

Als Präsentationssoftware steht in den Hörsälen Microsoft PowerPoint (Version 2003) zur Verfügung.Bitte achten Sie unbedingt auf die Einhaltung der Redezeiten, um einen Wechsel zwischen den Parallel-sessions zu ermöglichen.

5

Poster

Die Poster sind ebenfalls in Themenbereiche gegliedert. Die Poster sollten während der ganzen Ta-

gung aushängen. Bringen Sie daher Ihr Poster möglichst bereits am Sonntag an. Bitte bringen Sie bitte

zum Aufhängen der Poster ausreichend Pins mit. Die Posterpräsentationen finden auf drei Ebenen im

Melanchthonianum statt (s. Lageplan). Die Poster müssen bis spätestens Mittwoch, 14.00 Uhr entfernt

werden. Verbliebene Poster werden vernichtet. Die Nummerierung der Poster erfolgt fortlaufend. Von

den präsentierenden Autoren wird erwartet, dass sie sich während der Postersitzungen an ihren Postern

aufhalten, und zwar für die Poster mit ungerader Nummer am Montag und für die Poster mit gerader

Nummer am Dienstag. Am Ende der Tagung werden an von einer Jury ausgewählte Autoren Poster-

preise vergeben.

Workshops

Die vier auf dem Programm stehenden Workshops werden am Sonntag, den 7. März 2010, von 14.00

Uhr bis 17.30 Uhr in den dafür angegebenen Hörsälen des Melanchthonianums durchgeführt. Eine Kaf-

feepause für die Workshop-Teilnehmer wird in der Mensa „Tulpe“ am Universitätsplatz stattfinden.

Lunch-Seminare

Im Rahmen der diesjährigen DGMS-Tagung finden während der Mittagspause am Montag und Dienstag

in vier Hörsälen des Melanchthonianums acht Lunch-Seminare statt, zu denen Sie sich bei der jeweili-

gen Firma vorher anmelden müssen. Bitte kontaktieren Sie dazu eventuell einen Firmenvertreter an den

Firmenständen.

Veröffentlichung wissenschaftlicher Beiträge

Die Zeitschrift Analytical and Bioanalytical Chemistry (ABC) bietet allen interessierten Vortragenden

sowie Autoren von Posterpräsentationen der DGMS 2010 an, den entsprechenden Beitrag in einem

Sonderheft von ABC gemeinsam zu veröffentlichen. Als Gast-Editoren für dieses Sonderheft werden

A. Sinz, J. Schmidt und K.G. Heumann verantwortlich sein. Beiträge können in Form von kritischen

Reviews, kurzen Trends oder von Originalarbeiten eingereicht werden, die das übliche Reviewsystem

durchlaufen. Genauere Informationen über ABC sowie zur Manuskriptherstellung können über folgende

Internetseite erhalten werden: www.springeronline.com/journal/00216.

Es wird gebeten, Manuskripte ausschließlich elektronisch über http://mc.manuscriptcentral.com/abc ein-

zureichen und an entsprechender Stelle der elektronischen Maske unter "special issue" DGMS 2010 an-

zugeben. Deadline für die Einreichung von Beiträgen ist der 31. Mai 2010, so dass diese Beiträge dann

in einem der beiden Dezemberhefte 2010 erscheinen werden.

Für Fragen zur Einreichung oder zu anderen Punkten einer Publikation in ABC kann mit der verant-

wortlichen Mitarbeiterin, Frau Dr. Andrea Pfeifer, oder mit dem Editor der Zeitschrift, Herrn Prof. Klaus

Heumann, Kontakt aufgenommen werden.

Internetzugang

Am Tagungsort stehen in eine Computerpool mehrere internetfähige Computer zur Verfügung. Wer mit

einem eigenen Notebook einen Internetzugang möchte, kann dies über ein freigeschaltetes WLAN be-

kommen (Passwort am Tagungsbüro).

6

Abendveranstaltung

Das Konferenzdinner wird am Dienstagabend im Gotischem Gewölbe der Moritzburg stattfinden. Am

Friedemann-Bach-Platz (gegenüber der Moritzburg) gibt es beschränkt Parkmöglichkeiten. Die Moritz-

burg ist vom Audimax in etwa 10-15 Minuten zu Fuß zu erreichen. Dort erwartet Sie an diesem Abend

auch ein faszinierender Gang durch die sehenswerten Dauerausstellungen, einschließlich der Sammlung

Hermann Gerlinger („Die Brücke“). Bitte bringen Sie unbedingt Ihren Coupon für das Dinner mit.

Soweit noch Restplätze verfügbar sind, können Sie bis Dienstagmittag im Tagungsbüro noch Buchungen

vornehmen.

Mittagessen

Falls Sie nicht an einem der Lunchseminare teilnehmen, kann ein Mittagessen gegen Barzahlung in

der Mensa „Tulpe“ am Universitätsplatz oder in der Harz-Mensa (s. Lageplan) eingenommen werden.

Darüberhinaus verfügt Halles Innenstadt über zahlreiche Restaurants und Imbissmöglichkeiten, auch im

nahen Umfeld des Audimax, die teilweise spezielle Mittagsangebote haben.

Sonstiges

Unser Organisationsteam wird stets bemüht sein, Ihnen bei auftretenden Problemen und Fragen behilf-

lich zu sein. Sie erkennen die Mitarbeiter der Organisatoren an ihren roten T-Shirts oder den farbigen

Namensschildern.

7

Danksagung

Für die großzügige Unterstützung der 43. Jahrestagung der DGMS in Halle, ohne die diese Tagung nicht

möglich gewesen wäre, möchten wir uns bei den folgenden Firmen bedanken:

AB Sciex

Advion Biosciences Ltd

Agilent Technologies

Bruker Daltonik GmbH

CovalX AG

Dionex

GSGMess- und Analysengeräte GmbH

Ionics MSV Europe

Ionicon Analytik GmbH

JEOL GmbH

KR Analytical Ltd.

MasCom GmbH

Medicwave AB

Peak Scientific Instruments LTD

Pfeiffer Vacuum GmbH

Probiodrug GmbH

Proxeon Biosystems A/S

SeQuant GmbH

Shimadzu

SMS GmbH

SunChrom GmbH

Thermofisher Scientific

Vacuubrand GmbH + Co KG

Waters GmbH

8

Kurzvortragsbeiträge

Themenbereiche

Peptide und Proteine: V1 - V5 Seite: 37 - 41V11 - V15 Seite: 47 - 51V21 - V24 Seite: 59 - 62

Organische Massenspektrometrie: V6 - V10 Seite: 42 - 46V25 - V28 Seite: 63 - 68

Instrumentelle Entwicklungen: V16 - V20 Seite: 52 - 58V34 - V38 Seite: 74 - 79

Lipide und Kohlenhydrate: V29 - V33 Seite: 69 - 73MS – ausgewählte Themen: V39 - V42 Seite: 80 - 84

Posterbeiträge

Themenbereiche

Peptide und Proteine: P1 - P67 Seite: 91 - 159Naturstoffe/Metabolomics: P68 - P93 Seite: 160 - 186Lipide und Kohlenhydrate: P94 - P107 Seite: 187 - 203Instrumentelle Entwicklungen und Imaging: P108 - P128 Seite: 204 - 225Organische Massenspektrometrie: P129 - P160 Seite: 226 - 258Element- und Isotopenmassenspektrometrie: P161 - P167 Seite: 259 - 265

9

Sonntag, 07.03.2010

ab 12:00Audimax (Foyer)

Registrierung, Öffnung des Tagungsbüros

14:00 - 17:30Melanchthonianum

Workshops

W1: HS XVIII Elemental Mass Spectrometry and ProteomicsFit for Life Sciences and Environmental Research

W2: HS B 4. Workshop „Grundlagen der Massenspektrometrie“: Halle 2010W3: HS XV Computational Mass SpectrometryW4: HS XVI Bioaffinity Mass Spectrometry in Life Science and Biomedical Analysis18:00Audimax (Gr. HS)

Eröffnung der Tagung,Musik, Ehrungen

18:45Audimax (Gr. HS)

Wolfgang-Paul-Vortrag Seite: 24Peter Roepstorff (Universität Odense, DK)The role of mass spectrometry in protein studies, from peptide and protein characterization to proteomics

19:45 Empfang im Stadthaus (mit Buffet)

Montag, 08.03.2010


Plenarvortrag PL1 Seite: 25Julia Laskin (Richland, USA)Soft-landing of Complex Ions onto Self-Assembled Monolayer Surfaces

9:20 - 10.15Audimax (Gr. HS)

Verleihung der Wolfgang-Paul-Studienpreise

Dr. Philipp Grüne, Fritz-Haber-Institut der MPG, Berlin Seite: 22Dr. Bernd-Bastian Schaack, MPI für Kohlenforschung Mülheim (Ruhr) Seite: 23Nicolas Paul Richard Dietl, TU Berlin Seite: 226Vorträge der Promotionspreisträger

10:15Melanchthonianum

Kaffeepause

10:45 - 12:25 Session 1

„Peptide und Proteine I“

Session 2

„Organische Massenspektrometrie I“

Audimax (Gr. HS) Audimax (HS XXII)10:45 V1 Seite: 37

Targeted data acquisition for improved repro-

ducibility and robustness of proteomic mass

spectrometry assays

Mikhail Savitski, Frank Fischer, Toby Mathieson,Gavain Sweetman, Manja Lang, Marcus BantscheffCellZome Heidelberg, D

V6 Seite: 42GC-MS Analysis of Mars Relevant Oxygenates and

Methane from Photochemically Induced Reaction

of Carbon Dioxide and Water

Michael Bartoszek, Mike Wecks, Gunnar Jakobs, DirkMöhlmannLeibniz-Institut für Katalyse, Rostock, D

11:05 V2 Seite: 38Metal Ion-Masked LC-MS for Straight Detection

of Phosphopeptides

Joerg Seidler, Andreas Schlosser, Jutta Panke, NicoZinn, Martin E. Böhm, Dirk Bossemeyer, Wolf D.LehmannDKFZ Heidelberg, D

V7 Seite: 43Rapid metabolic profiling of Nicotiana tabacum

defense against Phytophthora nicotianae using

direct infrared laser desorption ionization mass

spectrometry

Alfredo J. Ibáñez, Philipp Bones, Judith Scharte,Alexander Pirkl, Franz Hillenkamp, Engelbert Weis,Klaus DreisewerdInstitute of Medical Physics and Biophysics, Univer-sity of Münster, D

11:25 V3 Seite: 39DBnovo: Optimierte Sequenzierung von Biopoly-

meren - Anwendungen und Aspekte

Andreas Kühn, Katja Tham, Stephan Heymann,Johann-Christoph Freytag, Michael W. LinscheidHumboldt-Universität Berlin, D

V8 Seite: 44Molekularer Fingerabdruck vonGetränkenmittels

pos/neg-switching LC-API-ToF-MS

Thomas LetzelTU München, D

11:45 V4 Seite: 40Frog skin peptides – sequence analysis using high

accuracy mass spectrometry

Markus Langsdorf, Alireza Ghassempour,; AndreasRoempp, Bernhard SpenglerJustus-Liebig-Universität Gießen, D

V9 Seite: 45Analyzing Complex Combustion Chemistry

of Nitrogenated Compounds with TOF-Mass-

Spectrometry

Arnas Lucassen, Patrick Osswald, Ulf Struckmeier,Katharina Kohse-Höinghaus, Tina Kasper, NilsHansen, Nicole Labbe, Phil R. Westmoreland, TerryA. CoolUniversität Bielefeld, D

12:05 V5 Seite: 41De Novo Sequencing Of Peptides On Single Resin

Beads by MALDI-FTMS

Reinhold Weber, Angelika Semmler, ValentinWittmann, Michael PrzybylskiUniversität Konstanz, D

V10 Seite: 46Investigating organocatalytic reactions: Mass spec-

trometric studies of aldol condensation reaction

catalyzed by TFA-morpholine

MohamedWasimAlachraf, Kristina Zumbansen, Ben-jamin List, Wolfgang SchraderMPI für Kohlenforschung, Mülheim, D

14


Mittagspause

Lunchseminare der Firmen (Teilnahme nach Anmeldung)14:00 - 15:40 Session 3

„Peptide und Proteine II“

Session 4

„Instrumentelle Entwicklungen I“


Challenges of big macromolecular soluble and

membrane protein complexes for ESI-MS

Nina Morgner, Helena Hernandez, Min Zhou, LauraLane, Carol V. RobinsonUniversity Cambridge, UK

V16 Seite: 52Explosives and chemical warfare agents - detection

and analysis with PTR-MS

Philipp Sulzer, Fredrik Petersson, Simone Jürschik,

Stefan Jaksch, Alfons Jordan, Gernot Hanel, Eu-

gen Hartungen, Hans Seehauser, Lukas Märk, Stefan

Haidacher, Ralf Schottkowsky,Tilmann D. Märk

Ionicon Analytik GmbH, Innsbruck, A

14:20 V12 Seite: 48

Structural insights into Munc13/calmodulin com-

plexes by photoaffinity labeling and mass spec-

trometry

Kalina Dimova, Nils Brose, Olaf Jahn

MPI für Experimentelle Medizin, Göttingen, D

V17 Seite: 54

Schneller Nachweis von Betäubungsmitteln

mittels SPI-Massenspektrometrie und Laser-

Ionenmobilitätsspektrometrie

Robert Laudien, Rainer Schultze

Optimare GmbH, Wilhelmshaven, D

14:40 V13 Seite: 49

Untersuchung der Raumstruktur von Proteinen

und Proteinkomplexen mittels Ionenmobil-

itätsspektrometrie

Marc Kipping, Iain Campuzano, James Langridge,

Frank Sobott, Brandon Ruotolo, Carol V. Robinson

Waters Corporation, Manchester, UK

V18 Seite: 55

Generation and Identification of Reactive Metabo-

lites and Phase II Adducts by Electrochemistry

Coupled On-line to Liquid Chromatography/Mass

Spectrometry

Sandra Jahn, Anne Baumann, Uwe Karst

Westfälische Wilhelms-Universität Münster, D

15:00 V14 Seite: 50

Quantitative mass spectrometry combined with a

covalent peptide approach for determination of

protein interactions

Sabine Lange, Eberhard Krause

Leibniz-Institut für Molekulare Pharmakologie (FMP)

Berlin, D

V19 Seite: 57

MS/MS/MS-Quantifizierung in einem hybri-

den Triple Quadrupol/Lineare Ionenfallen-

Massenspektrometer

Christof Lenz, Jan Lembcke, Axel Besa

AB Sciex, Darmstadt, D

15:20 V15 Seite: 51

Mass spectrometric measurement of protease activ-

ities with MALDI and ESI mass spectrometers

Maria Trusch, Diana Hildebrand, Xiaoxia Zhao,

Michael W. Linscheid, Hartmut Schlüter

Universitätsklinikum Hamburg-Eppendorf, D

V20 Seite: 58

Application of MALDI-imaging MS to monitor

surface changes of photo-oxidized poly(styrene)

films

Anna Crecelius, Ulrich Schubert

Friedrich-Schiller-Universität Jena, D

15:40 - 16:15

Melanchthonianum

Kaffeepause

16:15

Audimax (Gr. HS)

Plenarvortrag PL2 Seite: 26

Harald Meller (Halle, D)

Die Himmelsscheibe von Nebra und weitere sensationelle Funde aus Sachsen-Anhalt

17:30 - 19:30

Melanchthonianum

Firmenausstellungen und Poster-Session I (mit Brezeln und Bier)

Dienstag, 09.03.2010

8:30

Audimax (Gr. HS)

Plenarvortrag PL3 Seite: 27

Karl Mechtler (Wien, A)

Relative and Absolute Quantification of Protein Complexes

9:20 - 10:40 Session 5

„Peptide und Proteine III“

Session 6

„Organische Massenspektrometrie II“

Audimax (Gr. HS) Audimax (HS XXII)

9:20 V21 Seite: 59

Recombinant Isotope Labelled and Selenium

Quantified (RISQ) Protein Standards for Quantifi-

cation of Proteins and Their Degree ofModification

Nico Zinn, Sebastian Tittebrandt, Wolf D. Lehmann

DKFZ Heidelberg, D

V25 Seite: 63

Laser-Massenspektrometrische Untersuchung

von Trichlorbenzolen mit REMPI- und MATI-

Methoden

Frank Witte, Mikko Riese, Frank Gunzer, Jürgen

Grotemeyer

Christian-Albrechts-Universität Kiel, D

9:40 V22 Seite: 103

A new tandem mass spectrometry approach for the

detection and identification of O-GlcNAc-modified

peptides

Hannes Hahne, Bernhard Küster

TU München, D

V26 Seite: 64

Neue dissoziative Cross-Linker für die vereinfachte

massenspektrometrische Proteinstrukturauf-

klärung

Frank Dreiocker, Mathias Müller, Andrea Sinz,

Mathias Schäfer

Universität zu Köln, D

15

10:00 V23 Seite: 61Elucidation of Temporal and Spatial Protein Pat-

terns in Developing Barley Grains by Multiplexed

LC-MS and MALDI-imaging MS Approaches

Andrea Matros, Stephanie Kaspar, Udo Seiffert, Win-friede Weschke, Hans-Peter MockIPK Gatersleben, D

V27 Seite: 66Massenspektrometrie mit weicher Photo-ionisation

für die on-line Charakterisierung von organischen

Spurenkomponenten in Verbrennungs- und Pyro-

lysegasen

Thorsten Streibel, Alois Fendt, Martin Sklorz, Nico-laus Dahmen, Ralf Zimmermann Universität Rostock,

D

10:20 V24 Seite: 62

A predefined proteome signature for invasive duc-

tal breast carcinoma enables differentiation of can-

cer samples from controls - first results from a

prospective follow-up study

Claudia Röwer, Cornelia Koy, Hans Vissers, Marc

Kipping, Toralf Reimer, Bernd Gerber, Michael

Hecker, Björn Ziems, Hans-Jürgen Thiesen, MichaelO. GlockerProteom Center Rostock, University of Rostock, D

V28 Seite: 68Stars in MALDI - Tandem MS investigations of

star-shaped poly(e-caprolactone)

Katrin Knop, Anja Baumgaertel, Cornelia Bader,Anna C. Crecelius, Ulrich S. Schubert Friedrich-Schiller-University Jena, D

10:40 - 12.30Melanchthonianum

Firmenausstellungen und Poster-Session II (inklusive Kaffeepause)


Mittagspause

Lunchseminare der Firmen (Teilnahme nach Anmeldung)14:15 - 15:00 Plenarvortrag PL4 Seite: 28

Hans H. Maurer (Homburg, D)Current status of hyphenated mass spectrometry in clinical and forensic toxicology

15:00 - 15:20 Mattauch-Herzog-Förderpreis


Kaffeepause

15:50 - 17.30 Session 7

“Lipide und Kohlenhydrate”

Session 8

„Instrumentelle Entwicklungen II“


Spermabeurteilung mit MALDI-TOF-

Massenspektrometrie?

Karin Müller, Ulrike Jakop, Kristin Teuber, Mandy

Eibisch, Rosmarie Süß, Beate Fuchs, Uwe Paasch,

Jürgen Schiller

Universität Leipzig, D

V34 Seite: 74

Novel Routine Clinical Applications for Mass Spec-

trometry

Arnd Ingendoh, Wolfgang Pusch, Sebastian Goetz,

Guido Mix, Carsten Baessmann

Bruker Daltonik GmbH, Bremen, D

16:10 V30 Seite: 70

Multiparametric analysis by tandem mass

spectrometry- challenges for modern labora-

tory diagnostics

Uta Ceglarek, Georg-Martin Fiedler, Joachim Thiery

Universitätsklinikum Leipzig, D

V35 Seite: 75

In situ real time identification of biological tissue

with surgical tools combined with mass spectrome-

try

Karl Christian Schäfer, Tamás Szaniszló, Júlia Balogh,

Lajos Balogh, Júlia Dénes, Dániel Szalay, Lajos

Gödörházy, Zoltan Takats

Justus-Liebig-Universität Gießen, D

16:30 V31 Seite: 71

Lipidomics and Neurodegeneration

Dominik Schwudke, Sarita Hebbar

National Centre for Biological Sciences, TIFR, India

V36 Seite: 76

Direct Surface Sampling of Planar Tissues using a

Chip-Based Nano-Electrospray system

Vilmos Kertesz, Reinaldo Almeida, Jack Henion, Gary

J. Van Berkel Advion BioSciences, D/USA

16:50 V32 Seite: 72

The analysis of molecular lipid species by higher

energy collisional dissociation (HCD) on a LTQ

Orbitrap XL instrument

Kai Schuhmann, Ronny Herzog, Stefan R. Bornstein,

Andrej Shevchenko

Department of Internal Medicine III, TU Dresden, D

V37 Seite: 77

Speciation Analysis of Gadolinium Chelates in

Hospital Effluents and Wastewater Treatment

Plant Sewage by a Novel HILIC/ICP-MS Method

Uwe Karst


17:10 V33 Seite: 73

3-Aminochinolin als Matrix und

Derivatisierungsreagenz zur Oligosaccharid-

Analyse mittels MALDI-Massenspektrometrie

Marion Rohmer, Björn Meyer, Marko Mank, Bernd

Stahl, Ute Bahr, Michael Karas

Goethe-Universität Frankfurt, D

V38 Seite: 79

Differenzierung der isobaren Aminosäuren Leucin

und Isoleucin und schnelle Charakterisierung von

posttranslational modifizierten Peptiden durch

MALDI-Massenspektrometrie kombiniert mit

Stosskühlung

Jens Soltwisch, Klaus Dreisewerd


17:30

Audimax (Gr. HS)

Jahreshauptversammlung der DGMS

19:30 - 24:00 Konferenzdinner (Moritzburg)

16

Mittwoch, 10.03.2010


Plenarvortrag PL5 Seite: 29Renato Zenobi (Zürich, CH)Neue Perspektiven für die on-line-/on-site-Analytik mittels EESI-MS

9:20 - 10.00 Waters-Life-Science-Preis

Albert Heck (Utrecht, NL)

10:00 - 10:30 Kaffeepause

10:30 - 11:50 Session 9

„MS – ausgewählte Themen“

10:30 V39 Seite: 80

Auf der Suche nach deutschen Begriffen in der Massenspektrometrie

Jürgen Gross

Universität Heidelberg, D

10:50 V40 Seite: 81

Cyclization and Rearrangement Reactions of a Ions of Protonated Peptides

Bela Paizs, Benjamin Bythell, Philippe Maitre

DKFZ Heidelberg, D

11:10 V41 Seite: 82

High Performance MALDI Imaging

Bernhard Spengler, Andreas Römpp, Zoltan Takats, Sabine Günther, Oliver Schulz, Yvonne Schober

Justus-Liebig-Universität Gießen, D

11:30 V42 Seite: 84

Lucky Survivor zum Letzten - Experimentelle Bestätigung eines lange postulierten Modells

Thorsten W. Jaskolla, Michael Karas

Goethe-Universität-Frankfurt, D

11:50 - 12:35 Plenarvortrag PL6 Seite: 30

Wolf D. Lehmann (Heidelberg, D)

Beruf Massenspektrometrie

12:35 - 13:00 Finale

Vorstellung des Tagungsortes 2011

Verleihung der Posterpreise

Abschluss der DGMS 2010

17

Plenarvorträge

18

��

�

�� !"�� #�� $� ��% ��!� ��&� �� $� ��'%!&��!(��)� !"�� !� ��&� �� !�#�� $� ��)�*+*�+��$� �� #�� ,��$� �� -��&�� .�#��/��% ��+��0�1 �$�2��,� �� 3��)!4��$� ��/� ��&��3��5

��,�� + �� 5��!�� 1��-�6% �� 6�� 7��8��5

�� !

��3�.�� 9��#��,��$�� -� ��.��: �;� #��0��<�= �%�1��-�� >�� -�� $�� >�� # ��0� ��#�1� 9�#-��= �� 0�� #�1�� 9��#��!� �� .�� 2��,� ��9�0��7��1� #*

�"��3�� %�*��*��%��&�� 0�1 �$�2��,� ��

/� ��*�3�� *��*��%��*��*�%�� 1� #��+��2��,� ��'��

.��# ��%��/��(��?��

��##��$�� ;�'��(��@��)��/;��A��@�

��///��B��A��//��3��%�� $��

3��3�� %�� ?��

�1��1��

�� ;� � � �� :�#�� % �,��>�� C�� 9��#��!��#�� :�� *3��B ��% ��3��A�3��@�� +��#�>� ��! � ��#�� D��>��#

�� C�� + ��# ��1��- ��9 ��#� �*

3��@�3��7�� #�� E �F� �G�-�� -�� #�

3��3��E�1�� .��: � �� 9��#��

��

�

��

�� !�� "��#��$��#%&'�(��$��

��

�� )�*�� +!��!,�%��-!��.��/� �� # �,�� %�� ,�� +!��,� � '�� -�0��,� � +�� (!�� # �,�� +�� .��/��.��/��"��# ��(!��1��'� 2��3+4-�5�6�� *�� #�� &��!� ��(��

�!� "��#�! � �� !��"�� 7�� !� � � #�� 8�� +� �� +�� -�0��, � � � �!� � 7 �� #�� -!�� .�� /� �� +�� -�0��,��7#-./5+-��9 ��:;;��</��=4� ��<��>�>�'� 2��-�0��# �,��1��'-#��.� ��$�� !��#� �+��,�"� ��#!�� #�� -!��7 �� #�� '�� -�0��-7#'-��/�� $��?

�"��$�%�!��!� ��!��3 ��,�� 6!� ��%��$��

��&!��"�:;@@ 4%"9#� ��%��!��,� ��6� ��-�! ��,�A��

�� $�:;@�5:;@@ 4��!��B�� :;@; 6��5�� (��(��, �!��

(�� !��7 �� /��(�!��,�7/(�A��

�' !��!��(�� ) �� C��

� � ��!)��"� � ��) /��,��5�� 1� �� ,��! �1��

(�� (!��,�D�-�0��,��!��'��,�� /�� !��'��,�� (!��,��3 ��,�� 4�� !��.�� C0�� +�� -�0��,�� :;;$�/� ��!��)� �,�� 0��,��6+5/��"+5/�� !�� 5�� 5��!��7� ��!��77��!�� ! ��, �!��?

/ ,�� ! �� 6+5/��?

/��!��

(�� !��7 �� #�� -!��.��/� �� +�� -�0��,��7#-./5+-��$��5$��;�

��

�

�� !"#$ �� %��

��& ' �� (��)*+*$�� (��,��(� ��)**-$�� .��/�0��)**1$�� 2��/�0��)**+$�� ' �� .��(��!�� 3--4$�� 5�� (��3--6$

�� 2��7 ��#��'�� 3--8$�� 9� �� :��!�� 3--*$�� #��7 ��9��3--*$

�� !� ��"�� #�

�� );-(�� 0��/�� <�� =�� &5�"�!� �� 9��/��>��)**6

"�$%&��'��$&�'��(��&&�'��$��)�":�9�"�� :�� ?��/��$�:"7 �:�� "�� 7 ��/�$�"�� :�� ?0�� ":0,$� 9�9� ��9��9��$

��&!��9��0��@� ��3--1��":�9

0�� 7 �� A��7 ��!�� 9(��,��#��'�� 2'�$��

)*+6 ��9�� 2'�� )**- 0 �"��9��B��%�� B9 )**-C)**) �(�� B��%��0��

�� B��%�� )**3 5��,��(�� 20,!�!��)**4 �� 2'�� )**8 ��3--- ��3--3�3--1 � �� A��7 ��!��3--;�3--4 %�� <��#��<��$

)*66�)*6*7 ��B��%��=�<��)*6*�)*8-7 ��B��%��=��?�� " "�9��$)*8-�)*837 ��B��%��=�<��)*83 "��7 ��B��%��=�<��)*84 0��0 ��/�� 7 ��B��%��=�<��

0��"�<��/��)*84�)*8+B��%��=�<��#��?0 ��/�� 7 ��)*8+�)*+* ��5��

B��%��=��D��/�� 2��)*+6 ��0 �� 7 ��B��%��=��)*+*5�� "D,��)**;C ��0��B��%��=��

Wolfgang Paul Studienpreis

Structure and Reactivity of Metal and Semiconductor Clusters inthe Gas Phase

Grüne, Philipp

Fritz-Haber-Institut der MPG, Abteilung Molekülphysik

Einleitung: Clusters bridge the gap between single atoms and the bulk phase. Very often the physical andchemical properties of such particles depend dramatically on their size and differ from the corresponding bulkspecies. The possibility to tailor the characteristics of materials by carefully controlling their dimension has led tothe rise of nanoscience.The properties of a cluster are determined by its electronic and geometric structure. While the first can be probedexperimentally rather directly, for example by means of photoelectron spectroscopy, there has been a lack ofexperimental methods for the detailed study of the size-specific geometry of nanoparticles in the gas phase.Experimenteller Teil: Vibrational spectroscopy is inherently sensitive to the geometry of a given species, since itprobes the force constants of its bonds. We apply infrared multiple photon dissociation (IR-MPD) spectroscopy,making use of the Free Electron Laser for Infrared eXperiments (FELIX) coupled to a molecular-beam setup witha reflectron time-of-flight mass spectrometer, in order to obtain the size-selective vibrational finger print of metalclusters.Ergebnisse und Diskussion: This presentation focuses on the structures of transition-metal clusters. We haveinvestigated cationic clusters of the vanadium group elements. Common spectral features point to similar structuralmotifs throughout the whole group.[1] To date, IR-MPD is the only experimental technique that can probe thestructure of neutral metal clusters in the gas phase. We have applied IR-MPD to neutral gold clusters and haveconfirmed a pyramid structure for Au20. The lowering of the symmetry when a corner-atom is cut from the Au20cluster is directly reflected in the vibrational spectrum of Au19. For Au7 we show how the structure is different forthe cation, the neutral, and the anion.[2]IR-MPD can also be used to follow the chemistry of adsorbats on the cluster surface. We present a sistematicoverview on our findings for CO adsorption on transition-metal clusters, covering large parts of the periodictable.[3,4]Neue Aspekte: IR-MPD is an innovative and versatile method to probe the structure and reactivity of transition-metal clusters.Referenzen: [1] P. Gruene, A. Fielicke, G. Meijer, J. Chem. Phys. 2007, 127, 234307.; [2] P. Gruene, D. M.Rayner, B. Redlich, A. F. G. van der Meer, J. T. Lyon, G. Meijer, A. Fielicke, Science 2008, 321, 674.; [3] P.Gruene, A. Fielicke, G. Meijer, D. M. Rayner, Phys. Chem. Chem. Phys. 2008, 10, 6144.; [4] A. Fielicke, P.Gruene, G. Meijer, D. M. Rayner, Surf. Sci. 2009, 603, 1427.Thema: Instrumentelle EntwicklungKeywords: IR-MPD, Vibrational Spectroscopy, Clusters, Geometric StructureKontakt: [email protected]

22

Wolfgang Paul Studienpreis

The solid state formation of zeolitic materials: An analyticalchallenge for mass spectrometry

Schaack, Bernd Bastian; Schüth, Ferdi; Schrader, Wolfgang

Max-Planck-Institut für Kohlenforschung, Germany

Einleitung: Most properties of zeolites and related materials highly depend on their pore dimensions and topo-logy as well as the nature and presence of intra- or extraframework centers. To tailor the synthesis of zeolites it isnecessary to increase the knowledge of the fundamental steps during their solid state formation.However, analytically these steps are the most elusive ones in solid state chemistry. This is due to the size of theoccurring species, which lies between small molecules and the extended solid and the high salt loadings of thesynthesis mixtures.Herein, we use ESI-MS and ESI-MS/MS to study the formation mechanism of zeolites, e.g. ITQ-21 and zeolite A,both highly important for industrial and academic applications.Experimenteller Teil: Both zeolites were obtained from synthesis mixtures with the following overall molar com-position: 0.67 TEOS : 0.33 GeO2 : 0.5 SDAOH : 0.5 HF : x H2O.Therein SDAOH is the used structure directing agent in its hydroxide form, i.e. N(16)-methylsparteinium hy-droxide for ITQ-21 and 4-Methyl-2,3,6,7-tetrahydro-1H,5H-pyrido[3.2.1-ij]-quinolinium hydroxide for zeolite A.After combining the reactants the synthesis mixtures were filled into a stainless steel autoclave and heated up tothe required synthesis temperature. After defined time intervals, samples were taken and subsequently analyzed byESI-MS (Waters ZMD), ESI-MS/MS (Bruker Esquire 3000) as well as dynamic light scattering (DLS). Further-more, the obtained materials were characterized by X-ray diffraction (XRD).Ergebnisse und Diskussion: The structure of zeolite ITQ-21 and zeolite A are characterized by four ring (4R) anddouble four ring (D4R) building motives. Their solid state structure mainly differs in the way how these motivesare linked to each other. In order to yield insights into their formation mechanism, in particular to understand theinfluence of various reaction parameters on the silicate species formed, their synthesis were studied time resolvedby ESI-MS and DLS. It was found that in the lower till middle mass range (∼ 200-1000 Da) 4R and D4R speciesare predominant in solution. This is indeed due to the preferred incorporation of germanium into such species.However, immediately before first particles start to grow, large silicate oligomers (> 1000 Da) are present. Theirstructures as well as the germanium distribution in these species were determined by fragmentation experiments(ESI-MS/MS): The obtained results indicate that their structures already represent structural characteristics of thefinal materials. The detected silicate oligomers are built up by D4R and/or 4R subunits; in particular it was foundthat their mode of linkage changes by varying the synthesis conditions (the organic SDA). Thus, the formation ofsilicate species with structural characteristics of the final material already in solution could be the basis to yielddifferent zeolite phases.Neue Aspekte: ESI-MS and ESI-MS/MS have been applied to yield insights into the formation mechanism ofzeolitic materials.Thema: Isotopen-MassenspektrometrieKeywords: Nucleation, Zeolites, Silicate Species, ESI-MS, Inorganic CompoundsKontakt: [email protected]

23

The role of mass spectrometry in protein studies, from peptideand protein characterization to proteomics

Roepstorff, Peter

Department of Biochemistry and Molecular Biology, University of Southern Denmark

Einleitung: Mass spectrometry (MS) is one of the most versatile analytical methods. Originally it could only beused for the analysis of molecules which could be brought on the gas phase by heating. In the 1980s new ionizationmethods were developed which allowed non volatile molecules such as proteins to be analyzed byMS. This openedan entirely new era in protein studies and especially two new ionization methods, matrix assisted laser desorptionionization (MALDI) and electrospray ionization (ESI) have now resulted in MS being a key analytical technique inprotein studies. The most widespread use of MS in protein studies is for protein identification and characterizationof post translational modifications in proteomics. Proteomics is based upon combining the information fromgenome sequencing with the information generated by MS for large scale identification of proteins and theirmodifications. However MS is also a powerful tool in structural studies of novel peptides and proteins suchas toxins, antifungal or antibiotic peptides from species where no genomic information is available. Combiningtraditional protein chemistry with mass spectrometry allows highly sensitive analyses of such often highly modifiedpeptides. Two strategies dominate in proteomics, one based on separation or the proteins by 2D-PAGE prior toprotein identification and characterization by mass spectrometry, the other based on proteolytic digestion of all theproteins in a sample followed by separation and sequencing of the resulting peptides by multidimensional LC-MS.A number of intermediate strategies are also used. Different strategies will be described and their strengths andlimitations evaluated for the use on different levels in peptide and protein characterization and proteomics. Someconcepts that allow modification specific proteome analysis will be described as well as strategies for the use ofmass spectrometry to study protein interaction and conformational changes.

24

PL1

Soft-landing of Complex Ions onto Self-Assembled MonolayerSurfaces

Laskin, Julia; Hadjar, Omar; Hu, Peng Wang Qichi; Johnson, Grant

Chemical Sciences Division, Pacific Northwest National Laboratory, Richland, WA, 99352, USA

Einleitung: Preparatory mass spectrometry utilizes deposition of mass-selected ions on substrates also calledion soft-landing. Immobilization of complex molecules on solid supports plays an important role in a variety ofdisciplines including materials science, catalysis and biochemistry. Covalent binding of transition metal complexesto polymer surfaces is commonly used for preparation of environmentally benign catalysts for organic synthesis,while covalently immobilized biomolecules are used for identification of biologically active motifs in proteins,development of novel biosensors and substrates for improved cell adhesion. Soft-landing of mass-selected ionsintroduces unprecedented selectivity and specificity into the surface modification step by eliminating the effect ofsolvent and sample contamination on the quality of the film and enables preparation of high-purity uniform thinfilms on surfaces.We will discuss fundamental aspects of soft-landing and reactive landing of complex ions on self-assembled mono-layer surfaces (SAMs). Deposition of mass-selected ions on surfaces is accompanied by a number of processesincluding charge reduction, neutralization, covalent and non-covalent binding, and thermal desorption of ions andmolecules from the substrate. Factors that affect the competition between these processes will be discussed. Funda-mental principles derived from such studies of interaction of protonated peptides with hydrophobic or hydrophilicsurfaces are relevant to the understanding of the transport of biomolecules through membranes in living organismsand provides a clear pathway for highly-selective preparation of biological and catalytic surfaces.

25

PL2

Die Himmelsscheibe von Nebra und weitere sensationelle Fundeaus Sachsen-Anhalt

Meller, Harald

Direktor des Landesamtes für Denkmalpflege und Archäologie Sachen-Anhalt und des Landesmuseums für

Vorgeschichte Halle (Saale)

Einleitung: Die Himmelsscheibe von Nebra zählt zu den bedeutendsten archäologischen Funden des letzten Jahr-

hunderts. Sie ist Teil eines Fundensembles, das um 1600 v. Chr. auf dem Mittelberg bei Nebra in Sachsen-Anhalt

niedergelegt und dort im Juli 1999 illegal von Raubgräbern geborgen wurde. Die auf ihr abgebildeten komplexen

astronomischen Phänomene machen sie zu einem Schlüsselfund der europäischen Vorgeschichte, der Astrono-

miegeschichte sowie der frühen Religionsgeschichte. Sie ist damit einer der ältesten Belege für die erstaunlichen

astronomischen Kenntnisse des bronzezeitlichen Menschen. Im Zuge des Vortrages werden neben den astrono-

mischen Zusammenhängen auch die weitreichende archäologische und kulturhistorische Bedeutung des Fundes

beleuchtet und weitere aufsehenerregende Neufunde der letzten Jahre aus der Mitte Deutschlands präsentiert.

26

PL3

Absolute accurate quantification and stoichiometry determinationof protein complexes

Holzmann, Johann; Pichler, Peter; Madalinski, Mathias; Kurzbauer, Robert; Mechtler, Karl

Institute of Molecular Pathology (IMP), Wien, Austria

Einleitung: Determination of protein complex stoichiometry can be achieved by absolute quantification of theinteracting subunits based on isotope dilution mass spectrometry. Current available platforms for the generationof standard peptides are cost-intensive and deliver variable results concerning the equimolarity of the peptidesmixture. Here we describe a novel and cost-efficient method to generate such an equimolar mixture of internalstandard peptides applicable to absolute quantification of proteins and subsequent stoichiometry determination ofcomplex constituents. We call this method the Equimolarity through Equalizer Peptide (EtEP) strategy.All selected internal standard peptides are chemically synthesized concatenated to a N-terminal equalizer peptide,which is a short peptide with a high ionization efficiency and an optimized trypsin cleavage site. Trypsinization ofthe concatamers releases exact equimolar amount of standard peptides and the equalizer peptide. By normalizingall internal standard peptides to the signal generated by the equalizer peptide an equimolar mixture of standardpeptides of high accuracy can be generated and absolute quantification of any protein of interest can be achievablevia the equalizer peptide. Hence, the equalizer peptide is the only peptide species from which the exact amounthas to be determined by amino acid analysis. Additionally, to cut the costs for quantification, we make use of themTRAQ reagent to introduce the isotopic label.We used the EtEP strategy to determine the MP1-p14 complex stoichiometry of two different concentrations, onesimulating a condition following tandem affinity purification. Absolute quantification of MP1-p14 was performedon two different mass spectrometers, and the 1:1 stoichiometry was confirmed with high accuracy and precision.In summary the EtEP strategy allows the generation of an equimolar mixture of standard peptides and signifi-cantly decreases the costs of absolute quantification and is ideally suited for a higher throughput in stoichiometrydetermination of protein complexes.

27

PL4

Current status of hyphenated mass spectrometry in clinical andforensic toxicology

Maurer, Hans H.

Department of Experimental and Clinical Toxicology, Saarland University, D-66421 Homburg (Saar), Germany

Einleitung: The reliability of analytical methods helps to ensure the quality of analytical data needed for correctinterpretation of analytical findings in clinical and forensic toxicology thus helping to avoid wrong treatment of thepatient or analytical data being contested in court. The analytical strategy mostly includes screening, confirmationand identification followed by quantification of relevant compounds and pharmacokinetics-based interpretationof the results. Mass spectrometry coupled to gas chromatography (GC-MS) or liquid chromatography (LC-MS)is the gold standard in this field because of its universality, reliability, high sensitivity and specificity. BesidesGC-MS more and more LC-MS techniques are used for target and comprehensive screening, library-assistedidentification, and validated quantification of drugs, poisons and their metabolites in blood, urine or alternativematrices. Concepts and procedures using LC-MS techniques in the areas of toxicology and drug monitoring withspecial focus on multi-analyte procedures will be presented and discussed [1-5]. The presentation will close witha short discussion of the potential of high resolution mass analyzers and future perspectives of MS in these fields.Referenzen:

[1] H.H. Maurer. Position of chromatographic techniques in screening for detection of drugs or poisons in clinicaland forensic toxicology and/or doping control [review]. Clin. Chem. Lab. Med. 42, 1310-1324 (2004)[2] H.H. Maurer. Hyphenated mass spectrometric techniques - indispensable tools in clinical and forensic toxicol-ogy and in doping control [review]. J. Mass Spectrom. 41, 1399-1413 (2006)[3] H.H. Maurer. Current role of liquid chromatography-mass spectrometry in clinical and forensic toxicology[review]. Anal. Bioanal. Chem. 388, 1315-1325 (2007)[4] H.H. Maurer. Mass spectrometric approaches in impaired driving toxicology [review]. Anal. Bioanal. Chem.393, 97-107 (2009)[5] H.H.Maurer. Perspectives of Liquid Chromatography Coupled to Low and High ResolutionMass Spectrometryfor Screening, Identification and Quantification of Drugs in Clinical and Forensic Toxicology [Review]. Ther. DrugMonit. in press (2010).Kontakt: [email protected]

28

PL5

Neue Perspektiven für die on-line / on-site Analytik mittelsEESI-MS

Zenobi, Renato

Departement Chemie und Angewandte Biowissenschaften, ETH Zürich, CH-8093 Zürich / Schweiz

Einleitung: Extraktive Electrospray Ionizations – Massenspektrometrie (EESI-MS) ist eine neuartige und sehr

leistungsfähige Methode für rasches chemisches “Fingerprinting” und für die on-line Analytik. EESI-MS beruht

darauf, dass eine neutrale gasförmige oder Aerosol-Probe in einen durch Elektropray-Ionisation (ESI) erzeugten

Sprühnebel geladener Tröpfchen eingeleitet wird. Durch Wechselwirkung der Probe mit dem ESI-Sprühnebel wer-

den die in der Probe enthaltenen Substanzen geladen, und können anschliessend wie in einer normalen Atmosphären-

druck-Ionisation massenspektrometrisch analysiert werden. Aus diesem Grund kann EESI ohne grossen Aufwand

auf kommerziellen MS-Geräten implementiert werden.

EESI-MS kann auch für die Analyse von Oberflächen eingesetzt werden. In diesem Fall geschieht das Sampling

durch eine Anblasvorrichtung, mit welcher z.B. durch einen Stickstoff-Strahl Oberflächenmoleküle von der Probe

abgetragen und zur Ionisation in die EESI-Quelle geleitet werden.

Dieser Vortrag wird die verschiedenen Einsatzgebiete der EESI-MS-Technik präsentieren:

1. Medizinische Diagnostik durch Analytik der Atemluft

2. Analyse von gefrorenem Fleisch und Fisch auf bakteriellen Befall

3. Direkte Analytik von Käseprodukten, von Milch, und von viskosen Flüssigkeiten

4. Verfolgen des Reifeprozesses von Früchten durch “Headspace-Sampling”

5. Prozessanalytik für das Verfolgen chemischer Reaktionen

6. Oberflächenanalytik menschlicher Haut

Ferner werden jüngste Ergebnisse zum Mechanismus der Ionenbildung in der EESI-Methode präsentiert. Als Aus-

blick in die Zukunft wird die Möglichkeit einer feldtauglichen EESI-MSAnalytik mittels portabler, miniaturiseirter

Massenspektrometer skizziert.

Referenzen: H. Chen, Y. Sun, A. Wortmann, H. Gu, and R. Zenobi, Differentiation of Maturity And Quality of

Fruit Using Non-Invasive Extractive Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry, Anal.

Chem. 79, 1447-1455 (2007)

H. Chen, A. Wortmann, W. Zhang, and R. Zenobi, Rapid in-vivo Finterprinting of Non-volatile Compounds in

Breath by Extractive Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry, Angew. Chem. Inter-

nat. Ed. 119, 580-583 (2007)

H. Chen, A. Wortmann, and R. Zenobi, Neutral Desorption Sampling Coupled to Extractive Electrospray Ioniza-

tion Mass Spectrometry for Rapid Differentiation of Biosamples by Metabolic Fingerprinting (Special Feature:

Tutorial), J. Mass Spectrom. 42, 1123-1132 (2007)

H. Chen and R. Zenobi, Neutral Desoption Sampling of Biological Surfaces for Rapid Chemical Characterization

by Extractive Electrospray Ionization Mass Spectrometry, Nature Protocols 9, 1467 – 1475 (2008)

L. Zhu, G. Gamez, H. Chen, K. Chingin, and R. Zenobi, Rapid Detection of Melamine in Untreated Milk andWheat

Gluten by Ultrasound-assisted Extractive Electrospray Ionization Mass Spectrometry (EESI-MS), Chem. Comm.

559 – 561 (2009)

W. S. Law, H. Chen, J. Ding, S. Yang, L. Zhu, G. Gamez, K. Chingin, Y. Ren, and R. Zenobi, Rapid Characterization

of Complex Viscous Liquids at the Molecular Level, Angew. Chem. Intern. Ed. 48 8277 – 8280 (2009)

Kontakt: [email protected]

29

PL6

Beruf Massenspektrometrie

Lehmann, Wolf D.

Molecular Structure Analysis, DKFZ, Heidelberg

Einleitung: Aus den unterschiedlichen zentralen Anwendungsfeldern der Massenspektrometrie werden Gemein-samkeiten und Unterschiede extrahiert. Darauf aufbauend wird versucht gemeinsame Merkmale und Fähigkeiten

der mit Massenspektrometrie arbeitenden Spezialisten abzuleiten. Fragen zum beruflichen Umfeld und Selbstver-

ständnis der Massenspektrometrie-Gemeinschaft werden gestellt und vorschlagsweise beantwortet: Was ist der

kleinste und größte gemeinsame Nenner unserer Tätigkeiten? Warum treffen wir uns regelmäßig zu MS Fachta-

gungen? Gibt es eine Balance zwischen Methodik und Anwendung? Machen Automatisierung und Informatik

MS-Spezialisten überflüssig? Sind Core Facilities Existenznische oder Operationsbasis für MS in der Forschung?Ist die MS eine Methode wie viele andere auch oder doch etwas Besonderes? Gibt es einen speziellen Wissens-und Fähigkeitskanon für MS Spezialisten? Wie könnte die ideale Ausbildung für den Nachwuchs aussehen? Hatdie MS ein Image in der Öffentlichkeit? Und zum Ausklang Reaktionsvorschläge zur Frage: Was machen Sieeigentlich beruflich?

30

31

Abstracts der Vortragsbeiträge

36

V1

Targeted data acquisition for improved reproducibility androbustness of proteomic mass spectrometry assays

Savitski, Mikhail; Fischer, Frank; Mathieson, Toby; Sweetman, Gavain; Lang, Manja; Bantscheff, Marcus

Cellzome AG, Germany

Einleitung: Recently, we described a chemical proteomics approach using immobilized non-specific kinase in-hibitors (KinobeadsTM ) that capture a large fraction of the kinome1. In a competition binding assay bindingpotencies of ATP-competitive inhibitor compounds are determined by measuring relative amounts of affinity en-riched kinases as a function of inhibitor concentration using quantitative mass spectrometry (iTRAQ). Affinitiesof inhibitor compounds against >100 kinases are determined in a single LC-MS/MS run. Data acquisition viaconventional data dependent peptide fragmentation (shotgun approach) leads to limited reproducibility in kinaseidentification in studies containing larger panels of inhibitor molecules. Here we present a targeted data acquisitionapproach that significantly improves reproducibility and precision of quantification of this proteomic assay.Experimenteller Teil: Kinobeads experiments were performed as described1 and samples were analysed on aLTQ-Orbitrap mass spectrometer coupled to an Eksigent Nano-HPLC. Gradient elution was performed over 4hours at a flow rate of 200 nl/min. Shotgun runs were acquired using a repeat count of 1 and dynamic exclusionfor 120 s. Protein identification was performed using Mascot and data were imported into a Oracle data base.Inclusion lists were generated based on statistical analysis of 80 such analyses and contained approx. 1200 includemasses (up to 10 per kinase). Eluting peptides matching to charge state, retention time and precursor ion mass (±7.5 ppm) were selected for up to 4 consecutive MS/MS scans using either PQD or a combined CID/HCD approach.Ergebnisse und Diskussion: Quantitative mass spectrometry-based proteomic assays often suffer from a lack ofrobustness and reproducibility. We developed a targeted mass spectrometric data acquisition strategy for affinityenriched sub-proteomes - in our case the kinome - that enables a substantially improved reproducibility of detec-tion, and improved quantification via isobaric tags. Inclusion mass lists containing m/z, charge state and retentiontime were created based on a set of 80 shotgun-type experiments performed under identical experimental con-ditions. For each target protein, peptides were selected according to their frequency of observation and iTRAQreporter ion quality. Retention times of selected peptides were aligned using similarity driven pairwise alignmentstrategy yielding <1 minute standard deviation for 4hr gradients. Multiple fragmentation of the same peptides re-sulted in better statistics and more precise reporter ion based quantification without any loss in coverage. Overall,24% more target proteins were reproducibly quantified using the targeted data acquisition approach, and precision

of quantification improved by > 1.5-fold. We also show that a combination of higher energy collisional dissociation

(HCD) with collisional induced dissociation (CID) outperformed pulsed-Q-dissociation (PQD) on the OrbitrapXL.

With the CID/HCD based targeted data acquisition approach 10% more quantifiable target proteins were identified

and a 2-fold increase in quantification precision was achieved. We have observed excellent reproducibility between

different instruments, underlining the robustness of the approach. We concur that the inclusion mass list-driven

targeted data acquisition approach of isobarically labeled peptides will be advantageous for a variety of affinity

purification based mass spectrometry assays.

Neue Aspekte: Targeted data acquisition approach for improved reproducibility and precision of quantification

quantitative mass spectrometry-based proteomics assays.

Referenzen: [1] M. Bantscheff et al, Quantitative chemical proteomics reveals mechanisms of action of clinical

ABL kinase inhibitors.; Nat Biotechnol. 2007 Sep;25(9):1035-44

Thema: Proteine und Peptide

Keywords: Targeted data acquisition, chemical proteomics, affinity enrichment, quantitative mass spectrometry,

protein kinases


37

V2

Metal Ion-Masked LC-MS for Straight Detection ofPhosphopeptides

Seidler, Joerg (1); Schlosser, Andreas (2); Panke, Jutta (3); Zinn, Nico (1); Böhm, Martin E. (1);

Bossemeyer, Dirk (2); Lehmann, Wolf D. (1)

1: Molecular Structure Analysis, German Cancer Research Center, Heidelberg; 2: Proteomics Core Facility,

Center for Systems Biology, Freiburg; 3: Mechanisms of Biomolecular Interactions, German Cancer Research

Center, Heidelberg

Einleitung: Phosphopeptide analysis by LC-MS/MS has developed into a major discipline in analytical pro-

teomics. It has the reputation of being particularly challenging, since incomplete recovery of singly and especially

multiply phosphorylated peptides from RP columns is often observed. Metal ion contamination of RP columns

has been proposed as major cause for phosphopeptide trapping. The addition of metal chelating additives has

been demonstrated to improve phosphopeptide recovery in LC-MS [1], in particular for multiply phosphorylated

species. Here we present diagnostic approaches for identification and quantification of the type of metal ions

accumulating on RP LC columns in particular when used in combination with IMAC.

Experimenteller Teil: Metal-ion masked LC has been evaluated by analysis of a seven phosphopeptide-standard

containing singly to quadruply phosphorylated peptides. This sample was analyzed on three different LC-MS sys-

tems. (i) nanoAcquity-UPLC-QTOF2 system (Waters) with direct on-column sample injection; (ii) nanoAcquity-

UPLC system (Waters) with trapping column interfaced to an LTQ-Orbitrap (Thermo); (iii) 1200 series LC with

HPLC-Chip system interfaced to a 6500 QTOF (both Agilent).

Ergebnisse und Diskussion: To identify metal ions present on LC columns we selected the strong trivalent

metal ion chelator desferoxamine. Following injection of free desferoxamine DFO, elution of the corresponding

DFO-Fe(III) and DFO-Al(III) complexes were observed by LC-MS. By repeated injections of DFO, metal ion

depletion of the LC-columns was observed. Direct injection of IMAC enriched phosphopeptide fractions was

found to result in accumulation of the IMAC-metal ion on the LC column. For instance, following the use of

Ga(III)IMAC, injection of DFO resulted in the elution of DFO-Ga(III) complexes. Analyses of the 7-component

phosphopeptide mixture showed an improvement of LC phosphopeptide recovery in the presence of DFO. Since

the LC elution of DFO and its metal ion complexes may interfere with peptides and phosphopeptides, the use

of DFO is recommended to monitor the metal ion contamination status of the LC system. For analytical routine

workflows, citrate [1], ascorbate and imidazole were evaluated, since these additives lead to metal ion depletion of

the LC column and elute with the injection front. An enhancing effect on phosphopeptide recovery was observed

for all three additives, with citrate performing best. It is also demonstrated that citrate addition to the sample is

also effective in an LC-MS set-up with trapping column.

This technical improvement results in an equal LC recovery for peptides and phosphopeptides and thus creates

the instrumental basis for standard-free phosphorylation degree determination by LC-MS. This is demonstrated by

following the in vitro autophosphorylation kinetics of the intracellular domain of the c-KIT receptor.

Neue Aspekte: Characterization of LC metal ion contaminants and improvement of phosphopeptide recovery by

their removal.

Referenzen: [1] Winter D, Seidler J, Ziv Y, Shiloh Y, Lehmann WD. J Proteome Res 2009, 8, 418-424. Citrate

boosts the performance of phosphopeptide analysis by UPLC ESI-MS/MS.


Keywords: Phosphopeptide, Recovery, LC-MS, Metal


38

V3

DBnovo: Optimierte Sequenzierung von Biopolymeren -Anwendungen und Aspekte

Kühn, Andreas (1); Tham, Katja (2); Heymann, Stephan (2); Freytag, Johann-Christoph (2);

Linscheid, Michael W. (1)

1: Humboldt-Universität zu Berlin, Institut für Chemie, Germany; 2: Humboldt-Universität zu Berlin, Institut für

Informatik, Germany

Einleitung:Massenspektrometrische Analysen von Biopolymeren werden derzeit mit Instrumenten-Methoden au-

tomatisiert, die durch eine überwiegend starre Abfolge von Einzelmessungen charakterisiert sind. Die Möglich-

keiten einer Einflussnahme in ein laufendes Messexperiment sind bisher sehr beschränkt und basieren auf recht

einfachen, direkten Informationen aus bereits aufgenommenen Einzelmessungen. Viele aus den Messungen ab-

leitbare Aussagen bspw. zur Sequenzabdeckung der untersuchten Analyten werden bislang erst in einem dem

Messlauf nachgeschalteten Prozessierungsschritt ermittelt (post-processing).

Experimenteller Teil: Die bereits vorgestellte Methode, DBnovo, ermöglicht eine direkte datenbankgestützte De-

novo Sequenzierung und Datenanalyse zur Laufzeit und bietet einen Zugang zu Aussagen wie z.B. dem Grad

der Sequenzaufklärung mit ggf. undefinierten Sequenzabschnitten oder die prognostizierte Verfügbarkeitsdauer

jedes Analyten, direkt nach jeder Einzelmessung. Dies eröffnet die Möglichkeit eine Reihe von aussagekräftigen

Kriteren zu nutzen, um das Messgerät während eines Analyseexperimentes zielgerichtet und problembezogen zu

beeinflußen, solange die Analyten noch verfügbar sind.

Ergebnisse und Diskussion: Als ein Anwendungsbeispiel kann mit DBnovo je nach angestrebter Sequenzie-

rungsqualität durch Auswahl der notwendigen Vorläuferionen und der zu verwendenden Aktivierungsparametern,

sowohl eine Identifizierung mit gewünschter Validität (Scoring), als auch eine vollständige Sequenzierung mög-

lichst aller Analyten forciert werden. Vergleichende Messexperimente mit proteolysierten Proteinmischungen als

Modellsystem zeigten, dass unter Einsatz von DBnovo im Vergleich zu bisherigen Methoden lediglich 50% der

Einzelmessungen benötigt werden. Die gesuchten Analyt-Sequenzen wurden dabei mit einer mindestens genauso

hohen Verläßlichkeit identifiziert. Die durch Minimierung von redundaten Messungen nun zur Verfügung stehen-

den Scans konnten verwendet werden, um bisher nicht untersuchte, niederabundanten Analyten zu identifizieren

und aufzuklären. Ein weiterer, enorm resourcensparender Vorteil besteht darin, dass am Ende eines Messexperi-

mentes bereits eine umfassende Datenanalyse vorliegt und die für ein post-processing benötigte Zeit um mehr als

eine Größenordnung reduziert werden kann. Durch eine zeiteffiziente und dynamische Verteilung der Fragmentie-

rungsexperimente mittels DBnovo, konnten die Anzahl an Einzelmessungen pro Analyt in einem Messlauf auf ein

Minimum reduziert werden. Je nach Zielsetzung kann der Schwerpunkt entweder auf die Anzahl der mit einer ge-

wissen Validität identifizierten Analyten gelegt werden oder auf die Vollständigkeit der Sequenzierung und damit

eindeutigen Aufklärung.

Neue Aspekte: Problem- und zielorientierte Steuerung von Massenspektrometern zur optimierten Sequenzierung

von Biopolymeren.

Referenzen: [1] [ PatentNr: P550108WO | DE 10 2008 021 703.4 ]


Keywords: DBnovo, Optimierte Sequenzierung, Gerätesteuerung


39

V4

Frog skin peptides - sequence analysis using high accuracy massspectrometry

Langsdorf, Markus (1); Ghassempour, Alireza (2); Roempp, Andreas (1); Spengler, Bernhard (1)

1: Justus Liebig University, Giessen, Germany; 2: Shahid Beheshti University, Tehran, Iran

Einleitung: Skin secretions of amphibians represent a rich chemical pool of host-defence compounds. They arepart of their body’s own defence system against microorganisms and predators. Peptides from glands of the dorsalskin have a large variety of bioactive functions and are of pharmaceutical interest. Therefore, they are excellentcandidates for developing novel therapeutic agents. Our aim is to increase the reliability of de novo sequencing,since frog peptides are not listed in peptide/protein sequence databases. In this work, we used a non-databaseassisted sequencing strategy and specified fragmentation characteristics.Experimenteller Teil: Skin secretions were collected by mild electrical stimulation of frogs. After centrifugation,filtration and freeze-drying, peptides were separated and fractionated by reversed-phase micro high performanceliquid chromatography (microHPLC). Mass spectra were acquired by electrospray ionisation Fourier transformion cyclotron resonance mass spectrometry (ESI-FTICR-MS). NanoHPLC-MS was used to analyze peptides atlow concentration levels. Peptides were characterized by manual de novo sequencing and composition-based se-quencing (CBS). The latter method is a non-database assisted sequencing strategy and includes two steps. In thefirst step, the amino acid composition is determined by evaluation of accurate mass values without employing prob-ability functions. The second step determines the peptide sequence by scoring the agreement between observedand expected fragment ion signals of permuted sequences.Ergebnisse und Diskussion: We characterized twelve peptides from the skin secretion of the Middle East treefrog Hyla savignyi. Nine peptides were classified as tryptophyllins H. They share sequence similarities with pep-tides found earlier in the skin secretion of Australian tree frogs (genus Litoria), called tryptophyllins L. Threesequences occur both as free acid and acid amide at the C-terminus. It was observed that amide peptides yieldedlower amounts of y-type ions by collision induced dissociation (CID) compared to their acid analogues. Otherpeptides of Hyla savignyi were named as "savignins", according to the taxonomic name. One peptide contains the

modified amino acid hydroxyproline, which could be identified and localized by high accuracy FTICR-MS. By

means of the frog skin peptides, we investigated particular fragmentation features. A mechanism of CO2 elimina-

tion of deprotonated peptides in CID measurements is proposed. The formation of internal z-type ions after ECD

is described as well. CID spectra show high amounts of internal b-type ions. We proved that internal (two-step)

fragments can be formed via y-type ions releasing water or ammonia, respectively, and via b-type ions detaching

its N-terminal amino acid. We also exemplified phenomena such as the proline effect and the formation of non-

direct sequence ions due to sequence rearrangements. We observed that proline containing peptides show a favored

cleavage N-terminal to proline after CID. We showed that the proline effect, which commonly suppresses other ion

signals and impedes the spectrum evaluation, can be useful for sequence determination. We found that rearrange-

ment products that usually lead to misinterpretations can instead contain valuable information. Knowledge of the

formation process is important when employing non-direct sequence ions for sequence analysis, as shown in this

work. Determined peptide sequences were confirmed by CID spectra of synthesized peptides.

Neue Aspekte: Novel peptides were reliably characterized by database-independent sequencing strategies. Frag-

mentation artifacts in MSn measurements were specified.


Keywords: frog peptides, composition-based sequencing, peptide fragmentation


40

V5

De Novo Sequencing Of Peptides On Single Resin Beads byMALDI-FTMS

Weber, Reinhold (1); Semmler, Angelika (2); Wittmann, Valentin (2); Przybylski, Michael (1)

1: Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, University of Konstanz, Konstanz,Germany; 2: Laboratory of Bioorganic Chemistry, University of Konstanz, Konstanz, Germany

Einleitung: Combinatorial peptide libraries obtained by split-mix synthesis have a great impact in drug discovery.Despite the fast access to solid-phase bound peptide libraries, in which each resin bead carries a single product (socalled one-bead-one-compound libraries), the analysis of such a variety of compounds remains a bottleneck. Here,we report a direct mass spectrometric approach facing this challenge [1]. Using a photolinker between bead andpeptide [2], an optimised sample preparation procedure and MALDI FTICR mass spectrometry, peptides could bedirectly desorbed and analysed. Furthermore, increasing the laser power leads to extensive fragmentation, leadingto sequence-specific b- and y”-ions. In cases in which the laser-induced fragmentation is providing no sufficientsequence information, additional IRMPD fragmentation could be successfully employed.Experimenteller Teil: The peptides 1) (Boc)Cys(Mmt)-Ile-Lys(Aloc)-Pro-Gly-Lys(Aloc)-Ala-Cys(Mmt)-Lys(Boc)and 2) (Boc)Cys(Mmt)-Ile-Lys(Aloc)-Gly-Pro-Lys(Aloc)-Ala-Cys(Mmt)-Lys(Boc) were synthsised with an auto-mated peptide synthesizer (ABI-433A, Applied Biosystems, Foster City, USA) using Fmoc strategy on Hydroxy-ethyl-Photolinker NovaSyn TG resin (Novabiochem, Läufelfingen, Switzerland). Resin beads were separated un-

der a microscope, placed on a MALDI target, smashed, and covered with 2,5-DHB. MALDI-FTICR spectra were

recorded on an Apex II FTMS (Bruker Daltonik, Bremen, Germany) equipped with a 7T supraconducting magnet

and a Scout 100 MALDI source with pulsed nitrogen laser at 337nm. Experimental parameters:15 - 20 laser shots

per scan with total power of 64-70%, scanned mass range m/z=100-1500. For IRMPD experiments, ions of interest

were isolated using SWIFT, and fragmented with a CO2-laser (Synrad, Mukilteo, USA) at 10, 6µm.

Ergebnisse und Diskussion: The two isobaric peptides were randomly selected from a 56-membered combina-

torial library designed for multivalent presentation of carbohydrate epitopes attached to Lys side chains. Least

diversity was obtained by permutation of the two neighbouring amino acids Pro and Gly. The protecting groups

chosen with regard to possible scaffolds were not removed prior to mass spectrometric analysis to simulate amino

acid modifications. A nitrobenzyl linker photocleavable at the wavelength of the UV-MALDI laser (337nm) was

used for attachment to the resin. The development of a suitable sample preparation procedure proved to be cru-

cial for subsequent MALDI-MS analysis and sequence determination. Best results were obtained by swelling

and smashing single beads on a target and addition of matrix, due to most homogeneous matrix-analyte mixture.

MALDI-FTICR spectra show as most prominent signals the protonated peptide ions upon loss of the acid-labile

Mmt and Boc groups. Moreover, series of sequence-specific b- and y”-ions were found. As increasing the laser

power from 60% to 70% leads to a substantial increased yield of fragment ions, these products are formed laser-

induced. Attempts to enhance fragmentation by further increasing the laser power >70% led to a decreased yield

of b- and y”-ions, as polymeric ions origination from the PEG chain of the resin TentaGel become predominant. In

case of both peptides, y”6-ions are formed with highest yield, so cleavage C-terminal of the sterically demanding

Lys(Aloc) is favoured. The relative higher yield of y”6-ions in the spectrum of peptide 1 can be explained by

the proline-effect [3, 4]. In cases where no complete sequence representation could be achieved by laser-induced

dissociation, the [M+H]+ was isolated and fragmented using SORI/CID or IRMPD. In summary, direct desorption

and laser-induced fragmentation of peptides using a photolinker proved to be an efficient and sensitive approach

for molecular characterisation of peptide libraries.

Neue Aspekte: De Novo sequencing of combinatorial peptides using laser-induced fragmentation in MALDI-

FTICR mass spectrometry

Referenzen: [1] A. Semmler, R. Weber, M. Przybylski and V. Wittmann, De Novo Sequencing of Peptides on

Single Resin Beads by MALDI-FTICR Tandem Mass Spectrometry, J Am Soc Mass Spectrom (2010), in press;

[2] J. M. Gerdes and H. Waldmann, Direct mass spectrometric monitoring of solid phase organic syntheses, J

Comb Chem (2003), 6, 814-820; [3] M. Przybylski, I. Dietrich, I. Manz, H. Brückner, Elucidation of Structure andMicroheterogeneity of the Polypeptide Antibiotics Paracelsin and Trichotoxin A-50 by FAB MS in Combinationwith Selective In-Situ Hydrolysis, Biomed. Mass Spectrom. (1984), 569; [4] L. A. Breci, D. L. Tabb, J. R. Yates,3rd and V. H. Wysocki, Cleavage N-terminal to proline: analysis of a database of peptide tandem mass spectra,Anal Chem (2003), 9, 1963-1971Thema: Proteine und PeptideKeywords: Combinatorial libraries, laser-induced cleavage and fragmentation, MALDI-FTMSKontakt: [email protected]

41

V6

GC-MS Analysis of Mars Relevant Oxygenates and Methane fromPhotochemically Induced Reaction of Carbon Dioxide and Water.

Bartoszek, Michael (1);Wecks, Mike (2); Jakobs, Gunnar (1); Möhlmann, Dirk (3)

1: Leibniz-Institut für Katalyse e.V., Rostock; 2: Institut für Nichtklassische Chemie e.V. Leipzig;3: Institut für Planetenforschung, Deutsches Zentrum für Luft- und Raumfahrt, Berlin

Einleitung: Inspired by the discussion about possible sources of methane in the atmosphere of Mars, the trans-formation of carbon dioxide to oxygenates involving UV irradiation under laboratory conditions was investigated.The aim was to investigate relationships of available carbon dioxide and (at least temporarily present) liquid in-terfacial water on the one hand and the influence of semiconductor materials like hematite as relevant planetarysurface material on the other, with respect to the possible formation of oxygenate molecules under UV irradiation.Experimenteller Teil: Assuming that a series of oxygenates will occur a GC-MS was used to analyze the photo-catalytic reaction mixtures. The experiments were carried out under atmospheric pressure and room temperaturein a glass cell equipped with a quartz window. This reaction cell was directly connected as a sample loop to aquadrupol GC-MS-system. An internal standard was available by adding of small amounts of krypton to the cellvolume prior to UV radiation experiments.Ergebnisse und Diskussion: The irradiation of carbon dioxide with UV-light in the presence of water results in theformation of C, O, and H containing molecules Oxygenates with one carbon atom were found like formaldehydeand methanol, but also C2- and C3-species like acetaldehyde and acetone. The role of hematite seems to beambivalent. Hematite has a significant influence on the distribution of the products. Additionally, the conversionof carbon dioxide increases in the presence of hematite. Note, however, that most of the oxygenate species alsoappear without hematite in the reaction cell. Experiments with 18O-labelled water show a transfer of oxygenfrom water to carbon which results in labelled oxygenate molecules. Oxygenates could be formed from CO2

via photocatalytic reduction processes. The observed formation of C2- and C3-species might point to secondarycompetitive and consecutive reactions via complex and less selective mechanisms. All these results give hints todifferent reaction mechanisms, and support the assumption that both photocatalytic and radical reactions contributeto the formation of oxygenates from carbon dioxide and water by UV-irradiation. More importantly, the resultsgive indication of a potential formation of hydrocarbons such as methane via conversion of carbon dioxide andwater on Martian mineral interfaces. Nevertheless, it is certainly necessary to gain more and quantitative insightinto the contribution of photocatalytic processes to the apparent presence of oxygenates and hydrocarbons in thenear surface atmosphere of Mars.Neue Aspekte: Results give indication of a potential formation of hydrocarbons via conversion of carbon dioxideand water on Martian mineral interfacesThema: Organische Massenspektrometrie, Umweltanalytik und AerosoleKeywords: oxygenates, photocatalysis, mars, GC/MSKontakt: [email protected]

42

V7

Rapid metabolic profiling of Nicotiana tabacum defense againstPhytophthora nicotianae using direct infrared laser desorption

ionization mass spectrometry

Ibáñez, Alfredo J. (1); Bones, Philipp (2); Scharte, Judith (2); Pirkl, Alexander (1); Hillenkamp, Franz (1);

Weis, Engelbert (2); Dreisewerd, Klaus (1)

1: Institute of Medical Physics and Biophysics, University of Münster, Robert-Koch-Str. 31, D-48149 Münster,

Germany; 2: Institute of Botany, University of Münster, Schlossgarten 3, D-48149 Münster, Germany.

Einleitung: The defense of N.tabacum toward P.nicotianae includes a form of programmed cell death called

hypersensitive response (HR).1 For the proper development of a HR, complex and not completely understood

signaling processes must occur simultaneously on the infected tissue. Here, we monitored different families of

secondary metabolites using direct infrared laser desorption ionization orthogonal time-of-flight mass spectrometry

applied to small pieces of infected, mechanically-wounded, and healthy tobacco leaves. The time course of plant

defense was followed between 1 - 9 hours post infection. Principal component analysis was applied for spectra

interpretation and elucidating which metabolic pathways are involved in the plant defense mechanism, as well as

identifying possible biomarkers that can be used to predict the quality of the pathogen infection.

Experimenteller Teil: An Er:YAG laser (λ = 2.94 µm; τ = 150 ns, spot size = 150 × 200 µm2, pulse repetition

rate = 2 Hz) was used for IR-LDI MS analysis.2 Ions were desorbed into a buffer gas environment of ∼ 1 mbar

of N2and separated using an orthogonal time-of-flight (o-TOF) mass spectrometer, providing a mass accuracy of

∼ 10 ppm. Two cultivars of N.tabacum were studied: (i) Samsun (SNN, capable of developing strong HR)1 and

(ii) Xanthi (not capable of producing sizable HR)1. Blank, placebo, and infected samples were compared at time

points of 1, 3, 6, and 9 hrs post infection. ESI-triple quadrupole MS/MS analysis of plant extracts3was used to

validate the IR-LDI-o-TOF MS results.

Ergebnisse und Diskussion: IR-LDI-o-TOF mass spectrometry allowed rapid and simultaneous detection of a

wide range of naturally occurring secondary metabolites, both in neg. and pos. ion mode. Overall, about 60

putative metabolites (e.g. alkaloids, carbohydrates, lipids, small organic acids) were detected directly from disk

probes cut from leaf tissue ( 0.5 cm2).

A supervised principal component analysis (PCA) was employed to find correlations between the overall defense

response of N.tabacum and the different metabolic profiles obtained at different time points and sample treatment

conditions. A unidimentional projection of the PC1 vs PC2 plot obtained from positive ion mode spectra can be

used to generate a Biological Response Index (BRI). Comparison between this BRI and a dead cell count analysis

(measured between 3-9 hours post infection) demonstrates that the BRI can be employed to rapidly determine the

quality (strength) of theN. Tabacum response toward the pathogen infection. Further evaluation of the loading plots

associated with the PCA plot hints that the plant defense is correlated to metabolic changes in the Shikimic acid,

oxylipin and Abscisic acid degradation pathways. Validation of this correlation was done with LC-ESI MS/MS

by monitoring the content - in plant extracts - of phytohormones that are associated with the above mentioned

pathways (Salicylic acid, Jasmonic Acid, and Abscisic acid).3

Finally, the evaluation of the PCA data also show that two unknown metabolites with m/z values of 269.170 and

431.222, detected by IR-LDI MS, may be utilized as putative biomarkers for predicting the amount of tissue

damage by the HR.

Further work will focus on identifying these observed biomarkers as well as the involvement of the proposed

metabolic pathways by employing N.tabacum mutants.

Neue Aspekte: The work presented here combines a robust MS system with multivariate analysis tools for

metabolic pathway elucidation, and biomarker discovery.

Referenzen: [1] Scharte, J.; Schön, H.; Tjaden, Z.; Weis, E.; von Schaewen, A. Natl. Acad. Sci. U.S.A. 2009, 106,

8061-8066.; [2] Dreisewerd, K.; Draude, F.; Kruppe, S.; Rohlfing, A.; Berkenkamp, S.; Pohlentz, G. Anal. Chem.

2007, 79, 4514-4520.; [3] Heiling, S.; Schuman, M. C.; Schoettner, M.; Mukerjee, P.; Berger, B.; Schneider, B.;

Jassbi, A. R.; Baldwin, I. T. The Plant Cell in press

Thema: Naturstoffe, Metabolomics

Keywords: Metabolomics, Pathogen-Plant interaction, PCA, IR-LDI-MS.


43

V8

Molekularer Fingerabdruck von Getränken mittelspos/neg-switching LC-API-ToF-MS

Letzel, Thomas

TU München, Germany

Einleitung: Reinheit und Audentizitäten von Getränken zu betimmen ist aufgrund der häufig komplexen Matrix

auch heute noch eine Herausforderung. Möchte man die ‚Matrix‘-enthaltenen Moleküle allerdings für einen ein-

deutigen Fingerabdruck nutzen, so benötigt man Ergebnisse, die man nur durch Massenspektrometrie so detailliert

ermitteln kann.

Experimenteller Teil: In der Präsentation wird eine neu entwickelte LC-MS-Methode vorgestellt, mittels derer

eine sehr große Zahl organischer Moleküle in Getränken analysiert werden kann, ohne diese zuvor aufbereiten zumüssen. Es wurden 25 unterschiedliche Getränke qualitativ und teilweise quantitativ u.a. auf etwa 50 Polyphenolehin untersucht. Weitergehend wurde mit diesen Messungen auch ein Fingerabdruck erstellt, der durch den grossenMessbereich, der akkuraten Masse und der Ionisationseigenschaften zustande kam.Ergebnisse und Diskussion: Die Trennung der Analyten fand an phenyl-modifizierten Silicapartikel unter erhöh-ter Flussrate statt, da ein Pumpsystem verwendet werden konnte, das bis 600 bar stabil arbeitet.Die Ionisation derin der Lösung enthaltenen Analyten wurde mittels sog. Multi-Mode-Ionisation bewerkstelligt. Diese Ionisations-technik ermöglicht den Übertritt von in Lösung geladenen Ionen in die Gasphase und produziert gleichzeitig Ionen

ursprünglich ungeladener Analyten durch Chemische Ionisation unterAtmosphärendruck.Die Detektion wurde mit

einem Flugzeit-Massenspektrometer durchgeführt, das parallel positiv sowie negativ geladene Ionen erfassen kann.

Die Nutzung von Massenstandards und einem Detektionsbereich von m/z 50-3100 ermöglichte die Detektion aller

untersuchter Substanzen.Dieser große Bereich und die Flexibilität erlaubt es die Daten zukünftig auch auf noch

unbekannte Komponenten zu re-analysieren.Vor- und Nachteile dieses analytischen Ansatzes werden diskutiert

und mehrere Anwendungen präsentiert.

Neue Aspekte: Neue Screeningmöglichkeit komplexer Gemische durch Nutzung der chromatographischen und

massenspektrometrischen Eigenschaften von Molekülen sowie deren Ionisationsverhalten

Thema: Organische Massenspektrometrie, Naturstoffe

Keywords: Lebensmittel, Fingerabdruck, Screening,


44

V9

Analyzing Complex Combustion Chemistry of NitrogenatedCompounds with TOF-Mass-Spectrometry

Lucassen, Arnas (1); Oßwald, Patrick (1); Struckmeier, Ulf (1); Kohse-Höinghaus, Katharina (1);

Kasper, Tina (2); Hansen, Nils (2); Labbe, Nicole (3); Westmoreland, Phil R. (3); Cool, Terry A. (4)

1: Universität Bielefeld, Germany; 2: Sandia National Labs, USA; 3: Noth Carolina State University, USA;

4: Cornell University, USA

Einleitung: Transportation fuels are being produced from biomass as substitutes for conventional fossil fuels.

They are considered renewable and carbon-neutral, although a critical discussion also addresses less benign effects

as land and water use, for example. From a combustion-chemistry perspective, some concerns are warranted since

biomass contains heteroelements such as nitrogen, oxygen, sulfur and others. Fuel-bound N and O may give rise

to harmful pollutants such as aldehydes, nitric oxide, and cyanic acid. We have therefore investigated flames of N-

and O-containing model fuels under premixed low-pressure conditions by flame sampling molecular-beam mass

spectrometry.

Experimenteller Teil: Gas is sampled from low-pressure premixed flames of selected fuels with a molecular-

beam setup preserving the molecule and radical composition at the sampling position. Time-of-flight (TOF) mass

spectrometry is well suited for simultaneous and quantitative detection of all relevant combustion intermediates.

Also, complex fuel structures, especially N- and O-containing fuels, can result in a rich mixture of different

flame species which presents a challenge for the analysis. Ambiguities in species assignment can be overcome

by combining high energy resolution of a TOF mass spectrometer employing single-photon VUV photo-ionization

(E/∆E =0.04 eV) that enables the separation of isomers, with the high mass resolution of an electron-ionization

reflectron TOF mass spectrometer (m/∆m=4000)[1].

Ergebnisse und Diskussion: In this study, we have chosen morpholine (1-oxa-4-aza-cyclohexane), a heterocyclic

compound with the chemical structure O(CH2CH2)2NH, as a fuel to investigate the fuel decomposition and oxi-

dation pathways, with special attention to the formation of harmful intermediates and products[2]. Morpholine is

widely used in organic synthesis, as a fungicide in agriculture, as an impregnating agent of fruit, cardboard and pa-

per, an additive for corrosion protection in steam generation, a defoaming agent for paper and pulp, and is a major

constituent of some N-containing fuel additives. A slightly fuel-richΦ = 1.3 (C/O = 0.41) flat morpholine-oxygen-

argon flame at 40 mbar was investigated. Quantitative mole fraction profiles of stable and radical intermediates

and products of the combustion process were derived. A kinetic model developed to predict the combustion be-

havior of morpholine is compared to these experimental data. Furthermore, an approach to assess the behavior of a

complex fuel by analyzing its building blocks in laminar premixed flames is attempted. Ethylamine and dimethy-

lamine were investigated under similar conditions. Based on these data, detailed fuel destruction schemes are

proposed and the formation of possible pollutants as well as soot precursors is compared. For morpholine, ethanol,

dimethylether[3], dimethylamine and ethylamine, results are reviewed as potential starting points of a modular

approach for the understanding of the combustion chemistry of more complex fuels.

Neue Aspekte: Flames of model substances for biofuel components are analyzed with respect to potential pollu-

tant emissions.

Referenzen: [1] Kohse-Höinghaus, K., Oßwald, P., Struckmeier, U., Kasper, T., Hansen, N., Taatjes, C.A., Wang,

J., Cool, T.A., Gon, S., Westmoreland, P.R., Proc. Combust. Inst. 2007, 31 1119-1127.;

[2] Lucassen, A., Oßwald, P., Struckmeier, U., Kohse-Höinghaus, K., Kasper, T., Hansen, N., Cool, T.A., West-

moreland, P.R., Proc. Combust. Inst. 2009, 32 1269-1276.;

[3] Wang, J., Struckmeier, U., Yang, B., Oßwald, P., Kohse-Höinghaus, K., Kasper, T., Hansen, N., Westmoreland,

P. R. J. Phys. Chem. A 2008, 112 9255-9265.

Thema: Instrumentelle Entwicklungen, Umweltanalytik und Aerosole

Keywords: In-situ-MS, MBMS, TOF, Biofuel, Combustion


45

V10

Investigating organocatalytic reactions: Mass spectrometricstudies of aldol condensation reaction catalyzed by

TFA-morpholine

Alachraf, Mhd Wasim; Zumbansen, Kristina; List, Benjamin; Schrader, Wolfgang

Max-Planck-Institut für Kohlenforschung, Germany

Einleitung: In the last few years, organocatalysis has emerged as a new catalytic methods based on metal-

free organic molecules. In many cases, these small compounds give rise to extremely high enantioselectivities.

Usually the reactions can be performed under an aerobic atmosphere with wet solvents. The catalysts can be easily

synthesized in both enantiomerically pure forms and they are often more stable than enzymes or other bioorganic

catalysts. Also, these small organic molecules can be anchored to a solid support and reused more conveniently

than organometallic/bioorganic analogues[1]. Herein we present a mechanistic study of the organocatalytic aldol

condensation reaction by ESI-MS, and ESI-MS/MS.

Experimenteller Teil: The mechanism study of the organocatalytic aldol condensation reaction was studied by

ESI-MS using a Thermo TSQ Quantum Ultra AM triple quadrupole. Different aromatic and aliphatic aldehydes

were used with (20% mol) TFA-morpholine as a catalyst in acetone. The acetone is used as a reactant and solvent

at the same time. The reaction was performed at 75 °Cfor 24h [2]. Samples were taken after different time intervalsfor mass spectrometric study. The reaction was performed at different conditions and deferent techniques wereapplied (e.g. online monitoring reaction) to study the mechanism of this reaction.Ergebnisse und Diskussion: There are two plausible mechanisms for the organocatalyzed aldol condensationreaction. The first mechanism is an aldol mechanism, in which the catalyst build an enamine intermediate with theketone (here acetone). This intermediate reacts in the next step with the aldehyde to build the second intermediate,which is hydrolyzed in the last step to form the condensation product and the regenerated catalyst. The secondpotential mechanism is a Mannich mechanism, in which the catalyst reacts with the aldehyde to form iminiumcation as a first intermediate, an acetone molecule attacks this intermediate in a nucleophilic attack to build thesecond intermediate. In the last step the catalyst splits off to form the condensation product. We tried in thisinvestigation to confirm witch route the reaction takes place using mass spectrometry. The first step in determiningthe proper mechanism of such a reaction is to find selective intermediates that can be used as reaction markersfor each mechanism. Reaction markers of both pathways were discovered. Although the empirical formula andthe mass of the intermediates of both mechanisms is identical, it is able to determinate the intermediates structureby using ESI-MS/MS according to McLafferty rearrangement fragmentation. These intermediates showed to bea perfect reaction marker, until we tried to verify these results with a retro-reaction between the final product andthe catalyst. This reaction showed that the intermediates could be able to formed throw the retro-reaction. Themechanism then was determined by freezing the reaction before the final hydrolysis step (i.e. before the formingof the final product).The final results will be presented.Neue Aspekte: Development and application of mass spectromety methods for the investigation of organocatalyticreaction.Referenzen: [1] P. I. Dalko, L. Moisan, Angew. Chem. 2004, 116, 5248-5286;[2] (submitted): Zumbansen, K;List, B.Thema: Organische Massenspektrometrie, Ionen-Molekül-Reaktionen

Keywords: Organocatalysis, Mechanistic study, Mass spectrometry, Tandem mass spectrometry, Electrospray

ionization.


46

V11

Challenges of big macromolecular soluble and membrane proteincomplexes for ESI-MS

Morgner, Nina; Hernandez, Helena; Zhou, Min; Lane, Laura; Robinson, Carol V.

Uni Cambridge, United Kingdom

Einleitung: With increasing size and complexity of soluble and membrane protein assemblies to be studied by

MS, it is necessary to develop not only the instruments further, but the techniques to fully analyse the obtained

data. Even though measurements of intact complexes can already be quite challenging, determining the structure,

neigbour relations and unknown stoichiometries usually requires the investigation of subcomplexes. Solution

disruption into stable subcomplexes as well as subunit stripping via CID can reveal required information, but

often hidden in very complicated spectra. Peak broadening, overlap or low abundancies hamper mass assignments.

Deducing the correct subcomplex from a measured mass can be even more challenging, especially if many possible

subunit combinations differ in mass by less than can be resolved.

Experimenteller Teil: We optimize experimental conditions to produce spectra that contain variing sets of sub-

complexes, by means of different solution disruption or CID conditions. For the thorough spectra analysis, we

are developing a new LabVIEW based software tool (Massign), with which simulated spectra can be produced to

resemble the experimental spectra. Peak series can be detected and compatible subcomplexes determined, based

on subunit masses. Mass shifts can be taken into account, as well as differences in behaviour of soluble/membrane

complexes, dependencies of the charge state distributions of parent complexes, dissociated subunits and subcom-

plexes. Known restraints regarding allowed/forbidden subunit combinations can be included, to rule out incorrect

combinations. Compositional changes depending on time, collision energy etc can be followed automatically over

spectra series.

Ergebnisse und Diskussion: The approach of producing mass spectra of large complexes and their subcomplexes

and simulating them with Massign has been very successful for different biological systems. Up to now several

macromolecular complexes, such as different ATPases, Polymerases or the spliceosomal U1snRNP have been

analysed this way. The simulations can be used for qualitative, and where applicable, quantitative analysis of the

spectra. Simulation of the spectra enables a more complete analysis of all subcomplexes than is possible with

standard programs. Even peak series with low intensity and/or considerable overlap with other species can be

extracted from an experimental data set. Considering charge state distributions of parent complexes, CID sub

complexes and dissociated subunits is often the only way to assign a peak series to the correct subcomplexes,

particularly where several possible subunit combinations differ in mass by less than can be resolved.

The dissociation pathways of different complexes could be determined for several VoV1 as well as FoF1 ATPases

as well as Polymerase I and III. Conclusions about assembly pathways could be drawn and information about

the stoichiometry and conectivity of subunits could be deduced, where not known before. In the case of the

spliceosomal U1snRNP it could be shown that two of the subunits (SmB/B’ and U1C) are dynamic and their

interactions with the core are affected by binding to different isoforms of the subunit U1-70k. This was achieved

by a careful inspection of relative abundancies of CID subcomplexes, which vary depending on the isoforms

present. Such an interpretation was not possible using standard software packages.

Neue Aspekte: Aquire qualitative / quantitative conclusions about structure of complexes and function of subunits

by fitting simulated spectra to experimental data


Keywords: macromolecular protein complexes, data analysis, simulation


47

V12

Structural insights into Munc13/calmodulin complexes byphotoaffinity labeling and mass spectrometry

Dimova, Kalina; Brose, Nils; Jahn, Olaf

MPI Experimentelle Medizin, Germany

Einleitung: Munc13 proteins are essential regulators of synaptic vesicle priming and play a key role in adap-

tive synaptic plasticity phenomena. We recently identified and characterized the Ca2+-dependent interaction of

Munc13 and calmodulin (CaM) as the molecular mechanism linking changes in residual Ca2+ concentrations to

presynaptic vesicle priming and short-term plasticity [1, 2]. To gain first structural insights into this interaction, we

used here peptidic photoprobes covering the established CaM-binding motif of Munc13 for photo-affinity labeling

(PAL) of CaM, followed by structural characterization of the covalent photoadducts.

Experimenteller Teil: We characterized the photoadducts formed by Munc13-derived benzophenone photoprobes

and CaM at the amino acid level by applying an innovative analytical strategy based on isotopically labeled CaM

and HPLC/MALDI-TOF-MS. Inclusion of 15N-CaM as reference protein facilitated the identification of cross-

linked peptides, which were further analyzed by MS/MS sequencing and on-target CNBr cleavage. The resulting

contact sites between Munc13 and CaM were complemented by constraints obtained from chemical cross-linking,

integrated into structural models, and correlated with NMR data.

Ergebnisse und Diskussion: Our PAL-based analytical workflow revealed that, in the bound state, the hydropho-

bic anchor residue of the CaM-binding motif in Munc13 proteins contacts two distinct methionine residues in the

C-terminal domain of CaM. By combining these distance constraints with the ones obtained from complementary

chemical cross-linking approaches, we were able to gain insights into both, binding sites (mainly by PAL) and

overall conformations (mainly by chemical cross-linking) of CaM in complex with Munc13. Molecular modeling

indicated that short Munc13 peptides (22 amino acids) bind to the C-terminal domain of CaM in an antiparallel

orientation through a 1-5-8 motif and led to the preliminary conclusion that Munc13/CaM complexes resemble the

closed conformation of the NO synthase/CaM complex [3]. To investigate the presence of additional interactions,

we employed bifunctional photoprobes based on C-terminally elongated Munc13 peptides (34 amino acids). PAL

data indicated that the Munc13/CaM complex adopts a rather open conformation with a modular architecture, in

which the Munc13 peptide N-terminus is anchored to the CaM C-terminal domain while a conserved Trp residue

of the Munc13 peptide C-terminus is embedded in the hydropobic cleft of the CaM N-terminal domain. This

finding was in agreement with NMR data on the solution structure of the Munc13/CaM complex and led to the

identification of a novel 1-5-8-26 CaM-binding motif [4]. Our approach can effectively complement NMR and

X-ray crystallography studies as it rapidly yields structural information with a reasonable technical effort, and,

most importantly, enables the characterization of a peptide-protein interaction under physiological solvent and

concentration conditions.

Neue Aspekte: Innovative analytical strategy for the structural characterization of peptide-protein interactions and

its application to a novel calmodulin target.

Referenzen: [1] Junge, HJ et al. (2004). Cell 118:389-401.; [2] Dimova, K et al. (2006). Biochim. Biophys.

Acta - Mol. Cell Res. 1763:1256-1265.; [3] Dimova, K et al. (2009). Biochemistry 48:5908-5921.; [4] Rodriguez-

Castaneda, F et al. (2010). EMBO Journal, doi:10.1038/emboj.2009.373.


Keywords: Photoaffinity labeling, cross-linking, peptide synthesis, calmodulin, synapse


48

V13

Untersuchung der Raumstruktur von Proteinen undProteinkomplexen mittels Ionenmobilitätsspektrometrie

Kipping, Marc (1); Campuzano, Iain (1); Langridge, James (1); Sobott, Frank (2); Ruotolo, Brandon (3);

Robinson, Carol (3)

1: Waters Corporation, Manchester; 2: Oxford University, Dept Chemistry; 3: Cambridge University, Dept

Chemistry

Einleitung: Die Möglichkeiten bei der massenspektrometrischen Untersuchung nichtkovalenter Proteinkomplexe

haben sich seit der Etablierung der Elektrospray-Ionisationstechnik durch die Entwicklung neuer Massenspektro-

meter und Elektrospray-Quellen zunehmend verbessert. So ist es heute möglich große Protein/Proteinkomplexe

intakt in die Gasphase zu überführen und so zum Beispiel Informationen über deren Stöcheometrie zu gewin-nen. Durch die Kopplung von Ionenmobilitätsspektrometrie (IMS) und Massenspektrometrie können durch Be-stimmung der Collisional Cross Section auch direkt Informationen über die Raumstruktur der Proteinkomplexegewonnen werden.Experimenteller Teil: Wir verwenden hier ein orthogonales Quadrupol-Tof-Massenspektrometer, welches miteiner Travelling-Wave (T-Wave) Ionenmobilitätszelle ausgestattet ist (Synapt HDMS, Waters, Manchester, UK).Alle Proben wurden unter nichtdenaturierenden Pufferbedingungen (10-100mM Amonium-Acetat, pH 6.8) mittelsDirektinfusion aufgegeben. Die gebildeten Gasphasen-Ionen passieren den Quadrupol-Massenfilter und gelangenanschließend in die IMS Sektion des Spektrometers. Diese besteht aus drei T-Wave Ion Guides. Im ersten werdendie Ionen gesammelt und anschließend paketweise freigesetzt. Im zweiten T-Wave Ion Guide werden die Ionen-pakete bei unterschiedlicher Mobilität in der Gasphase (unterschiedlicher Collisional Cross Section) getrennt. Derdritte T-Wave Ion Guide dient hier zur Weiterleitung der getrennten Ionen in den orthogonal angeordneten ToF-Analysator.Ergebnisse und Diskussion: Bei der Untersuchung der Collisional Cross Section des Proteinkomplexes von Tryp-tophan RNABinding Protein (TRAP) konnte gezeigt werden, dass die 11 Untereinheiten beinhaltende Ringstrukturdes Komplexes bei Übergang in die Gasphase erhalten bleibt. Außerdem konnte gezeigt werden, dass sich die Mo-lekülgröße durch Bindung eines spezifischen RNA-Moleküls vergrößert, während die Stabilität des Komplexeszunimmt. Die mittels IMS gemessene Collisional Cross Section des TRAP-Komplexes stimmt sehr gut mit denDaten der Röntgenstrukturanalyse überein. Zusätzlich zeigen wir ein Beispiel, bei dem die schlechte Kristalli-sierbarkeit eines Proteins durch das Vorliegen eines Gleichgewichtes mehrerer stabiler Konformationen bedingtist, das sich deutlich und schnell durch die Kopplung von Ionenmobilitätsspektrometrie und Massenspektrometrienachweisen lässt.Neue Aspekte:Der direkte Nachweis der Erhaltung der räumlichen Struktur eines Proteinkomplexes bei Übergangin die Gasphase nach Elektrospray-Ionisation wird gezeigt.Referenzen: [1] Anston et al., Nature, 401, 1999, 235-242.; [2] Ruotolo et al., Science, 310, 5754, 2005, 1658-1661.Thema: Proteine und PeptideKeywords: Protein, Proteinkomplex, RNA, Raumstruktur, IonenmobilitätsspektrometrieKontakt: [email protected]

49

V14

Quantitative mass spectrometry combined with a covalentpeptide approach for determination of protein interactions

Lange, Sabine; Krause, Eberhard

Leibniz-Institut für Molekulare Pharmakologie (FMP Berlin), Germany

Einleitung: Protein-protein interactions are often mediated by short peptide sequences. This gives the possibility

to use synthesized peptides on the basis of natural occurring proteins as well-defined baits for protein interac-

tion studies. Here we used peptides covalently coupled to agarose beads in combination with quantitative mass

spectrometry (metabolic and enzymatic labeling) to investigate phosphorylation-mediated interactions during T

cell receptor stimulation. Stable isotope labeling with amino acids in cell culture (SILAC) and enzymatic label-

ing with 18O water led to the identification and quantification of various interaction partners of the adhesion and

degranulation promoting adapter protein (ADAP).

Experimenteller Teil: Phosphorylated peptides containing the ADAP phosphorylation sites 595, 625, and 771 and

the corresponding non-phosphorylated analogs were synthesized and immobilized covalently on agarose beads.

Preparation of the peptide baits resulted in 20-30 nmol bound peptide per mg agarose which was determined by

quantitative amino acid analysis. Pulldown experiments (Tyr-phosphorylated versus non-phosphorylated peptide)

were performed with lysates from Jurkat T-cells. Proteins were separated by SDS-PAGE and identified by nanoLC-

MS/MS with an LTQ-Orbitrap instrument. For relative quantification of proteins, stable isotope labeling with

amino acids in cell culture (SILAC) and enzymatic labeling with 18O water was used. Specific interaction partners

were identified and quantified using the MaxQuant algorithms or Mascot Distiller Quantitation Toolbox.

Ergebnisse und Diskussion: For unambiguous differentiation between phosphorylation-specific protein interac-

tions and non-specific binding to ADAP peptide sequences, we employed two different stable isotope labeling

techniques. First, stable isotope labeling with amino acids in cell culture (SILAC) and second, enzymatic labeling

with 18O water, both in combination with high-resolution mass spectrometry was performed. With each peptide

sequence covering Tyr595, Tyr625, and Tyr771, we performed at least two experiments in a cross-over manner

leading to the relative quantification of more than 1000 proteins per experiment. Isotope ratios of identified pro-

teins indicate the dominance of non-specifically bound proteins with abundance ratios (phosphopeptide vs. non-

phosphorylated peptide) of about 1. For both labeling strategies an isotopic ratio above 5 was taken as evidence

of a specific interaction. This resulted in the identification of a total number of 12 phospho-specific binding pro-

teins. All of these contain SH2 domains which are known to bind specifically to phosphorylated tyrosine motifs.

Seven proteins bind phospho-specifically to all phosphotyrosine sequences studied, such as the proto-oncogene

C-crk (CRK) or 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-1 (PLCG1). In contrast, few

proteins like the crk-like protein (CRKL) or the proto-oncogene tyrosine-protein kinase FER (FER) were found to

bind phosphorylation-dependent to exclusive tyrosine motifs. This fact indicates the sequence-specific contribution

of tyrosine phosphorylation to the formation of ADAP-protein complexes in T cell receptor signaling. In conclu-

sion, these results show that quantitative mass spectrometry in combination with a covalent peptide approach is

well suited for determination of specific protein-protein interactions in the presence of an excess of non-specifically

bound protein background.

Neue Aspekte: Quantitative mass spectrometry with a covalent peptide approach gives insights into protein

interactions of the larger T cell receptor complex.


Keywords: quantitative mass spectrometry, peptide-protein interactions, SILAC, 18O-labeling, T cell signaling


50

V15

Mass spectrometric measurement of protease activities withMALDI and ESI mass spectrometers

Trusch, Maria (1); Hildebrand, Diana (1); Zhao, Xiaoxia (1); Linscheid, Michael (2); Schlüter, Hartmut (1)

1: Universitätsklinikum Hamburg-Eppendorf, Germany; 2: Humboldt-Universität zu Berlin

Einleitung: For the investigation of proteolytic activities, we developed the mass spectrometry based enzyme

screening (MES) method [1,2]. The assay comprises the immobilisation of a protein fraction onto chromatographic

beads and the incubation with a defined peptidic substrate. Originally the assay was combined with MALDI mass

spectrometry offering several advantages, such as the possibility to follow the fate of the substrate. Since it can

be very laborious to establish a relative quantification of the proteolytic reaction products with MALDI, the MES

method was additionally combined with the selected reaction monitoring (SRM) ESI-MS. The combined MALDI

and SRM-ESI approach is demonstrated on the development of an MES assay for the measurement of urotensin-

II-generating activity.

Experimenteller Teil: The proteins of the fraction containing the target protease are covalently immobilized onto

an affinity chromatography material. All substances that negatively interfere with MS can then be removed by

washing. For the determination of the protease activity the reaction specific probe - a peptide or protein, which

is identical with the endogenous substrate - is incubated with the immobilized proteins. After defined incubation

times aliquots are removed and analysed by MALDI-MS. The transitions for the ESI-SRM-MS are detemined by

anMS/MS analysis of the reaction product (urotensin-II). By applying LC-ESI-SRM-MS the relative quantification

of urotensin II was performed. The MALDI-MES and ESI-SRM-MES were applied for guiding the purification of

urotensin-II-generating enzymes and the measurement of urotensin-II-generating activities in blood plasma

Ergebnisse und Diskussion: The MES method in combination with MALDI-MS ensures high sensivities and

easily interpretable spectra since the substrate and the proteolysis products are the only molecules dissolved in the

incubation mixture, even if crude raw extracts can be tested for proteolytic activities. The presence of further

signals appearing at m/z not identical with those of the substrate or expected reaction products indicates the

presence of other proteases accompanying the target protease.

The determination of the urotensin-II-generating activity with a MES assay combined with LC-ESI-SRM-MS

yielded reliable and reproducible results. The results of both, the MALDI- MES and the ESI-SRM-MES approach,

demonstrate that they ideally complete each other for the development of MES protease assays and investigation

of proteolytic activities with MES.

In summary, the combined MES assay is a powerful tool for developing protease assays and measuring proteolytic

activities even in complex protein mixtures like blood plasma. It can be applied for a wide range of applications in

the investigation of proteases.

Neue Aspekte: Qualitative and quantitative analysis of proteolytic activities in complex biological samples.

Referenzen: [1] H. Schlüter, J. Jankowski, J. Rykl, J. Thiemann, S. Belgardt, W. Zidek, B. Wittmann, T. Pohl. De-

tection of protease activities with the mass-spectrometry-assisted enzyme-screening (MES) system. Anal Bioanal

Chem. 2003 Dec;377(7-8):1102-7;

[2] H.Schlüter, D. Hildebrand, C. Gallin, A. Schulz, J. Thiemann, M. Trusch. Mass spectrometry for monitoring

protease reactions. Anal Bioanal Chem. 2008 Nov;392(5):783-92.


Keywords: Proteases, MALDI, SRM, MES, relative quantification


51

V16

Explosives and chemical warfare agents - detection and analysiswith PTR-MS

Sulzer, Philipp (1); Petersson, Fredrik (1); Jürschik, Simone (1); Jaksch, Stefan (1); Jordan, Alfons (1);

Hanel, Gernot (1); Hartungen, Eugen (1); Seehauser, Hans (1); Märk, Lukas (1); Haidacher, Stefan (1);

Schottkowsky, Ralf (1); Märk, Tilmann D. (1,2)

1: Ionicon Analytik GmbH, Technikerstr. 21a, 6020 Innsbruck, Austria; 2: Institut für Ionenphysik und

Angewandte Physik, Leopold-Franzens Universität Innsbruck, 6020 Innsbruck, Austria

Einleitung: The broad range of advantages that proton-transfer-reaction mass spectrometry (PTR-MS) offers, e.g.

i) real-time analysis, ii) very low detection limits, iii) high selectivity, iv) no sample preparation, etc. seems to be

ideal for the detection of illicit substances. In the present work we demonstrate proof-of-principle investigations

on all common solid explosives and several chemical warfare agent (CWA) analogues. It is shown that not only

the sensitivity of the used PTR-MS instruments is sufficient to detect even the explosives with the lowest vapor

pressures (HMX), but it also provides a selectivity that allows for unambiguous identification and therefore avoids

false positives or false negatives.

Experimenteller Teil: We utilized two recently developed high sensitivity PTR-MS instruments equipped with

high resolution time-of-flight mass analyzers (see [1] for a description of the instrument) for detailed investigations

on explosives and chemical warfare agents (CWAs) [2,3]. With the PTR-TOF 8000 it is possible to record full mass

spectra with a mass resolution of up to 8.000m/∆m and down to a detection limit in the single digit pptv level.

The very recently developed PTR-TOF 2000 shows a somewhat lower mass resolution of about 2.000m/∆m and a

factor of five better sensitivity compared to the PTR-TOF 8000 instrument.

The solid explosives used were the commonly used ones: TNT, RDX, PETN, HMX and Semtex, whereas for

CWAs we used analogues for lab safety reasons.

Ergebnisse und Diskussion: We show that with the utilized PTR-MS instruments it is possible to identify solid

explosives (RDX, TNT, HMX, PETN and Semtex A) by analyzing the headspace above small quantities of samples

at room temperature and also from trace quantities not visible to the naked eye placed on surfaces. As the

mentioned solid explosives possess very low vapor pressures, the main challenge for detecting them in the gas

phase is to provide an instrument with a sufficient sensitivity.

CWAs on the other side have very high vapor pressures but are difficult to identify unambiguously as their nom-

inal molecular masses are usually comparably small and therefore hard to distinguish from harmless everyday-

compounds (e.g. mustard gas: 159 g/mol). It has to be mentioned that the analogue of mustard gas showed a small

impurity of "real" mustard gas, which we could detect without any difficulty. This leads us to the assumption that

the results are fully applicable to real CWAs.

In summary we can say that we cannot only detect a broad range of dangerous substances, ranging from the CWA

mustard gas to the explosive HMX, but also identify and distinguish them from other compounds with an extremely

high accuracy level due to a mass resolution of up to 8.000 m/∆m (FWHM).

Neue Aspekte: Two very recently developed PTR-MS instruments are used for the detection of illicit substances.

Referenzen: [1] A. Jordan, S. Haidacher, G. Hanel, E. Hartungen, L. Märk, H. Seehauser, R. Schottkowsky, P.

Sulzer, T.D. Märk, Int. J. of Mass Spec., 286 (2009), 122-128.;

[2] F. Petersson, P. Sulzer, C.A. Mayhew, P. Watts, A. Jordan, L, Märk and T.D. Märk, Rapid Commun. Mass

Spectrom. 23 (2009), 3875-3880.;

[3] C.A. Mayhew, P. Sulzer, F. Petersson, S. Haidacher, A. Jordan, L. Märk, P. Watts, T.D. Märk, Int. J. of Mass

Spec., 289 (2009), 58-63.

Thema: Instrumentelle Entwicklungen, Ionen-Molekül-Reaktionen

Keywords: PTR-MS, PTR-TOF, illicit substances, explosives, detection


52

V17

Schneller Nachweis von Betäubungsmitteln mittelsSPI-Massenspektrometrie und Laser-Ionenmobilitätsspektrometrie

Laudien, Robert; Schultze, Rainer

Optimare GmbH, Germany

Einleitung: Optimare entwickelt Messsysteme zur schnellen Detektion sicherheitsrelevanter Substanzen wie Be-

täubungsmittel und Sprengstoffe; zwei Geräte werden in diesem Beitrag präsentiert. Ein Messsystem basiert auf

einem Ion-Trap-Massenspektrometer (ITMS) mit Einzelphotonenionisation (SPI). Die Ionenquelle erlaubt eine

schonende Ionisation der Zielanalyten und somit die Anreicherung und empfindliche Detektion charakteristi-

scher Ionen (zumeist Molekülionen), so dass auf zeitaufwendige Probenvorbereitungen verzichtet werden kann.Zur Einzelphotonenionisation wird eine elektronenstrahlgepumpte Excimerlichtquelle eigener Fertigung einge-setzt (ELux). Dieses System wurde bereits erfolgreich in einem stillgelegten Drogenlabor getestet.Das zweite Detektionssystem basiert auf einem Ionenmobilitätsspektrometer mit resonanter Multiphotonenionisa-

tion (REMPI). Mit dem zur Ionisation eingesetzten frequenzvervierfachten Nd:YAG-Laser ist ein selektiver und

empfindlicher Nachweis aromatischer Verbindungen möglich. Durch Einsatz geeigneter Dopants können auchnichtaromatische polare Verbindungen über Ionenreaktionen wie Protonentransfer oder Komplexbildung nachge-wiesen werden.Experimenteller Teil: Zum Aufbau des SPI-ITMS-Systems wurde ein Ionenfallenmassenspektrometer der FirmaVarian (MS 4000) so modifiziert, dass zur Einzelphotonenionisation VUV-Licht der Excimerlichtquelle über eineSpiegeloptik in die Vakuumkammer vor die Ionenfalle fokussiert wird. Zur Erzeugung der VUV-Strahlung wirdder Elektronenstrahl einer Kathodenstrahlröhre durch eine dünne Membran (SiN) in ein Edelgas geführt. Dabeikönnen Ar (126 nm), Kr (147 nm), Xe(176 nm) oder verschiedene Gasgemische eingesetzt werden.Beim Ionenmobilitätsspektrometer werden die Ionen bei Atmosphärendruck gegen einen Gasstrom auf eine Fara-

dayplatte zu bewegt. Vakuumtechnik oder teure Multikanalplatten sind nicht erforderlich, was einen kompakten

und preiswerten Aufbau des Gerätes ermöglicht. Die Probenahme kann einerseits direkt aus der Umgebungsluftz.B. über ein Endoskop erfolgen. Zudem wurde ein Thermodesorptionssystem entwickelt, das eine Analyse auchschwererflüchtiger Substanzen erlaubt.Ergebnisse und Diskussion: Für den eindeutigen Nachweis sicherheitsrelevanter Substanzen mit dem SPI-ITMS-System werden die erzeugten Ionen zunächst selektiv angereichert und durch die Aufnahme von MS/MS-Spektren

nach Stoßfragmentierung identifiziert. Der effektive Einsatz dieses Verfahrens setzt eine selektive Ionisation und

Bildung charakteristischer Ionen voraus. Bei Drogen und Drogenvorläufern wird dies mit Krypton als Gasfüllungder VUV-Lampe erreicht. Viele Sprengstoffe weisen hingegen höhere Ionisationsenergien auf und erfordern denEinsatz von Argon oder speziellen Gasmischungen.Drogen und Drogenvorläufer, als oft schwererflüchtige Substanzen, können mit Wischtüchern genommen undüber einen Thermodesorber zugeführt werden. Dafür wurden unterschiedliche Konstruktionen getestet und Unter-suchungen zum Zeitverhalten, Totvolumen oder zur thermischen Fragmentierung durchgeführt.Das SPI-ITMS-System wurde durch die Untersuchungen verschiedener Drogen wie Cannabis, Heroin, Kokain,verschiedene Amphetamine und von Drogenvorläufern charakterisiert, wobei insbesondere das Fragmentierungs-verhalten bei SPI- und Elektronenstoßionisation verglichen und die Möglichkeit zur selektiven Anreicherung un-tersucht wurde. Anders als bei EI konnten durch SPI für diese Substanzen stets charakteristische Ionen, oft dasMolekülion erzeugt, angereichert und identifiziert werden. Bei einer Messkampagne in einem ehemaligen Drogen-labor konnten geringe Mengen an MDMA direkt in Proben von Wänden, Decken und Einrichtungsgegenständennachgewiesen und so die Möglichkeiten des Systems bei Vor-Ort Untersuchungen gezeigt werden.Zum Vergleich sind ausgewählte Zielanalyten der Betäubungsmittel und Drogenvorläufer auch mit dem Laser-Ionenmobilitätsspektrometer vermessen worden. Aufgrund ihrer aromatischen Struktur können die meisten Ver-bindungen direkt über resonante Multiphotonenionisation nachgewiesen werden. Andere Substanzen sind nichtdirekt ionisierbar, können aber über Protonentransfer, etwa von einem zuvor gebildeten Toluolradikalkation, io-nisiert und der Analyse zugänglich gemacht werden. Die Nachweisgrenze für alle Verbindungen liegt im unterenppb-Bereich. Die leichtflüchtigen Substanzen können aus der Umgebungsluft in das Ionenmobilitätsspektrome-ter eingebracht werden, schwererflüchtige Verbindungen mittels des schon erwähnten Thermodesorbers. Laser-Ionenmobilitätsspektrometer sind ein geeignetes Messsystem zum Nachweis von Betäubungsmitteln vor Ort ingeringen Konzentrationen. Sie stellen eine günstige Alternative oder sinnvolle Ergänzung des SPI-ITMS-Systemsdar.

53

Neue Aspekte:Vor-Ort-Nachweis von Betäubungsmitteln durch Einsatz der Einzelphotonen-Massenspektrometrie.Nachweis von Drogen und Drogenvorläufern mittels Laser-Ionenmobilitätsspektrometrie.Thema: Instrumentelle Entwicklungen, Ionen-Molekül-Reaktionen

Keywords: Einzelphotonenionisation, resonanteMultiphotonenionisation, Ionenfallenmassenspektrometer, Ionen-

mobilitätsspektrometer, BetäubungsmittelKontakt: [email protected]

54

V18

Generation and Identification of Reactive Metabolites and PhaseII Adducts by Electrochemistry Coupled On-line to Liquid

Chromatography/Mass Spectrometry

Jahn, Sandra; Baumann, Anne; Karst, Uwe

Westfälische Wilhelms-Universität Münster, Germany

Einleitung: Metabolism is generally regarded as a process contributing to the detoxification of xenobiotics. Duringthis process, xenobiotics such as drugs are converted into more polar compounds that are easily eliminated from thebody. In many cases, the resulting metabolites are less toxic than the corresponding parent drug. However, somedrugs undergo metabolic reactions that lead to the formation of reactive species ("metabolic activation"). These re-

active intermediates may modify proteins and DNA by covalent binding to such intercellular macromolecules and,

thus, cause drug-induced toxicity and cell damage. Consequently, it is very important to elucidate the metabolic

fate of new drug candidates in the human body at an early stage of development.

Experimenteller Teil: The main route of drug elimination is an enzymatic biotransformation which is frequently

initiated by oxidation reactions ("phase I metabolism") being catalyzed by enzymes of the cytochrome P450

superfamily. Nevertheless, the detection of reactive metabolites using conventional in vivo and in vitro techniques

is often hindered because the intermediately formed reactive species are prone to covalent binding to cellular

macromolecules making their detection very difficult. Therefore, the application of improved methods is required.

Electrochemistry (EC) is one of the classical methods to induce oxidation reactions. Thus, it seems obvious to

employ EC as simulation technique in drug metabolism studies. The on-line coupling of an electrochemical cell to

liquid chromatography/electrospray mass spectrometry (EC/LC/ESI-MS) allows for the direct detection of reactive

metabolites.

Ergebnisse und Diskussion: Three model compounds were investigated: dobutamine, a sympathomimetic drug,

galantamine, an alkaloid used in the treatment of Alzheimer’s disease, and lycorine, a toxic alkaloid found in

several plant species. As it is supposed that these substances are mainly metabolized in the liver by cytochrome

P450, EC/LC/MS experiments were carried out and compared to incubations with liver cell microsomes. The

electrochemical oxidation was accomplished using an amperometric thin-layer cell with a working electrode made

of boron-doped diamond (BDD) or glassy carbon (GC). Several reactive phaseI metabolites were found in this

electrochemical simulation of oxidative metabolism reactions. After being generated, the primary metabolites of

each compound were separated and identified on-line within a purely instrumental system using reversed-phase

liquid chromatography and electrospray mass spectrometry. In further studies, these metabolites were reacted

with conjugation agents such as glutathione (phaseII metabolism) or cysteine in order to evaluate their affinity

to endogenous nucleophiles. The obtained results were finally compared to the microsomal approach. Thus, the

particular significance of the presented electrochemical method as a rapid screening technique for the simulation of

oxidative metabolism reactions and as a beneficial complementary tool to conventional invivo and in vitro studies

is nicely demonstrated in this work.

Neue Aspekte: Reactive metabolites of xenobiotics are on-line generated, identified and reacted with trapping

agents by a purely instrumental method.

Referenzen: [1] W. Lohmann, U. Karst, Anal. Bioanal. Chem. 2008, 391, 79-96

Thema: Instrumentelle Entwicklungen, Organische Massenspektrometrie

Keywords: Electrochemistry, oxidative metabolism, xenobiotic, liquid chromatography, mass spectrometry


55

V19

MS/MS/MS-Quantifizierung in einem hybriden TripleQuadrupol/Lineare Ionenfallen-Massenspektrometer

Lenz, Christof; Lembcke, Jan; Besa, Axel

AB Sciex, Germany

Einleitung:Multiple Reaction Monitoring (MRM) ist als massenspektrometrische Standardmethode für die quan-

titative Bestimmung organischer Analyten in komplexen Matrices etabliert. MRM bezieht seine Selektivität aus

der Überwachung substanzspezifischer MS/MS-Fragmentierungsreaktionen in einer Quadrupol-Kollisionszelle. Inhochkomplexen Matrices reicht diese Selektivität jedoch vielfach nicht aus. Hybride Triple Quadrupol/LineareIonenfallen-Massenspektrometer (QqLIT, QTRAP) ermöglichen, im Anschluss an die Kollisionszellen-Fragment-ierung einen weiteren Fragmentierungsschritt in der linearen Ionenfalle in Quadrupol Q3 durchzuführen. Bei ge-eigneter Auswahl der MS3-Fragmente lässt sich die Selektivität der Detektion so weiter steigern. In dieser Prä-sentation diskutieren wir technologische Fortschritte der neuesten Generation von QqLIT-Systemen, mit denen dieSelektivität und Empfindlichkeit der MS3-Fragmentierung zur Quantifizierung in komplexen Gemischen deutlichverbessert wird.Experimenteller Teil: Alle Analysen wurden auf hybriden QqLIT-Massenspektrometern der neuesten Generationmit Turbo V-ESI-Quelle bei analytischen Flussraten durchgeführt (AB SCIEX QTRAP 5500 LC/MS/MS System).Das Fragmentierungsverhalten der Analyten wurde durch Infusion gereinigter Standards optimiert, insbesondereim Hinblick auf Interface-Parameter, Kollisionsenergie in Q2, Anregungsamplitude und -dauer in Q3. Zur Quanti-fizierung wurde jeweils eine LC/MS/MS on-line Kopplung mit Agilent 1200 Chromatographiesystemen etabliert.Detektions- und Quantifizierungslimits, der lineare Bereich der Quantifizierung sowie die Kompatibilität mit ana-lytischen HPLC-Trennsystemen wurden für zwei Anwendungen etabliert und mit der traditionellen MRM-Technikverglichen: a) Quantifizierung von Proteinen in humanem Serum nach tryptischem Verdau und b) Quantifizierungvon THC-Carbonsäure direkt aus Haaren.Ergebnisse und Diskussion:Die MS3-Fragmentierung auf QTRAP-Massenspektrometern unterscheidet sich zumTeil deutlich von der MS3-Fragmentierung in Ionenfallen-Massenspektrometern [1]:1) Zeitneutrale Isolierung des Vorläuferions in Q1 statt in der Ionenfalle2) Durchführung des ersten Fragmentierungsschritts in einer Quadrupol-Kollisionszelle statt in der Ionenfalle3) Verwendung einer Schmalbandanregung zur Durchführung des zweiten Fragmentierungsschrittes in der linearenIonenfalle statt der in kommerziellen Ionenfallen etablierten Breitbandanregung, was die Selektivität des zweitenFragmentierungsschrittes erhöht4) Verwendung eines gepulsten Gasventils in der linearen Ionenfalle, um den Kollisionsgasdruck für den zweitenFragmentierungsschritt lokal zu erhöhen [2].In Verbindung mit der signifikanten Erhöhung der Empfindlichkeit der Linearen Ionenfalle gegenüber früherenBaureihen ist die Quantifizierung per MS3 („MRM3") damit eine äußerst selektive Alternative zur etabliertenMRM-Technik. Dies wird an zwei Beispielen exemplarisch diskutiert. Die Quantifizierung niedrig abundanterProteinbiomarker aus humanem Serum oder Plasma per MRM stellt nachwievor eine massive Herausforderungdar, da es sich um Proben von extremer Komplexität handelt. Die MRM-Analyse klinisch relevanter Proteinbio-marker wie z.B. PSA wird derzeit vor allem durch hohen Untergrund limitiert. In einer Pilotstudie konnte gezeigtwerde, dass MS3 das Signal zu Rausch-Verhältnis auf klinsichen Proben signifikant verbessert, so dass vor allem inder Nähe des Quantifizierungslimits (Bereich ng/ml) eine robuste Quantifizierung erzeilt werden kann [3]. Tetrahy-drocannabinolcarbonsäure (THC-COOH), wird in den Haaren eingelagert und eignet sich somit hervorragend zurBeobachtung des THC-Konsumverhaltens. Haare stellen eine Matrix von äußerst komplexer Zusammensetzungdar. MRM-Analysen in dieser Matrix sind oft von starken Interferenzen geprägt, so daß sie trotz der hohen Selekti-vität des MRM oft nicht auswertbar/quantifizierbar sind. Selbst aufwendige Derivatisierungen führen aufgrund derstrukturelle Verwandschaft von THC-COOH zu einigen Fettsäuren kaum zu einer Minimierung der Interferenzen.Die Verwendung vonMS3 zur Quantifizierung bietet hier ein Mittel, die Selektivität massiv zu erhöhen, ohne dabeiein vermindertes Signal-Rausch-Verhältnis in Kauf zu nehmen.Neue Aspekte: Verwendung von MS3 Scanfunktionen in hybriden Triple Quadrupol/Lineare Ionenfallen-Massen-spektrometern zur Quantifizierung

56

Referenzen: [1] Collings BA, (2007) Fragmentation of ions in a low pressure linear ion trap. J. Am. Soc. MassSpectrom. 18, 1459; [2] Collings BA and Romaschin AR, (2009) MS/MS of ions in a low pressure linear ion trapusing a pulsed gas. J. Am. Soc. Mass Spectrom. 20, 1714; [3] Fortin T et al, (2009) Multiple Reaction MonitoringCubed for Protein Quantification at the Low Nanogram/Milliliter Level in Nondepleted Human Serum. Anal.Chem. 15, 9343.Thema: Instrumentelle Entwicklungen, Proteine und PeptideKeywords: Quantifizierung, QTRAP, Proteomics, ForensikKontakt: [email protected]

57

V20

Application of MALDI-imaging MS to monitor surface changes ofphoto-oxidized poly(styrene) films

Crecelius, Anna (1,3); Schubert, Ulrich (1,2,3)

1: Friedrich-Schiller-University, Germany; 2: Eindhoven University of Technology, The Netherlands;3: Dutch Polymer Institute, The Netherlands

Einleitung: Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-imaging MS) is arelative new technique, which has been developed by Richard Caprioli’s research group, and is nowadays appliedin a variety of fields, such as proteomics [1], peptidomics [2], lipidomics [3], and metabolomics [4]. However, upto now, this technique has not been exploited to the surface analysis of polymer films. Therefore, the aim of thiscontribution is the usage of this new technique to monitor changes occurring at the surface of polymer films.Experimenteller Teil: A size exclusion chromatography (SEC) standard poly(styrene) (PS) from PSS PolymerStandards Services GmbH (Mainz, Germany) was used throughout these studies (Mn=4755 g/mol, Mw=4992g/mol, PDI=1.05). Films were prepared by spin coating a mixture of 1:3:1 (v/v/v) containing 10 mg/mL PSdissolved in toluene, 30 mg/mL trans-[2-3-(4-tert-butylphenyl)-2-methyl-2-propen-ylidene]malononitrile (DCTB)dissolved in tetrahydrofuran (THF), and 100 mg/mL AgTFA dissolved in THF. The photo-oxidation of PS wascarried out in a UV box (6W, 254 nm) at room temperature. MALDI-TOF MS measurements were performed withan Ultraflex III TOF/TOF (Bruker Daltonics, Bremen, Germany) mass spectrometer equipped with a Nd:YAG laserand a collision cell.Ergebnisse und Diskussion: PS films were prepared by spin coating the polymer with the matrix DCTB and theionization salt AgTFA. The thickness of the resulting films was in the nm range (100-200 nm). The films weretreated with UV radiation at 254 nm for different time intervals (1, 24, 48, and 72 h) laying an aluminum maskwith holes of 6 mm diameter on top of the films. The ion intensity maps constructed after the MALDI-imaging MSexperiments from different repeating units of PS showed that the PS films were destroyed at the area of irradiation.Possible photo-oxidized products were not detected. The distribution of intact PS depends on the time intervalof UV irradiation. Future experiments will be concentrated on plasma-oxidized PS films and compared with theresults obtained by photo-oxidation.Neue Aspekte: MALDI-imaging MS of polymer filmsReferenzen: [1] A. C. Crecelius, D. S. Cornett, R. M. Caprioli, B. Williams, B. M. Dawant, B. Bodenheimer, J.Am. Soc. Mass Spectrom., 2005, 16, 1093.;[2] K. Sköld, M. Svensson, A. Nilsson, X. Zhang, K. Nydahl, R. M. Caprioli, P. Svenningsson, P. E. Andrén, J.Proteome Res., 2006, 5, 262.;[3] K. E. Burnum, D. S. Cornett, S. M. Puolitaival, S. B. Milne, D. S. Myers, S. Tranguch, H. A. Brown, S. K. Dey,R. M. Caprioli, J. Lipid Res., 2009, 50, 2290.;[4] D. Hölscher, R. Shroff, K. Knop, M. Gottschaldt, A. Crecelius, B. Schneider, D. G. Heckel, U. S. Schubert, A.Svatos, Plant J., 2009, 60, 907.Thema: ImagingKeywords: MALDI-imaging MS, polymer films, poly(styrene), surface analysisKontakt: [email protected]

58

V21

Recombinant Isotope Labelled and Selenium Quantified (RISQ)Protein Standards for Quantification of Proteins and Their

Degree of Modification

Zinn, Nico; Tittebrandt, Sebastian; Lehmann, Wolf D.

Deutsches Krebsforschungszentrum, Germany

Einleitung: The Identification of proteins with mass spectrometry is a standard technique. Currently the focuschanges to quantitative proteomic studies. While relative quantification between two cell states can be carriedout by differential labelling procedures, absolute quantification is more challenging since it requires an absolutelyquantified reference peptide or protein. Here a novel, widely applicable method for the production of absolutelyquantified proteins is described, which can be used as internal standards for quantitative proteomic studies basedon mass spectrometry. These standards are recombinant isotope labelled and selenium quantified proteins andtherefore named RISQ proteins.Experimenteller Teil: For recombinant protein expression, assembly of expression vectors fitted to cell-freeprotein synthesis was conducted using the gateway technology which offers fast access to a variety of genes viaopen reading frame libraries and an easy shuttling of genes between vectors. RISQ proteins are generated bycell-free expression in a medium in which methionine is exchanged against selenomethionine which is used forabsolute quantification by element mass spectrometry and at least one amino acid is exchanged by a highly stableisotope labelled analogue. The heavy amino acids in the RISQ protein serve as reference in subsequent analysesby LC-ESI-MS or MALDI-MS.Ergebnisse und Diskussion: Apolipoprotein A1 was synthesized as RISQ protein and its use as internal standardled to quantification of a commercial reference sample with an accuracy of ±10 %. Owing to their cell-freeexpression, RISQ proteins do not contain posttranslational modifications. Thus, correct quantitative data by ESI-or MALDI-MS are restricted to the use of peptides derived from unmodified regions of the analyte protein. Onthe other hand, this property shows that RISQ proteins are standards with two functions supporting (i) absolutequantification of a target protein and (ii) unbiased recognition of modified protein regions and their degree ofmodification.Neue Aspekte: Cell free synthesis of isotope labelled and selenium quantified protein standards for proteomics.Thema: Element-Massenspektrometrie, Proteine und PeptideKeywords: Quantitative proteomics, protein standards, RISQKontakt: [email protected]

59

V22

A new tandem mass spectrometry approach for the detection andidentification of O-GlcNAc-modified peptides

Hahne, Hannes; Kuster, Bernhard

TU München, Germany

Einleitung: The modification of serine or threonine residues with N-acetylglucosamine (O-GlcNAc) is an emerg-

ing dynamic PTM on nuclear and cytoplasmic proteins, but their large-scale mass spectrometry-based identification

still remains challenging. This is especially due to high cycling rate of O-GlcNAc sites, their substoichiometric

occupancy as well as the lability of the beta-O-glycosidic bond in the gas phase. Aiming for a robust and sensitive

strategy for the detection and identification of O-GlcNAc peptides, we compared and characterized different frag-

mentation modes on an LTQ Orbitrap XL ETD mass spectrometer using a synthetic O-GlcNAc peptide library.

Based on that, we developed a two-step pseudo precursor scanning approach for the detection and identification of

O-GlcNAc peptides in highly complex proteomic samples at the attomol level.

Experimenteller Teil: Using standard Fmoc solid phase peptide synthesis (Intavis, Cologne) we have generated

a synthetic 72 member O-GlcNAc peptide library. This peptide library was first used to evaluate fragmentation

techniques (CID, PQD, HCD, ETD) and data-dependent acquisition modes (MS2, NLMS3, MSA) on an LTQ

Orbitrap XL mass spectrometer. Based on the same library we also developed a two-step pseudo precursor ion

scanning approach comprising a discovery LC-MS/MS run using PQD fragmentation, followed by the extraction

of O-GlcNAC specific reporter m/z values and retention times of candidate O-GlcNAc peptides followed by a

candidate identification LC-MS/MS run using ETD. This approach was assessed by spiking a complete tryptic E.

coli protein digest with known amounts of O-GlcNAc-modified alpha-crystallin from bovine lens.

Ergebnisse und Diskussion: Like phosphopeptides, CID spectra of O-GlcNAc peptides are dominated by strong

peptide and fragment neutral loss species thus eliminating information on the precise modification site. On the

other hand, these spectra can contain intense diagnostic reporter ions (e.g. m/z 204.08665), but these are only

occasionally observed due the low mass cut-off in the ion trap. In addition, many amino acid combinations lead

to fragment ions, which interfere with the most intense m/z 204 reporter ion. The detection of this reporter ion

only becomes unambiguous when the MS/MS ions are detected with 30k resolution and high mass accuracy in

the Orbitrap after HCD fragmentation. However, lower resolution (7.5k or 15k) still provides an accurate mass

of the dominating species and concomitantly increases scan speed. Another fragmentation technique without

low mass cut-off is PQD. By optimizing PQD parameters (normalized collision energy, Q value, delay time),

possibly interfering fragments can (almost completely) be suppressed, thus offering a mode for pseudo-precursor

ion scanning on an LTQ Orbitrap mass spectrometer. In contrast to the aforementioned fragmentation techniques,

ETD spectra often contain sufficient sequence ions retaining the O-GlcNAc moiety for an unambiguous assignment

of the PTM site. Aiming for a sensitive and robust O-GlcNAc peptide detection and identification strategy,

we evaluated a two-step pseudo-precursor ion scanning approach which combines a discovery LC-MS/MS run

using PQD fragmentation at low normalized collision energy, extraction of m/z and retention time of candidate O-

GlcNAc peptides along with a final identification LC-MS/MS run using ETD fragmentation. A spiking experiment

revealed a limit of detection and identification in the range of 1ng O-GlcNAc modified protein in the presence of

2000ng of a complete tryptic E. coli digest.

Neue Aspekte: comparison of fragmentation modes for O-GlcNAc peptides, detection and identification of O-

GlcNAc peptides by a two-step pseudo precursor scanning approach


Keywords: proteomics, O-glycosylation, tandem MS, precursor ion scanning


60

V23

Elucidation of Temporal and Spatial Protein Patterns inDeveloping Barley Grains by Multiplexed LC-MS and

MALDI-imaging MS Approaches

Matros, Andrea (1); Kaspar, Stephanie (1); Seiffert, Udo (2); Weschke, Winfriede (1); Mock, Hans-Peter (1)

1: IPK-Gatersleben, Germany; 2: IFF Magdeburg, Germany

Einleitung: Recent developments in analytical techniques have enabled high trough-put acquisition of data as apre-requisite to characterize biological systems in a comprehensive manner. Especially the assessment of pro-teins has gained substantial impact from the advances in mass spectrometric techniques, which made these largemolecules better accessible for qualitative and quantitative analysis. We are interested in the proteome analysisof barley tissue. In particular we want to have a closer insight into changes of the proteome during barley graindevelopment, because better understanding of cereal seed development is one of the prerequisites for improvementof human nutrition.Experimenteller Teil: Protein extracts from developing barley grains of various stages were separated on ananoUPLC combined with ESI-Q-TOF MS. Data acquisition was performed by multiplexed LC-MS. For dataprocessing and protein identification the ExpressionE software (Waters) was utilised. To elucidate statisticallysignificant and objective kinetic patterns and for biomarker identification multivariate statistics was applied. Wealso want to gain a closer insight into the special protein distribution. Therefore we started adaptation of the fairlynew MALDI-imaging MS technique to cryo-dissected grain material. Initial data showing the applicability ofMALDI-imaging MS for analysing barley grains will be presented.Ergebnisse und Diskussion: Complex tryptic peptide mixtures from developing barley grains (3, 5, 7, 10, and 16days after flowering (DAF)) were analysed as mentioned above. Acquired data sets were processed and proteinsidentified with the ExpressionE system solution (Waters) processing the intensities of molecular ions for quan-tification and the fragment and molecular ions for identification. Besides, quantification allows also grouping ofpreliminary unidentified peptides. For an elucidation and visualisation of temporal changes in protein patternsand for biomarker identification multivariate statistics was applied. Prior to this, data pre-processing and initialvisualization was performed to ensure the quality of the data and the appropriateness of the subsequently appliedclustering algorithm. A number of computational intelligence based clustering algorithms, such as Self-OrganizingMaps (SOM) and Neural Gas (NG), that have proven to be highly suitable in a similar context, were applied forthe clustering task. Results indicated three major classes of changes in protein patterns during developmentalprocesses: i) high abundance at latest developmental stages (10 and 16 DAF), ii) transiently increased abundanceat 7 DAF, and iii) almost unchanged with slightly increased abundance at 5 DAF. Examples will be presented.Moreover, we want to gain a closer insight into local protein/peptide distributions. For this purpose cross sectionsfrom developing barley grains were prepared and analysed for metabolites and proteins/peptides by MALDI imag-ing mass spectrometry technique. Initial data are presented, which reveal distinct local distribution of individualproteins/peptides as well as metabolites. The future task will be to transfer the developed analytical methods fromthe scale of the whole organ down to the level of cellular regions to monitor spatio-temporal changes of proteindistributions and relate the patterns to distinct cellular and physiological events.Neue Aspekte: Multiplexed LC-MS and MALDI-imaging MS approaches enabled the elucidation of temporaland spatial protein patterns in developing barley grains.Thema: Proteine und PeptideKeywords: multiplexed LC-MS, MALDI-imaging MS, barley, seed developmentKontakt: [email protected]

61

V24

A predefined proteome signature for invasive ductal breastcarcinoma enables differentiation of cancer samples from controls

- first results from a prospective follow-up study.

Röwer, Claudia (1); Koy, Cornelia (1); Vissers, Hans (2); Kipping, Marc (2); Reimer, Toralf (3); Gerber, Bernd

(3); Hecker, Michael (4); Ziems, Björn (4); Thiesen, Hans-Jürgen (4); Glocker, Michael O. (1)

1: Proteom Center Rostock, University of Rostock, Germany; 2: WATERS Corp. MS Technologies Centre,Manchester, United Kingdom; 3: Department of Obstetrics and Gynecology, University of Rostock, Germany;

4: IndyMed GmbH, Rostock, Germany

Einleitung: With an incidence of one million per year world-wide breast cancer is still the most frequent malignanttumor in women [1]. Early diagnosis and individualized therapies are necessary to reduce the mortality of breastcancer. We had established a proteome signature [2] using label-free protein quantification by nanoscale LC-MS inwhich differential expression of 60 proteins were analyzed in order to distinguish invasive ductal breast carcinomatissues, the most frequent breast tumor, from control samples [3]. This proteome signature has now been appliedin a follow-up study on seven breast cancer patients.Experimenteller Teil: Tissue samples from invasive ductal carcinoma and control tissue originating from thesame breasts were taken from seven individuals after mastectomy. Proteins were extracted and subjected to 2Dgel electrophoresis. Samples were run in duplicate, resulting in 14 gels from tumor tissue and 14 gels fromcontrol (gland) tissue, respectively. After Coomassie staining, gels were scanned and subjected to image analysiswith Progenesis PG200, Version 2006 (Nonlinear Dynamics Ltd) prior to Principle Component Analysis (PCA)using normalized spot volumes. Protein spots of interest were excised, tryptically digested and peptide mixtureswere analyzed with a Reflex III MALDI ToF mass spectrometer (Bruker Daltonik) for protein identification. Forvalidation, quantitative analysis of immunohistochemical stained proteins was performed with Definiens Architect7 (Definiens).Ergebnisse und Diskussion: The evaluation of our pre-defined proteome signature in a prospective manner wasperformed with tissue samples from seven individuals that suffered from breast carcinoma. Protein mixtures fromtumor and control (gland) tissue pieces were subjected to 2D gel electrophoresis and image analysis. In total,773 spots were matched in the 28 gels and quantified by densitometry. From them 559 spots were selectedfor picking and 360 spots gave an MS identification result corresponding to 210 non-redundant proteins. 22proteins (corresponding to 60 spots) were found in the list of our 60 signature proteins. A Principle ComponentAnalysis (PCA) performed with the normalized quantitative data (spot volumes) of these 60 spots again resultedin a reliable separation of tumor samples from control samples. These findings confirm our predefined proteomesignature for invasive ductal breast carcinoma. Samples from other forms of breast cancer (invasive lobar andinvasive solid) were also investigated and showed an independent distribution in PCA plots. Protein expressiondifferences were validated by western blot analysis for exemplary proteins, substantiating the quantitative 2Dgel analysis. Furthermore, correlation of protein abundance differences with immunohistochemical data wasshown by quantitative analysis of immunohistochemical stainings of distinct proteins on tissue slices of breastcarcinoma and control samples, respectively. Also, the Human Protein Atlas (www.proteinatlas.org) provides 830tumor images and 80 control images from 47 patients representing 21 selected differentially expressed proteinsfrom our signature. These images were also analyzed in a quantitative manner and proved our previous findings.This combination of quantitative information from protein expression profiling and histology / pathology enablesthe identification of candidate proteins serving as key-marker for diagnostics and/or prognostics, or as futuretherapeutic targets in the treatment of breast cancer.Neue Aspekte: We suggest an invasive ductal breast carcinoma proteome signature based on label-free quantifi-cation by nanoscale LC-MS suitable for clinical use.Referenzen: [1] Reimer, T., Koczan, D., Müller, H., Friese, K., Thiesen, H.-J., Gerber, B., Breast Cancer Res,2002, 4(5): R9.; [2] Seike, M., Kondo, T., Fujii, K., Yamada, T., Gemma, A., Kuhdo, S., Hirohashi, S., Pro-teomics, 2004, 4:2776-2788.; [3] Röwer, C., Vissers, P. C., Koy, C., Kipping, M., Hecker, M., Reimer, T., Gerber,B., Thiesen, H.-J., Glocker, M. O., Anal Bioanal Chem, 2009, 395:2443-2456.Thema: Proteine und PeptideKeywords: breast carcinoma, proteome signature, label-free quantification, 2D gel electrophoresis, mass spec-trometryKontakt: [email protected]

62

V25

Laser-Massenspektrometrische Untersuchung vonTrichlorbenzolen mit REMPI- und MATI-Methoden

Witte, Frank (1); Riese, Mikko (2); Gunzer, Frank (3); Grotemeyer, Jürgen (1)

1: Christian Albrechts Universität Kiel, Kiel, Germany; 2: Photon Science Institute, Manchester, United

Kingdom; 3: German University in Cairo, Cairo, Egypt

Einleitung: Die Ionenspektroskopie ist eine der wichtigsten Methoden, um detaillierte Informationen über die

ionischen Eigenschaften von Molekülen zu erhalten. Die Substitution eines oder mehrerer H-Atome des Benzols

kann zu signifikanten Änderungen in der Elektronenverteilung führen. Dadurch können die Anregungsenergie fürden Übergang vom Grundzustand in den ersten angeregten Zustand, die Ionisationsenergie und die molekulareGeometrie geändert werden. Aufbauend auf den Ergebnissen für Dichlorbenzole [1,2] sollen hier einige Aspekteder gewonnenen Ergebnisse für Trichlorbenzole vorgestellt werden. Der erste angeregte Zustand wurde dabei mitder REMPI-, der ionische Grundzustand mit der MATI-Spektroskopie untersucht. Zusätzlich wurden die erhaltenenErgebnisse mit quantenchemischen Rechnungen verglichen.Experimenteller Teil: Das Experiment besteht aus einer einstufigen Ionenquelle und einem Standard-ReToF.Die Probemoleküle werden über ein gepulstes Ventil als Molekularstrahl in die Ionenquelle expandiert. Die An-regung bzw. Ionisation erfolgt unter feldfreien Bedingungen durch Multiphotonen-Absorption. Hierfür werdenzwei Farbstofflaser, die vom selben Nd:YAG-Laser gepumpt werden, verwendet. Im MATI-Betrieb wird nacheiner Zweiphotonen-Anregung in hochliegende Rydberg-Zustände ein kleines elektrisches Bremsfeld angelegt,um prompte Ionen von Rydberg-Neutralen zu trennen. Die Rydberg-Neutralen werden anschließend durch einenHochspannungspuls feldionisiert und in das ReToF beschleunigt. Im REMPI-Betrieb entfällt das Bremsfeld unddie gebildeten Ionen werden direkt in das ReToF beschleunigt. Die Detektion der Ionen erfolgt über einen MCP-Detektor, dessen Signal mit einem Digital-Oszilloskop ausgelesen wird.Ergebnisse und Diskussion: Mit der REMPI- und MATI-Spektroskopie konnten die Anregungsenergien undIonisationsenergien von 1,2,3-, 1,2,4- und 1,3,5-Trichlorbenzol sehr genau bestimmt werden. Weiterhin zeigte sichin den Spektren ein detailliertes Bild, welche Schwingungen bei der Ionisation der Moleküle aktiv sind. Währendder Anregung und Ionisation ändern die Moleküle ihre Geometrie in Richtung bestimmter Schwingungen ( z.B.7b1, 9a1 und 17b1 beim 1,2,3-Trichlorbenzol sowie 7b1 und 6b1 beim 1,2,4-Trichlorbenzol).Mit 37Cl isotopiertes 1,3,5-Trichlorbenzol zeigt im REMPI-Spektrum aufgrund des Symmetriebruchs eine Auf-spaltung der im Normalisotopomer entarteten Schwingungsmoden 6a und 6b. Bei diesem Molekül konnte bislangjedoch keine signifikante Jahn-Teller-Aufspaltung im Ion beobachtet werden. Dies steht im Gegensatz zu einererwarteten Jahn-Teller-Verzerrung bei D3h-Symmetrie und den Ergebnissen, die mit MATI-Spektroskopie für dasanaloge 1,3,5-Trifluorbenzol erhalten wurden [3].Neue Aspekte: Geometrieänderung bei Anregung und IonisationReferenzen: [1] A. Gaber, M. Riese, J. Grotemeyer, J. Phys. Chem. (2008), 112, 425.; [2] A. Gaber, M. Riese, J.Grotemeyer, Phys. Chem. Chem. Phys. (2008), 10, 1168.; [3] C. H. Kwon, M. S. Kim, J. Chem. Phys. (2004), 121,2622.Thema: Organische MassenspektrometrieKeywords: Ionenspektroskopie, Ionisationsenergie, Anregungsenergie, SchwingungsstrukturKontakt: [email protected]

63

V26

Neue dissoziative Cross-Linker für die vereinfachtemassenspektrometrische Proteinstrukturaufklärung.

Dreiocker, Frank (1); Müller, Mathias (2); Sinz, Andrea (2); Schäfer, Mathias (1)

1: Department für Chemie, Universität zu Köln, Germany; 2: Department für Pharmazie,

Martin-Luther-University Halle-Wittenberg, Germany

Einleitung: Chemisches Cross-Linking in Kombination mit Massenspektrometrie ist eine vielversprechende Me-

thode zur Aufklärung von tertiären- bzw. quaternären Proteinstrukturen. Die analytische Herausforderung stellt

hierbei die eindeutige und selektive Identifikation der verbrückten Peptide neben einer viel größeren Anzahl unde-

rivatisierter Peptiden dar. [1] Soderblom undGoshe entwickelten 2006 einige durch Kollisionsaktivierung spaltbare

Cross-Linker für die sensitive tandem-massenspektrometrische Detektion. [2] Die selektive Spaltung dieser Cross-

Linker beruht auf der bevorzugten Protonierung des im Linker eingebauten Prolins und der ebenfalls enthaltenen,

benachbarten Asparaginsäure unter Dissoziation der labilen Asp-Pro-Amidbindung. Diese favorisierte Spaltung

liefert Produktionen mit einem definierten Massenversatz, welche selektiv und automatisiert detektiert werden

können und in nachfolgenden MSn-Experimenten untersucht werden. Das Konzept für dissoziative Cross-Linker

wurde weiter entwickelt und wir präsentieren nun neue Cross-Linker und deren Anwendung.

Experimenteller Teil:Wir haben verschiedene homo-bifunktionelle N-Hydroxysuccinimid (NHS)-Aktivester her-

gestellt. Diese NHS-Cross-Linker reagieren vorzugsweise mit Amin-Funktionen (N-Terminus oder Lysin) in Pro-

teinen. Die selektive Identifizierung von verbrückten Peptiden erfolgt über definierte Verluste von Neutralteilchen

(Constant Neutral Losses, CNL) in MS/MS-Experimenten, die durch eine selektive Spaltung der Cross-Linker si-

chergestellt werden. MS und MS/MS-Experimente wurden an ESI-LTQ-Orbitrap (LTQ-OrbitrapXL, ThermoFis-

her Scientific, Bremen)- und MALDI-TOF/TOF (Ultraflex III, Bruker Daltronik, Bremen)-Massenspektrometern

durchgeführt.

Ergebnisse und Diskussion: Im Tagungsbeitrag wird die effiziente Synthese von verschiedenen Cross-Linkern

präsentiert. Zum Proof of principle und zur Bestätigung der zugrundeliegenden Fragmentierungsprozesse der

Cross-Linker-Reagenzien wurden diese mit verschiedenen Peptiden (z.B. Substanz P, LHRH) umgesetzt und die

verbrückten Peptide massenspektrometrisch (MS und MS/MS) untersucht. Des Weiteren wurde das Protein Lyso-

zym mit unseren Cross-Linkern derivatisiert und die nach der tryptischen Spaltung erhaltenen Peptide ebenfalls

mittels MS und MS/MS analysiert. Die Identifikation der verbrückten Produkte erfolgte über definierte Massen-

versätze und charakteristische Neutralverluste, welche durch die prädestinierte Fragmentierung der Cross-Linker

gebildet werden. Die effektive Dissoziation der schwefelhaltigen Cross-Linker beruht auf bevorzugten nukleophi-

len Angriffsstellen innerhalb des Moleküls, wobei es zu einem gezielten Bindungsbruch kommt. Der Mechanismus

dieser Fragmentierung und die entsprechenden Produktionen wurden durch exakte Bestimmung der Ionenmassen

analysiert.

Die gefundenen Neutralverluste erlauben eine zweifelsfreie Unterscheidung zwischen intramolekularen und parti-

ell hydrolysierten („dead-end") Cross-Links. Neben der Aminosäuresequenz kann der genaue Ort der Verbrückung

im Peptid in MSn-Experimenten ermittelt werden. Die grundlegenden Studien der Reaktivität und des Fragmen-

tierungsverhalten lieferten sowohl mit Peptiden als auch mit Proteinen vielversprechende Resultate. [3] Das neue

Konzept sollte eine Verbesserung in der Proteinstrukturaufklärung mittels Cross-Linking und Tandem-Massen-

spektrometrie ermöglichen.

Neue Aspekte: Neues Strukturkonzept für kollisionsinduziert spaltbare Cross-Linker zur vereinfachten Bestim-

mung von Proteinstrukturen mittels Tandem-Massenspektrometrie.

Referenzen: [1] A. Sinz. Mass Spectrom. Rev. 2006, 25, 663-682.; [2] E. J. Soderblom, M. B. Goshe, Anal.

Chem., 2006, 8, 8059-8068.; [3] F. Dreiocker, M. Q. Müller, A. Sinz, M. Schäfer, J. Mass Spec. 2009, DOI

10.1002/jms.1702.

Thema: Organische Massenspektrometrie, Proteine und Peptide

Keywords: Cross-Linker, Peptide, Kollisionsinduzierte Dissoziation (CID), Proteinstrukturaufklärung, Neutral-

verlust (CNL)


64

V27

Massenspektrometrie mit weicher Photoionisation für die on-lineCharakterisierung von Organischen Spurenkomponenten in

Verbrennungs- und Pyrolysegasen

Streibel, Thorsten (1); Fendt, Alois (2); Sklorz, Martin (1); Dahmen, Nicolaus (3); Zimmermann, Ralf (1,4)

1: Universität Rostock, Germany; 2: Universität Augsburg, Germany; 3: KIT Karlsruhe, Germany; 4: HelmholtzZentrum München

Einleitung: Pyrolyse- und Verbrennungsgase sind komplexe Systeme, die mehrere hundert organische Speziesin Spurenmengen enthalten können. Eine direkte Echtzeitanalyse dieser Substanzen gestaltet sich oft schwierig,da speziell bei Verbrennungsprozessen schnelle Veränderungen in den Konzentrationsniveaus auftreten. DirekteMassenspektrometrie bietet die Möglichkeit einer schnellen Echtzeitanalytik, sofern weiche Ionisationsmethodenverwendet werden, die Fragmentierungen der Molekülionen unterbinden. In diesem Beitrag wird die Flugzeit-massenspektrometrie in Verbindung mit Photoionisationsmethoden eingesetzt, um die organische Signatur bei derPyrolyse von Biomasse sowie der Verbrennung von Nadelbaumrinde in einer Papierfabrik zu untersuchen. Bei derBiomassepyrolyse stehen die Abbauprodukte der enthaltenen Biopolymere im Blickpunkt, die anschließend zu ei-nem Öl kondensiert werden. Die Detektion von polyzyklischen aromatischen Kohlenwasserstoffen (PAK) vor undnach Zugabe von Schwefel stand im Fokus der Rindenverbrennung.Experimenteller Teil: Zwei verschiedene Photoionisationstechniken wurden verwendet, zum einen die Resonanceenhanced multi photon ionisation (REMPI) generiert mit UV-Laserpulsen sowie die Single photon ionisation (SPI)mit VUV-Photonen, welche kontinuierlich mit einer neuartigen elektronenstrahlgepumpten Edelgasexcimer Lam-pe (EBEL) erzeugt werden. Die Probenahme erfolgt direkt in den Pyrolyse- und Verbrennungsgasen mit beheiz-ten inerten Quarzglaslanzen. Eine Gasprobenahmepumpe saugt einen konstanten Gasstrom über Quarzfaserfilter,die für die Abtrennung der im Gas enthaltenene Partikel unabdingbar sind. Die Verbindung zur Ionenquelle desFlugzeitmassenspektrometers erfogt über eine deaktivierte Quarzglaskapillare mit 200 µm i.d.. Der gesamte Pro-benahmeweg ist auf 250°Cgeheizt. REMPI ist aufgrund seines Ionisierungsschemas selektiv für die Detektion vonaromatischen Verbindungen, mit SPI sind zusätzlich aliphatische sowie sauerstoff- und stickstoffhaltige Kohlen-wasserstoffe zugänglich.Ergebnisse und Diskussion: Mit den oben beschriebenen massenspektrometrischen Systemen wurden zunächstorganische Primärprodukte aus der Schnellpyrolyse von Biomasse analysiert. Dieses wurde an einer Technikums-anlage am KIT Karlsruhe, Campus Nord durchgeführt. Die Schnellpyrolyse ist der erste Schritt im dort entwi-ckelten BtL-Prozess (Biomass to Liquid). Unterschiedlichste Biomassen wie Weizen- und Reisstroh, Weich- undHartholz, Rapsschrot, Weizenkleie und Energiepflanzen wie Miscanthus wurden untersucht. Das Muster der Pyro-lyseprodukte war spezifisch für jede Biomasse und kann somit als Unterscheidungsmerkmal herangezogen werden[1]. Zum Beispiel treten bei Weichholz bestimmte phenolische Ligninabbauprodukte im Gegensatz zu Hartholznicht auf, da sich der Aufbau des Lignins unterscheidet. Die Spektren des stickstoffhaltigen Rapsschrots zeigenviele N-haltige Spezies wie Indol, Pyrrol, Amine. Neben den Phenolderivaten werden Furanderivate detektiert,Abbauprodukte der Zellulose. Das Spektrum von Miscanthus ähnelt dem von Weizenstroh und Holz, was die Ver-mutung nahelegt, dass die kondensierten Pyrolyseprodukte ebenfalls eine ähnliche Zusammensetzung aufweisen.Letzteres ist wichtig für die weitere Verarbeitung der Bioöle zu Kraftstoffen und chemischen Plattformchemikalienoder zur Co-Verbrennung mit fossilen Brennstoffen.Die zweite Anwendung beschäftigte sich mit den Auswirkungen der Zugabe von Schwefel auf die PAK-Emissioneneiner Rindenverbrennung. Diese dient in einer Papierfabrik zur Bereitstellung von Dampf für die Trocknung desZellulosebreis. Das Verbrennungsgas wurde dafür bis zu acht Stunden kontinuierlich mit REMPI-TOFMS analy-siert. Schwefel wurde entweder als wässrige Lösung von Ammoniumsulfat oder als elementare Schwefelpelletsin die Verbrennungskammer zudotiert. Es zeigte sich, dass die Addidition von Schwefel zu einer signifikantenAbnahme der PAK-Konzentrationen führte, unabhängig davon, in welcher Form der Schwefel zugegeben wurde.Diese ging einher mit einer deutlichen Abnahme der Anzahl von Emissionsspitzen, die mit gleichzeitig auftre-tenden Erhöhungen der CO-Konzentration zusammenfielen und gestörte Verbrennungskonditionen anzeigen. Beidiesen Messungen wurde ein neuer Ansatz zur Quantifizierung der PAH versucht, der auf der Bestimmung vonIonisationswirkungsquerschnitten relativ zu Toluol, beruht, dessen Konzentration absolut über ein Kalibriergasbestimmt wird.Neue Aspekte: Umfassende Untersuchung semivolatiler organischer Spezies bei Pyrolyse und Verbrennung inEchtzeit mit der Möglichkeit zum Monitoring über längere Zeiträume

65

Referenzen: [1] T. Streibel, A. Fendt, R. Geißler, E. Kaisersberger, T. Denner, R. Zimmermann, Thermal analy-sis/mass spectrometry using soft photo-ionisation for the investigation of biomass and mineral oils, J. Therm. Anal.Cal. 97 (2009) 2 615-619Thema: Instrumentelle Entwicklungen, Organische MassenspektrometrieKeywords: Photoionisierung, Flugzeimassenspektrometrie, Echtzeitdetektion, Biomassepyrolyse, AromatenKontakt: [email protected]

66

V28

Stars in MALDI - Tandem MS investigations of star-shapedpoly(e-caprolactone) -

Knop, Katrin (1,2); Baumgaertel, Anja (1,2); Bader, Cornelia (1); Crecelius, Anna C. (1,2);Schubert, Ulrich S. (1,2,3)

1: Laboratory of Organic and Macromolecular Chemistry, Friedrich-Schiller-University Jena, Humboldtstr. 10,07743 Jena, Germany; 2: Dutch Polymer Institute (DPI), John F. Kennedylaan 2, 5612 AB Eindhoven, TheNetherlands; 3: Laboratory of Macromolecular Chemistry and Nanoscience, Eindhoven University of

Technology, Den Dolech 2, 5600 MB Eindhoven, The Netherlands

Einleitung: In the emerging field of drug delivery, star-shaped block copolymers of i.e. poly(e-caprolactone)promise large potential for designing vectors with improved delivery efficiency.[1,2]Biological applications of poly-mers require a very solid and profound characterization beyond NMR spectroscopic methods and size exclusionchromatography (SEC). Mass spectrometry (MS) arose to a versatile technique to acquire detailed information ofthe end group nature.To enable a complete understanding of the polymer structure beyond total end group mass determination, tandemmass spectrometric methods have been developed and applied to different synthetic polymers like PMMA, PSor aromatic and aliphatic copolyesters.[3−5] MALDI-TOF/TOF-collision induced dissociation MS studies wereperformed with linear and different star-shaped poly(e-caprolactone)s to investigate the possibility of structuredetermination by tandem MS methods.Experimenteller Teil: Poly(e-caprolactone) was synthesized by stannous octoate catalyzed ring-opening polymer-izations of e-caprolactone performed at 100 °Cfor 8 h. The polymers were purified by precipitation into ice-cold

methanol, resulting in powdery products. The products were characterized by 1H-NMR spectroscopy, SEC and

MALDI-TOF mass spectrometry. An Ultraflex III TOF/TOF (Bruker Daltonics, Bremen, Germany) was used for

the MALDI-TOF-MS and MS/MS analysis. For the MS/MS mode, argon was used as the collision gas at a pres-

sure of 2 × 10−6 mbar. For the sample preparation, 1 µL of the poly(e-caprolactone) (10 mg/mL), 10 µL of DCTB

(10 mg/mL) and 5 µL of lithium chloride or sodium bromide (100 mg/mL) (all solutions in THF) were mixed and

the dried-droplet sample preparation method was applied.

Ergebnisse und Diskussion: Linear poly(e-caprolactone) was synthesized with 6-bromohexanol as initiator and

investigated by MALDI-TOF-MS and MS/MS methods, to develop and establish the mechanisms of collision

induced decomposition under high energy conditions. The main losses of small mass fragments in the upper part

of the tandem mass spectrum after the parent ion are formed by HBr loss (corresponding a loss of m/z 80/82),

e-caprolactone loss (corresponding a loss of m/z 114) and 6-hydroxyhexanoic acid (corresponding a loss of m/z

132). The monomer region in the CID spectrum is dominated by acylium cations formed by the 6-hydroxyhexanoic

acid (corresponding m/z 115) or its dehydration product (corresponding m/z 97). The middle part of the spectrum

shows uniform series mainly formed by McLafferty+1 rearrangements (1-5 hydrogen rearrangements) of the poly-

(e-caprolactone).

Subsequently, star-shaped poly(e-caprolactone)s were investigated by MALDI-TOF/TOF CID, which were pre-

pared as reported prior with erythritol (for the four arm stars) and 2-(hydroxymethyl)-2-methylpropane-1,3-diol

(for the three arm stars) as initiator.

In principle, the same fragmentation mechanisms were observed, except the HBr elimination, as neither of the

star-shaped (e-caprolactone)s contains bromine atoms. In contrast, whereas the MS/MS spectrum of linear macro-

molecules is dominated by prominent losses in the upper molar mass region and high intense monomer fragments,

the MS/MS spectrum of the star-shaped macromolecules is characterized by two distributions forming two hill-

like patterns. The one following directly the parent ion is formed by the star-fragments while the one in the lower

molar mass region is formed by the arm fragments. Hence, the question arose whether these two distributions can

provide further information regarding the star structure (uniformly initiated at each arm or preferred propagation

at one or two steric unhindered arms). By varying the number of possible arms and changing the molar mass of

the star-shaped parent ion this problem is investigated.

Neue Aspekte: An understanding of the polymer structure by CID beyond end group mass and sequence determi-

nation is aimed for star-shaped polymers.

67

Referenzen: [1] S. M. Grayson, W. T. Godbey, J. Drug Target. 2008, 16, 329-356.; [2] O. G. Schramm, G. M.Pavlov, H. P. van Erp, M. A. R. Meier, R. Hoogenboom, U. S. Schubert, Macromolecules 2009, 42, 1808-1816.; [3]J. H. Scrivens, A. T. Jackson, Int. J. Mass Spec. 2000, 200, 261-276.; [4] A. C. Crecelius, A. Baumgaertel, U. S.Schubert, J. Mass. Spectrom. 2009, 44, 1277-1286.; [5] S. M. Weidner, J. Falkenhagen, K. Knop, A. Thünemann,Rapid Commun. Mass Spectrom. 2009, 23, 2768-2774.Thema: Organische MassenspektrometrieKeywords: MALDI-TOF/TOF, CID, poly(e-caprolactone), star-shaped polymersKontakt: [email protected]

68

V29

Spermabeurteilung mit MALDI-TOF Massenspektrometrie?

Müller, Karin (1); Jakop, Ulrike (1); Teuber, Kristin (2); Eibisch, Mandy (2); Süß, Rosmarie (2); Fuchs, Beate (2);

Paasch, Uwe (3); Schiller, Jürgen (2)

1: Leibniz-Institut für Zoo- und Wildtierforschung, Berlin, Germany; 2: Universität Leipzig, Med. Fak., Med.

Phys. Biophys., Germany; 3: Uniklinik Leipzig, Klinik für Dermatologie, Venerologie und Andrologie

Einleitung: Die Sicherung des Nachwuchses ist ein zunehmendes Problem bei Mensch und Tier (insbesondere bei

gefährdeten/bedrohten Tierarten aber auch bei Nutztieren). Obwohl beim Menschen ungewollte Kinderlosigkeit

zu nahezu gleichen Teilen durch Dysfunktionen bei Mann und Frau bedingt ist, beklagen Andrologen in den

letzten Jahrzehnten eine erschreckende Abnahme der Qualität des männlichen Spermas. Eine sichere und objektive

Einschätzung der Spermaqualität ist daher für die Therapie ungewollter Kinderlosigkeit von höchster Relevanz,

insbesondere wenn Methoden wie die "in-vitro-Fertilisation"(IVF) zum Einsatz kommen sollen, wo man gezielt

auf potente Spermien zurückgreifen möchte.

Wir möchten am Beispiel unterschiedlicher Spezies zeigen, daß die massenspektrometrisch bestimmbare Phos-

pholipidzusammensetzung von Spermien und insbesondere deren pathologische Veränderungen infolge oxidativer

Prozesse zuverlässige und sensitive Kriterien für die Qualität von Spermien darstellen.

Experimenteller Teil: Tierische Ejakulate wurden in Besamungsvatertierstationen bzw. im Verlauf von Maßnah-

men zur assistierten Reproduktion gewonnen, während humane Ejakulate im Rahmen der andrologischen Sprech-

stunde anfielen. Nach Abtrennung des Seminalplasmas wurden die Spermien mit organischen Lösungsmitteln ex-

trahiert und die PL-Zusammensetzung mittels MALDI-TOF MS (Bruker ¨Autoflex¨, Reflektor-Modus) bestimmt.

Die Aufnahme der Spektren erfolgte mit DHB (Positiv-Ionen-Spektren) bzw. mit 9-AA (Negativ-Ionen-Spektren)

als Matrixsubstanzen [1]. Aufgrund der teilweise sehr geringen Mengen an verfügbarem Ejakulat konnte nur sel-

ten eine zusätzliche chromatographische Reinigung vorgenommen werden. Diese erfolgte dann mit direkt kombi-

nierter TLC/MALDI-TOF MS nach einem kürzlich etablierten Verfahren [2]. Einige ausgewählte Proben wurden

zusätzlich mittels 31P-NMR-(nuclear magnetic resonance)-Spektroskopie charakterisiert [3].

Ergebnisse und Diskussion: Spermien-Phospholipide (PL) sind in der Regel durch einen hohen Gehalt an (a)

hochungesättigten Fettsäureresten (Docosahexaen- und Docosapentaensäure) und einen hohen Anteil an (b) Plas-

malogenen, d.h. Alkenyl-Acyl-PL, charakterisiert. Beides begründet die Tatsache, daß Spermien-PL hochgradig

oxidationsempfindlich sind. Dies führt neben verschiedenen intermediären Produkten zur Bildung von Lysophos-

phatidylcholinen (LPC) aus Diacyl-PL bzw. von Formyl-LPC aus Plasmalogenen. Lysolipide entstehen aus PL

durch Verlust eines Fettsäurerestes. Beide Verbindungen können mittels MALDI-TOF MS-selbst im Gemisch und

ohne Zusatz eines internen Standards-sehr einfach detektiert und ihr relativer Anteil im Vergleich zu den übri-

gen Lipiden bestimmt werden. Es ergaben sich dabei Korrelationen zu etablierten zellbiologischen bzw. andro-

logischen Parametern, so daß wir die genannten Lysoverbindungen als Marker für die Schädigung von Spermi-

en vorschlagen und dies anhand ausgewählter Proben diskutieren. Plasmalogene sind nicht nur gegen Sauerstoff

sehr empfindlich sondern auch gegen kleinste Mengen an Säuren [4]. Bemerkenswerterweise genügt bereits der

Kontakt mit der Oberfläche einer TLC-Platte (saure Gruppen des Kieselgels), um partielle Hydrolyse auszulö-

sen. Dies ist z.B. deutlich an der Anwesenheit eines LPC-Spots in der Phosphatidylcholin-Fraktion erkennbar.

Obwohl dies eigentlich ein unerwünschter Effekt ist, gestattet er uns, über die entstandene Lysoverbindung die

Zuordnung der Alkenyl-Funktion zur sn-1 oder sn-2-Position am Glycerol-Rückgrat. Schließlich werden wir am

Beispiel der Spermien von katzenartigen Tieren im Vergleich zu Spermien von Wiederkäuern zeigen, daß sich die

Lipid-Zusammensetzung der Spermienund insbesondere ihr Plasmalogengehalt möglicherweise vor dem Hinter-

grund des Fettstoffwechsels, der Ernährung, der Umwelt- und Lebensbedingungen etc. zwischen unterschiedlichen

Tiergruppen wesentlich unterscheiden können. Ob die unterschiedlichen Phospholipidmuster eine Anpassung an

oder die Ursache für artspezifische Eigenschaften des Reproduktionssystems sind, bleibt jedoch bisher offen.

Neue Aspekte: Lysophospholipide werden als einfach bestimmbare Marker für die Spermaqualität vorgestellt.

Referenzen: [1] Sun, G., et al.: Anal. Chem. 2008, 80:7576-7585.; [2] Fuchs, B., et al.: Anal. Bioanal. Chem. 2007,

389:827-834.; [3] Schiller, J., et al.: Curr. Anal. Chem. 2007, 3, 283-301.; [4] Fuchs, B., et al.: Theriogenology

2009, 71, 568-575.

Thema: Lipide

Keywords:MALDI-TOF MS, Phospholipide, Lysophospholipide, Spermien, TLC


69

V30

Multiparametric analysis by tandem mass spectrometry-challenges for modern laboratory diagnostics

Ceglarek, Uta; Fiedler, Georg-Martin; Thiery, Joachim

Universitätsklinikum Leipzig, Germany

Einleitung: The application of mass spectrometry becomes increasingly important as analytical tool in clinicallaboratory diagnostics. In the last decade there has been an exponential growth in clinical laboratory, pharmacol-ogy, and toxicology applications. Powerful analytical techniques like liquid chromatography coupled to tandemmass spectrometry (LC-MS/MS) offers a rapid, effective and economical way to analyze metabolic alterations ofpre-defined target metabolites in biological samples [1]. Novel hyphenated technical approaches like the couplingof tandem-mass spectrometry with linear ion trap (QTrap mass spectrometry) enables both the identification andquantification of known and unknown metabolic targets [2].Experimenteller Teil: We describe new concepts and developments of mass spectrometry based multi-targetmetabolome profiling in the field of clinical diagnostics and research. Today, LC-MS/MS is already used in routineclinical laboratory to screen for inherited metabolic disorders in newborns from dried blood, for therapeutic drugmonitoring, or for steroid hormone profiling in EDTA whole blood and plasma [3,4]. In addition the use ofLC-MS/MS opens new diagnostic possibilities for multiplex analysis in the field of metabolomics (e.g. sterols,phospholipids and eicosanoids) [2]. Standardization protocols of blood sampling, processing and storage weredefined to minimize in-vitro data variability of metabolomic studies.Ergebnisse und Diskussion: The presentation will give an overview of the current status of tandem mass spec-trometry techniques and its preanalytical and analytical challenges in clinical laboratory diagnostics. Particularly,the experiences from newborn screening provided important insights about the diagnostic potential of metaboliteprofiling arrays [5]. Perspectives for future developments in laboratory medicine directing to the clinical aim ofpredictive, preventive and personalized medicine by metabolomic and proteomic approaches will be depicted.Neue Aspekte: Importance of LC-MS/MS in laboratory medicine; perspectives for preventive and personalizedmedicineReferenzen: [1] Fiedler GM, Ceglarek U, Lembcke J, Baumann S, Leichtle A, Thiery J. J Lab Med 2004; 28:185-94; [2] Kortz L, Geyer R, Ludwig U, Planert M, Bruegel M, Leichtle A, Fiedler GM, Thiery J, Ceglarek U.J Lab Med 2009;33(6); [3] Ceglarek U, Lembcke J, Fiedler GM, Werner M, Witzigmann H, Hauss JP, Thiery J;Clin. Chim. Acta 2004; 346:181-90; [4] Ceglarek U, Kortz L, Leichtle A, Fiedler GM, Kratzsch J, Thiery J. .Clin Chim Acta. 2009, 401:114-8; [5] Ceglarek U, Leichtle A, Brügel M, Kortz L, Brauer R, Bresler K, Thiery J,Fiedler GM. Mol Cell Endocrinol. 2009, 301:266-71.Thema: Lipide, MetabolomicsKeywords: LC-MS/MS; clinical applications, metabolomicsKontakt: [email protected]

70

V31

Lipidomics and Neurodegeneration

Schwudke, Dominik; Hebbar, Sarita

National Centre for Biological Sciences, TIFR, India

Einleitung: Recently developed mass spectrometric approaches enabled to investigate the role of lipid metabolismin neurodegeneration. Drosophila melanogaster was chosen as model due to its combined advantage of a shortlife span, easy accessibility to nervous system, and sophisticated genetic tools. The sample preparation procedureswere optimized to support the analysis of a minimal amount of biological material. Single organism, body sectionand dissected organs were analyzed by direct infusion experiments and LC-MS/MS. The wealth of quantitativeinformation consisting of 15 lipid classes and more than 150 lipids was correlated to genetics, imaging, aging andbehavioral assays. Our results indicate the concurrence of degenerative phenotypes with sphingolipid metabolicperturbations in the brain.Experimenteller Teil: Samples were extracted with an optimized MTBE based extraction protocol as describedpreviously [1]. Mass spectrometric analysis was performed on a hybrid LTQ Orbitrap XL mass spectrometer(Thermo Fisher Scientific, Bremen, Germany) equipped with a robotic nanoflow ion source TriVersa (AdvionBioSciences Ltd, Ithaca NY) [2,3]. Liquid chromatography was performed on a 1200 micro-LC-system (AgilentTechnologies, Waldbronn, Germany) coupled to the MS-system via the Triversa ion source. Mass spectrometrydata interpretation was performed with proprietary software LipidX and Xcalibur (Thermo Fisher Scientific, Bre-men, Germany). Lipofuscin accumulation and synaptic morphology were examined as markers of degeneration inthe nervous system. Imaging was carried out on an Olympus FV100 Confocal Microscope (Olympus Corporation,Japan).Ergebnisse und Diskussion: Lipidomics analysis of bonafide models of neurodegeneration in D. melanogaster

was performed using spinster [4] and bluecheese [5] genes. These results were compared to transgenic flies inwhich sphingolipid metabolic pathways are manipulated in the brain using the UAS-Gal4 system. Single organism,body sections and dissected brains were analyzed by direct infusion experiments and LC-MS/MS. More than 150lipids of 15 classes were identified and quantified to correlate genotypes, imaging, aging and behavioral assays.Consistent with progressive degenerative processes, accumulation of lipofuscin (yellow-brown pigment granulescomposed of lipids and oxidized protein residues) in the brains was observed. Likewise, changes in synapticmorphology were also observed. Our results indicate the concurrence of degenerative phenotypes with lipidmetabolic perturbations in the brain, in particular on ceramide species with unusual high degree of unsaturation.Neue Aspekte: Our results correlate neurodegenerative phenotypes with perturbations of sphingolipid metabolismin the brain.Referenzen: [1] Matyash V, Liebisch G, Kur zchalia TV, Shevchenko A, Schwudke D. Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics. J Lipid Res. 2008 May;49(5):1137-46.;[2] Schwudke D, Liebisch G, Herzog R, Schmitz G, Shevchenko A. Shotgun lipidomics by tandem mass spec-trometry under data-dependent acquisition control. Methods Enzymol. 2007;433:175-91.;[3] Schwudke D, Hannich JT, Surendranath V, Grimard V, Moehring T, Burton L, Kurzchalia T, Shevchenko A.Top-down lipidomic screens by multivariate analysis of high-resolution survey mass spectra. Anal Chem. 2007Jun 1;79(11):4083-93.;[4] 18.16. Nakano Y, Fujitani K, Kurihara J, Ragan J, Usui-Aoki K, Shimoda L, Lukacsovich T, Suzuki K, SezakiM, Sano Y, Ueda R, Awano W, Kaneda M, Umeda M, Yamamoto D. Mol Cell Biol 2001 21:3775-3788.;[5] Lim A, Kraut R. The Drosophila BEACH Family Protein, Blue Cheese, Links Lysosomal Axon Transport withMotor Neuron Degeneration. Journal of Neuroscience 2009, Jan 28;29(4): 951-963Thema: Lipide, MetabolomicsKeywords: Lipidomics, Neurodegeneration, GeneticsKontakt: [email protected]

71

V32

The analysis of molecular lipid species by higher energy collisionaldissociation (HCD) on a LTQ Orbitrap XL instrument

Schuhmann, Kai (1); Herzog, Ronny (2); Bornstein, Stefan R. (1); Shevchenko, Andrej (2)

1: Department of Internal Medicine III, TU Dresden, Dresden, GERMANY; 2: Max Planck Institute CBG,Dresden, GERMANY

Einleitung: Shotgun lipidomics aims at the identification of individual molecular species [1, 2]. They are usuallyidentified by accurate m/z of their precursor and corresponding fatty acid moieties produced via CID in the negativeion mode. Higher energy collisional dissociation (HCD) is a new powerful feature of LTQ Orbitrap instruments.Applying HCD for molecular species analysis brings the advantage of detecting acyl anions at the very highresolution and mass accuracy, which improves lipid profiling in organisms that produce a broad palette of unusualfatty acids, such as C. elegans. Importantly, both top-down and bottom-up shotgun lipidomics can now use LTQOrbitrap as a single instrumentation platform.Experimenteller Teil: Stock solutions of total lipid extracts of bovine heart (Avanti Polar Lipids) were dilutedwith isopropanol:methanol:chloroform (v:v:v - 4:2:1) containing 7.5 mM ammonium formiate. DDA of HCD-MS/MS spectra was performed on LTQ Orbitrap XL (Thermo Fisher Scientific) equipped with NanoMate roboticnanoflow ion source (Advion BioSciences). Spectra were acquired at a targeted resolution of 100.000 (for MS) and30.000 (for MS/MS). Each DDA cycle consisted of one FT-MS survey scan followed by five FT-MS/MS spectra.Spectra were processed by LipidX software developed in-house.Ergebnisse und Diskussion: Acyl anions of fatty acids with low m/z (< 0.25 x [precursor m/z]) are not detectablein CID spectra, while PQD often lacks the sensitivity on Orbitrap instruments. Both PQD and CID methodsdetected fragment peaks with low mass resolution typical for ion traps. In comparison, HCD allows the detectionof all small m/z fragments and increased the sensitivity. Importantly, precursors can be fragmented completely andacyl anions, along with other informative structural fragments, detected at the typical Orbitrap mass resolution ofup to 100.000 (FWHM). Pathways of HCD fragmentation and required collision energies (CE) were lipid classdependent. However, because of CE normalization, the yield of fragments did not depend on m/z. The abundanceof fragment peaks was enhanced by using an offset in precursor selection and increasing the precursor isolationm/z window to ±0.9 Da. MS/MS spectra were acquired with little or no noise (N < 50). Since HCD spectra wereacquired at high resolution, the CO2 loss (m/z 283.243) from the acyl anion of docosahexaenoic acid (FA 22:6)was clearly distinguishable from acyl anion of stearic acid (FA 18:0, m/z 283.264), which much improved theprofiling of biologically important polyunsaturated lipids.We validated the method by comparing the lipid profile obtained by direct unbiased FT-MS analysis of bovine heartextracts with molecular species profiles relying upon the abundance of fatty acid anions. Both profiles correlatedwell (r = 0.985). HCD analysis was enhanced by higher scanning speed of LTQ Orbitrap Velos. We furtherdemonstrated that HCD profiling enabled efficient determination of molecular species of new lipid class in C.elegans, in which the composition of fatty acid moieties was exceedingly complex.Neue Aspekte: Molecular lipid species profiling by HCD on LTQ OrbitrapReferenzen: [1] Schwudke, D. et al. (2006), Anal. Chem., 78, 585-595; [2] Ejsing, C.S. et al. (2006), Anal.Chem., 78, 6202-6214Thema: LipideKeywords: Shotgun lipidomics, LTQ Orbitrap, HCDKontakt: [email protected]

72

V33

3-Aminochinolin als Matrix und Derivatisierungsreagenz zurOligosaccharid-Analyse mittels MALDI-Massenspektrometrie

Rohmer, Marion (1); Meyer, Björn (1); Mank, Marko (2); Stahl, Bernd (2); Bahr, Ute (1); Karas, Michael (1)

1: Goethe-Universität Frankfurt, Germany; 2: Danone Research Centre for Specialised Nutrition, Friedrichsdorf,Germany

Einleitung: In den letzten Jahrzehnten hat sich die Massenspektrometrie als die vorherrschende Methode in derOligosaccharid-Analyse etabliert.1 Dank ihrer herausragenden Sensitivität und Schnelligkeit wird vor allem dieMALDI-TOF-Massenspektrometrie gerne verwendet.2 Da underivatisierte Oligosaccharide meist eine ungenü-gende Ionisierungseffizienz aufweisen, wird häufig von diversen Derivatisierungsmethoden Gebrauch gemacht.2

Allerdings schließen sich an solche Derivatisierungen oft aufwändige und zeitraubende Aufreinigungsschritte an,die zu Probeverlusten führen und dadurch den Sensitivitätsgewinn relativieren. Obwohl 3-Aminochinolin (3-AQ)eine vielfältige und leistungsstarke MALDI-Matrix darstellt,3 wurde sie in der Vergangenheit selten verwendet.Dies liegt an ihrer Fähigkeit zur Bildung von Schiffschen Basen (Iminen) mit reduzierenden Oligosacchariden, diebisher als ungewollte Nebenreaktion betrachtet wurde. In unserer Methode soll diese ungewollte Nebenreaktionals Derivatisierung genutzt werden, die die strukturelle Analyse von Oligosacchariden erleichtert.Experimenteller Teil: Standardoligosaccharide mit verschiedenen strukturellen Elementen und einer breiten Mas-senverteilung wurden ebenso analysiert wie komplexe, mehrfach fucosylierte Humanmilcholigosaccharide (HMOS).Dabei wurden unterschiedliche Reaktionsbedingungen getestet, um eine hunderprozentige Umwandlung in die 3-AQ-derivatisierten Oligosaccharide sicherzustellen. Die Spektren wurden an einem Ultraflex I MALDI TOF/TOF-Massenspektrometer (Bruker Daltonics, Bremen) mit einem Stickstofflaser (337 nm) und einem ABI 4800MALDITOF/TOFTM Analyzer (Applied Biosystems, Darmstadt) mit einem Nd-YAG laser (355 nm) aufgezeichnet.Ergebnisse und Diskussion: Die Vollständigkeit und Reproduzierbarkeit der Derivatisierung mit 3-AQ wur-de durch die Optimierung der Reaktionsbedingungen und der Matrixlösung erreicht. Die Derivatisierung mit

3-Aminochinolin erfolgte direkt auf dem MALDI-Target; es wurde keine anschließende Aufreinigung benötigt.

Dabei bildeten sich 3-AQ-Oligosaccharide, die von einem einzelnen Spot sowohl im Positiv- als auch im Negativ-

modus vermessen werden konnten. Weiterhin wurden die Nachweis- und Fragmentierungsgrenzen im Vergleich

zu Standardmatrices verbessert. Die MALDI-TOF/TOF-Fragmentspektren im Positivmodus lieferten schnell und

einfach Informationen über die Oligosaccharid-Sequenz, während die Fragmentspektren im Negativmodus Auf-schluss über Verknüpfungen und Verzweigungen gaben. Außerdem wurde die Derivatisierungsmethode auf zweiisomere Oligosaccharide aus humanerMuttermilch, Trifucosyl-lacto-N-hexaose und Trifucosyl-para-lacto-N-hexa-ose, angewendet. Die MALDI-TOF/TOF-Fragmentierung im Positiv- und Negativmodus lieferte Spektren mit ho-hem Informationsgehalt, welche die Strukturanalyse und Unterscheidung der beiden Isomere ermöglichten. Die

vielfältigen Möglichkeiten, die diese neue Derivatisierungsart bietet, sollten einen großen Gewinn für die Oligo-saccharidanalyse darstellen, da darin die Vorteile von vielen einzelnen Präparationen vereint werden.Neue Aspekte: Die Derivatisierung mit der Matrix 3-AQ ist schnell, benötigt keine Aufreinigung und erleichtert

die Strukturanalyse von neutralen Oligosacchariden.

Referenzen: [1] Zaia, J. Mass Spectrom. Rev. 2004, 23 (3), 161-227.; [2] Harvey, D. J. Mass Spectrom. Rev. 1999,

18 (6), 349-450.; [3] Metzger, J. O.; Woisch, R.; Tuszynski, W.; Angermann, R. Fresenius. J. Anal. Chem. 1994,

349 (6), 473-474.; [4] Stahl, B.; Thurl, S.; Zeng, Z. R.; Karas, M.; Hillenkamp, F.; Steup, M.; Sawatzki, G. Anal.

Biochem. 1994, 223 (2), 218-226.

Thema: Kohlenhydrate

Keywords: Oligosaccharide, Derivatisierung, MALDI-TOF/TOF, Humanmilcholigosaccharide (HMOS)


73

V34

Novel Routine Clinical Applications for Mass Spectrometry

Ingendoh, Arnd; Pusch, Wolfgang; Goetz, Sebastian; Mix, Guido; Baessmann, Carsten

Bruker Daltonik GmbH, Germany

Einleitung: Mass spectrometry is widely used in clinical routine applications already. Numerous methods basedon GC/MS and LC/MS/MS are established, e.g. the newborn screening of acylcarnitines or therapeutic drugmonitoring of patients. Nevertheless, routine patient analysis is still dominated by other, more established methods,mostly due to cost reasons and the limited automation / robustness of MS.With new applications, MS makes anincreased entry into clinical chemistry. Key requirements are ease-of-use and reliable results, in particular withregard to false positive rates for patient samples. Presented here will be two new applications for patient analysis,both based on mass spectra libraries. One is the identification of microorganisms by MALDI-TOF, the other isacute clinical toxicology screening with an ion trap.Experimenteller Teil: Based on a reference mass spectra library, MALDI-TOF allows the reliable identificationand classification of microorganisms, such as bacteria, archaea, yeasts or fungi within minutes. Following a fast andsimple sample preparation protocol, species-specific signal patterns, mostly from ribosomal peptides and proteins,are obtained by mass spectrometry. A database containing thousands of reference entries enables the secureidentification of unknown samples using sophisticated pattern matching algorithms. In contrast to contemporarybiochemical methods no initial data input is requested - every sample, including non-fermenters, is processed andanalyzed the very same way. The system answers most of the important conditions for clinical chemistry, i.e. isvery simple to use, robust, reliable, fast and cost effective.Ergebnisse und Diskussion: A new approach for acute clinical toxicology screening is based on an ion trap.Clinical emergency rooms are often faced with patients showing acute toxicological symptoms. Frequently thesepatients are not able to disclose the identity of the causing toxin or drug. In these cases blood or urine samplesare taken and analyzed to quickly enable a proper treatment of the patients. Existing screening methods in clinicalroutine labs often rely on spectral comparisons of LC-UV-DAD data. Instead of this, the identification of toxiccompounds is done here on an ion trap equipped with an MSn spectra library and a search algorithm. Thistechnique is already successfully applied in forensic and food testing applications and turned out to be a veryeffective and more information-rich alternative compared to just UV-data. It is designed here in a way thatreduces manual interaction to the lowest level. Clinical samples of patients with acute toxicological symptomsare extracted by SPE and subsequently analyzed by ultra high performance liquid chromatography (UHPLC)coupled to the ion trap. The UHPLC system ensures short analysis times (gradient time < 15 minutes) with highchromatographic resolution (peak widths < 5 sec FWHM). For both approaches, comparisons with the currentlyestablished techniques are presented to illustrate benefits and drawbacks.Neue Aspekte: Anwendung der Massenspektrometrie auf neue Felder in der klinischen RoutinediagnostikThema: Instrumentelle Entwicklungen, Proteine und PeptideKeywords: Mikroorganismen-Identifizierung, akute klinische Toxikologie, MALDI-TOF, Ionenfalle, Spektren-bibliothekKontakt: [email protected]

74

V35

In situ real time identification of biological tissue with surgicaltools combined with mass spectrometry

Schäfer, Karl Christian (1); Szaniszló, Tamás (2); Balogh, Júlia (2); Balogh, Lajos (3); Dénes, Júlia (1);

Szalay, Dániel (2); Gödörházy, Lajos (2); Takats, Zoltan (1)

1: Institut für Anorganische und Analytische Chemie Universität Giessen, Germany; 2: MediMass Ltd.,

Budapest, Hungary; 3: "Frédéric Joliot-Curie" National Research Institute for Radiobiology and Radiohygiene

Einleitung: The combination of established surgical methods with mass spectrometry leads to a powerful tool in

medical diagnostics. The mass spectrometer provides objective data in this combination which can be used for

the differentiation of different tissue types including tumor tissue and healthy tissue. Established MS methods

like MALDI can be used for this purpose. Sample preparation and measurement conditions however limit this

applications to the histopathological field. Therefore a method which provides data in real-time, directly from the

operating table during surgical intervention is a step forward in using mass spectrometry in surgical operations.

Experimenteller Teil: Experiments were performed on a Thermo Scientific LTQ-Orbitrap Discovery equipped

with a home-built ion source. Ions were produced with an electrosurgical unit (radioSURG®2200, Meyer-Haake)

and a surgical CO2 Laser (DreampulseIII, DSE-Daeshin). Cutting power was set to 60 W max and laser energy

was set to 10 W max in pulsed mode. For ion collection and delivery to the mass spectrometer a VAC-100 venturi

pump (Veriflo, Parker instruments) and a 1/8" O.D. 2 mm I.D. PTFE tubing were used. The gas flow for the venturi

pump was set to 20 L/min. Samples were placed on the counter electrode of the electrosurgical unit. The original

handpiece was used for cutting and the transfer tubing was placed directly over the cutting area.

Ergebnisse und Diskussion: The spectra mainly contain phospholipid signals and decomposition products of

those. The phospholipid pattern was found to be characterisitc for the tissue type. We have tested a number of

tissue types from different species including healthy and tumor tissue. In combination with PCA software we

managed to differentiate between different tissue types in real time during the experiment. The accuracy of the

identification of tissue type is around 97% depending on the tissue type. It is possible to get molecular information

with both electrosurgical and laser cutting tool in positive and negative ion mode. In both cases a characteristic

phospholipid pattern is obtained. The comparison of data originating form laser and electrosurgical experiments

from the same tissue type reveal differences in the phospholipid patterns.

Neue Aspekte: In-situ real-time tissue analysis

Referenzen: [1] Schafer, K. C.; Denes, J.; Albrecht, K.; Szaniszlo, T.; Balog, J.; Skoumal, R.; Katona, M.; Toth,

M.; Balogh, L.; Takats, Z., Angewandte Chemie-International Edition 2009, 48 (44), 8240-8242

Thema: Instrumentelle Entwicklungen

Keywords: REIMS, realtime, tissue analysis


75

V36

Direct Surface Sampling of Planar Tissues using a Chip-BasedNano-Electrospray system

Kertesz, Vilmos (1); Almeida, Reinaldo (2); Henion, Jack (3); Van Berkel, Gary J. (1)

1: Oak Ridge National Laboratory, Oak Ridge, TN , USA; 2: Advion BioSciences, Germany; 3: AdvionBioSciences,I thaca, NY,U SA

Einleitung: Metabolite distributions directly in tissues require atmospheric pressure surface sampling/ionizationof small sample volumes, where analytes can be directly analyzed from a variety of surface types without samplepreparation. A direct analyte extraction from a surface was recently developed, by contacting the surface witha liquid micro junction surface sampling probe (LMJ-SSP). This processwas mimicked and automated using acommercially available fully automated chip-based nano-electrospray source.A small solvent droplet was placedon the tissue area of interest for analyte extraction and then aspirated into a conductive pipette tip for automatedchip based nano electrospray infusion.Experimenteller Teil: Instrumentation: TriVersa NanoMate nanospray emitter system (Advion BioSystems,Ithaca, NY) coupled to a Synapt HDMS time-of-flight (Waters Corp., Milford, MA) mass spectrometer.Methodoptimization: Verapamil and propranolol standards were prepared in 50/50 (v/v) acetonitrile/water. Alpha cyano-4-hydroxycinnamic acid was dissolved in 56/36/8 (v/v/v) acetonitrile/methanol/water at a concentration of 3mg/mL. MALDI matrix was mixed 1:1 with analyte and 1 uL samples were deposited on a stainless steel 96well MALDI plate used as the sampling surface. The dried samples were extracted with 80/20/0.1 (v/v/v) acetoni-trile/water/formic acid. Verapamil and propranolol were monitored in selected reaction monitoring mode. Tissueslice analyses: Sulforafane (SFN), Propranololand its metabolites where analysed on mice tissue 3 hours post dose.The extraction solvent was 80/20/0.1 ACN/H20/FAErgebnisse und Diskussion: Verapamil samples in a MALDI matrix (1.5 mg/mL) were deposited on, let dry, sam-pled off of a typical MALDI plate insert and analyzed using the already built-in hardware and software elementsof a TriVersa NanoMate nanospray emitter system. Quantitation of verapamil in the 5-1000 ng/mL concentrationrange was accomplished using 1000 ng/mL propranolol as internal standard. The obtained plot showed excellentlinearity with R2=0.998. Thin mouse tissue slices pre dosed with sulforafane or propranolol, were investigated bythis method for analysis of drug and drug metabolite distribution of whole body and compared with control tissue.The parent drug ( SFN / Propranolol),and the phase II metabolites (SFN-GSH,SFN-NAC / Hydroxypropranolol-glucuronide) were screenedin MRM mode in different thin tissue sections ( lung, liver, kidney, brain ) from thedose and control mouse. Excellent signal to noise ratio could be achieved. Further more other planar surfaces likedry blood spots and analytes on TLC plates are explored.Neue Aspekte: Automated ambient pressure surface sampling/extraction and ionization of analytes on a planartissue surface.Thema: Instrumentelle Entwicklungen, ImagingKeywords: Surface Sampling,Extraction,Electrospray,ESI ImagingKontakt: [email protected]

76

V37

Speciation Analysis of Gadolinium Chelates in Hospital Effluentsand Wastewater Treatment Plant Sewage by a Novel

HILIC/ICP-MS Method

Karst, Uwe

Universität Münster, Germany

Einleitung: Since their introduction in the 1980s, the application of Gd-based contrast agents increased rapidly.

Today, almost every second MRI procedure is enhanced by contrast agents, resulting in about 20,000,000 appli-

cations per year worldwide. For the application as contrast agent, Gadolinium is complexed by chelating agents.

In general, contrast agent formulations are highly concentrated. This leads to a very high input of anthropogenic

Gd into the environment. Therefore, Gd chelates are among the most important emerging environmental contam-

inants. Although this has already caused a strong anomaly for Gd concentrations in surface waters, only the sum

concentration of Gd is typically determined by ICP-MS. However, there is little knowledge about the concentration

of various Gd species in wastewaters and surface waters.

Experimenteller Teil: This work describes method development and speciation analysis of gadolinium-based

MRI contrast agents in wastewaters. This novel approach comprises the hyphenation of hydrophilic interaction

chromatography (HILIC) with inductively coupled plasma mass spectrometry (ICP-MS). HILIC/ICP-MS exhibits

high separation efficiency for the simultaneous separation of the five predominantly applied MRI contrast agents

and the required selectivity and sensitivity for trace determination in wastewater samples. Sample preparation was

performed using silanized glass vessels to prevent adsorption effects of some of the analytes on glass surfaces.

A zwitterionic HILIC stationary phase allowed the separation of the five most important contrast agents. A

quadrupole ICP-MS instrument provided quantification independent on the Gd species.

Ergebnisse und Diskussion: The time of analysis for a single HILIC run was 30 minutes. The limits of detection

(LOD) were determined by means of a signal to noise ratio of three (S/N=3), whereas the limits of quantification

(LOQ) were determined by means of a signal to noise ratio of ten (S/N=10). For all components, the LOD was as

low as 1·10−9 mol/L and the LOQ was 3.3·10−9 mol/L, respectively. Linearity was verified in the concentration

range of 1·10−9 mol/L up to 1·10−6 mol/L by triplicate measurements of ten calibration solutions, containing

all five contrast agents. The resulting regression coefficients were 0.999 (Gd-BOPTA), 0.993 (Gd-DTPA-BMA),

0.998 (Gd-BT-DO3A), 0.999 (Gd-DOTA) and 0.998 (Gd-DTPA) respectively. The relative standard deviations

(RSD) of peak areas were in the range of 0.6-14.5% with an average of 4.8% over the complete calibration range.The mean RSD of retention times ranged from 0.1-0.8% with an average of 0.4%.A total of 14 samples was taken from hospital effluent, wastewater treatment plant effluent and at different po-sitions within the sewage treatment plant. All water samples were not only analyzed for the particular contrastagents by triplicate HILIC/ICP-MS measurements, but also total Gd was determined by ICP-MS for validationpurposes. The concentrations of the individual Gd species, the standard deviation of the mean values of the tripli-cate measurements, the sum of all Gd species determined by HILIC/ICP-MS as well as the total concentration ofGd determined by ICP-MS. It was demonstrated that the developed method is suitable for the speciation analysisof the five most important MRI contrast agents in real water samples of different origin.Neue Aspekte: The first method for speciation analysis of various Gd-based MRI contrast agents in wastewatershas been developedThema: Element-Massenspektrometrie, Umweltanalytik und AerosoleKeywords: contrast agents, wastewater, speciation analysis, LC/ESI-MS, LC/ICP-MSKontakt: [email protected]

77

V38

Differenzierung der isobaren Aminosäuren Leucin und Isoleucin

und schnelle Charakterisierung von posttranslational modifizierten

Peptiden durch MALDI-Massenspektrometrie kombiniert mit

Stosskühlung

Soltwisch, Jens; Dreisewerd, Klaus

Institut für Medizinische Physik und Biophysik, Westfälische Wilhelms-Universität Münster

Einleitung: Obwohl die MALDI ein „sanftes"Desorptions-/Ionisationsverfahren ist, werden nicht nur intakte Mo-

lekülionen erzeugt, sondern ein Teil der Ionen fragmentiert in Sekundärprozessen. Primär werden zwei Arten von

Tochterionen erzeugt. Die korrespondierenden Prozesse werden als In-Source Decay (ISD)und als Post-Source

Decay (PSD) bezeichnet. Die erzeugten ISD-Fragmente stellen hoch angeregte Spezies dar, die in der Folge weiter

fragmentieren können [1]. Durch Stoßkühlung in einem orthogonal-extrahierenden Flugzeitmassenspektrometer

mit Feinvakuum-Ionenquelle können diese wirkungsvoll stabilisiert werden. Wir zeigen hier, dass unter diesen Be-dingungen auch d- und w-Fragmentionen von Peptiden nachgewiesen werden, welche die wichtige Differenzierungisobarer Aminosäuren erlauben. Durch Variation des Stoßgasdruckes lassen sich auch posttranslationale Modifika-tionen untersuchen (z.B. von Phospho- und Glykopeptiden). Als drittes Beispiel wird die Strukturaufklärung einesDisulfidbrücken-gebundenen Peptids gezeigt.

Experimenteller Teil: Die Experimente wurden mit einem orthogonal-extrahierenden Flugzeitmassenspektrome-

ter [1] im pos. oder neg. Ionenmodus durchgeführt. Zur Anregung diente eine N2-Laser (337 nm, 3 ns, Strahlprofil

auf der Probe : 200 x 230 µm2, Fluenz: 900-1000 J/m²). Als Matrix wurde 2,5-Dihydroxybenzoesäure (DHB)

verwendet; 10 pmol an Peptidsubstanz wurden für eine Messung aufgetragen. Es werden Beispiele gezeigt für

die Analyse von Renin Substrat, Fibrinopeptid A, phosphoryliertes Angiotensin II, glykosyliertes EPO, sulfatier-

tes Cholecystokinin (26-33), sowie Disulfidbrücken-gebundenes Vasopressin. Die MALDI-Ionen wurden in einer

Feinvakuum-MALDI-Ionenquelle bei einem einstellbaren N2-Hintergasdruck von ∼ 0.05 - 2 mbar erzeugt. Die

Massenauflösung (FWHM) des Geräts betrug ca. 10.000, die Massenbestimmungsgenauigkeit ca. 10 mbar (beiinterner Eichung).Ergebnisse und Diskussion: Für alle untersuchten Peptide wurden weitgehend vollständige Aminosäuresequen-

zen erhalten. Bei hohem Druck ( 1,0 mbar) werden primär „ISD-Fragmenteërhalten, die aus einer chemischen Ak-tivierung der Analytmoleküle durch Übertrag eines Wasserstoffs der Matrix erzeugt werden, bei niedrigem Drucksolche, die auch in der MALDI-PSD-Analytik gefunden werden und aus einem unimolekularen Zerfall thermischangeregter Ionen stammen.Bemerkenswerterweise werden bei hohemDruck (also optimaler Stoßkühlung) und erhöhter Fluenz auch d- und w-Fragmentionen nachgewiesen. Deren Erzeugung schließt einen hochenergetischen Bindungsbruch in den Amions-äureseitengruppen ein. Dies erlaubt die wichtige Differenzierung der isobaren Aminosäuren Leucin und Isoleucin.Experimente mit zwei Derivaten der Peptide, bei denen Leucin durch Isoleucin ausgetauscht wurden, bestätigendiese spannende Beobachtung. Bei niedrigem Druck gehen die d- und w-Fragmente verloren. Dies legt nahe, dasses sich um hochangeregte Spezies handelt, die in sekundären Prozessen weiter zerfallen.Unter optimalen Stoßkühlungsbedingungen macht die Intensität der [M-SG+H]+-Signale (SG = labile Seiten-gruppe) der posttranslational modifizierten Peptide nur 2-3% des Molekülionensignals [M+H]+ aus). Keines derc- und/oder z-Fragmente zeigt einen messbaren SG-Verlust. Die Zuordnung der Bindungsstellen wird unterstütztdurch eine Analyse bei niedrigem Druck, einhergehend mit einer signifikant verstärkten Abspaltung der labilenGruppen. Das Beispiel von Ala-Gly-[Arg8]-Vasopressin zeigt, dass durch eine Variation des Druckes auch dieStrukturaufklärung eines über eine interne Disulfidbrücke stabilisierten Peptids möglich wird.Die beschriebene Methodik kann mit Standard-o-TOF-Massenspektrometern realisiert werden, wenn diese einenStoßgasdruck in der MALDI-Ionenquelle von etwa 1 mbar erlauben. Im Hinblick auf die Erzeugung der d-/w-Fragmente stellt die Technik eine mögliche Alternative zur TandemMSmit stoßinduzierter Hochenergie-Anregung(heCID) dar. Sie stellt auch eine interessante Alternative zur Tandem-Massenspektrometrie mit ECD/ETD dar. ImGegensatz zu diesen Verfahren lässt sie sich auf einfach geladene und auf negativ geladenen Ionen anwenden underlaubt zwanglos die Durchführung von High-Throughput-Analysen.Neue Aspekte: Nachweis von d- und w-Fragmenten unter MALDI-ISD-Bedingungen; schnelle Identifizierungvon posttranslationalen Modifikationen in Peptiden, einschließlich der von Disulfidbrücken

78

Referenzen: [1] Soltwisch, J., Souady, J., Berkenkamp, S., Dreisewerd, K., Effect of gas pressure and gas type onthe fragmentation of peptide and oligosaccharide ions generated in an elevated pressure, Anal. Chem. 81 (2009)2921.Thema: Instrumentelle Entwicklungen, Proteine und PeptideKeywords:MALDI-MS, ISD, Peptidsequenz, Stosskühlung, Wasserstofftransfer


79

V39

Auf der Suche nach deutschen Begriffen in der

Massenspektrometrie

Gross, Jürgen

Universität Heidelberg, Germany

Einleitung: Unsicherheiten im Gebrauch deutscher Fachbegriffe der Massenspektrometrie erfahren wir immer

wieder, denn Berufsausbildung, universitäre Lehre, innerbetriebliche Weiterbildung und Zeitschriftenbeiträge ver-

langen deutschsprachige Kommunikation. Andererseits lesen und publizieren wir fast ausschließlich auf Englisch.

Selbst bei unserer DGMS-Tagung in Halle wird wieder ein erheblicher Teil der Beiträge auf Englisch präsen-

tiert. In der englischsprachigen Literatur gibt es mehrere Quellen, die sich den Termini der Massenspektrometrie

und damit auch ihren Schreibweisen und Akronymen widmen,[1-3] im Deutschen herrscht dagegen eine gewisse

Beliebigkeit. Sucht man die Begriffe Atmosphärendruck-Ionisation oder Atmosphärendruck-Ionisierung bzw. At-

mosphärendruckionisation oder Atmosphärendruckionisierung im Index der Angewandten Chemie [4] bleibt die

Suche ohne Treffer. Mit dem Suchbegriff Elektrospray findet man selbst bei der für ihre Errungenschaften um die

deutsche Wissenschaftssprache geehrten Zeitschrift verschiedene Schreibweisen.

Experimenteller Teil: Zuerst wurde die Empfehlung der IUPAC für die englischen Begriffe zugrunde gelegt,

soweit diese darin aufgeführt sind. Zahlreiche weitere englische Begriffe wurden aus verschiedenen Quellen,

insbesondere aus Indizes von Büchern, zusammengetragen, um annähernde Vollständigkeit der Sammlung zu

erreichen. Aus der in geringem Maße existenten deutschsprachigen Fachliteratur, z.B. [5], wurden die englischen

Begriffe mit Deutschen korreliert. Für die englischen Begriffe wird hier durchgängig American English verwendet.

Ergebnisse und Diskussion: Eine umfangreiche Zusammenstellung von rund 450 englischen Begriffen samt ihrer

Akronyme aus dem Bereich der Massenspektrometrie und unmittelbar verwandter Bereiche ist erstellt und mit

Übersetzungen versehen. Für einen nennenswerten Teil läßt sich ein deutscher Begriff finden, für den allerdings

manchmal verschiedene Schreibungen denkbar sind. In Grenzfällen wurde ein Übersetzungsvorschlag aufgenom-men, einige wenige Begriffe haben aktuell noch keine Übersetzung erhalten. Die Übersetzung eines Begriffs umjeden Preis wurde vermieden, denn die Absichten dieses MS-Wörterbuchs liegen mehr in einer Bestandsaufnahmeund der Erreichung einer einheitlichen, korrekten Wissenschaftssprache als in einer Vorgabe oder Neuschöpfungvon Wörtern. Oberste Priorität hat die Schaffung einer Basis für effektive und korrekte wissenschaftliche Kommu-nikation, was nicht zuletzt in der Lehre von enormer Bedeutung ist.Die Mitglieder der DGMS sind eingeladen, diese Liste kritisch zu prüfen, alternative Übersetzungen oder Schreib-weisen vorzuschlagen und die Liste aus ihrem speziellen Arbeitsfeld abzurunden. Dazu ist die Arbeitsversion derListe ab Tagungsbeginn im Internet unter http://www.rzuser.uni-heidelberg.de/ bl5/deutsch/begriffe.html abrufbar.Beiträge zur deutschen Terminologie in der MS sind bis 31. Juli 2010 an die dort angegebene Korrespondenzadres-se erbeten.Neue Aspekte: Initiative zur einheitlichen Begriffsfindung bei Beiträgen zur Massenspektrometrie in deutscherSprache mit dem Vorschlag langfristiger Pflege durch die DGMS.Referenzen: [1] Sparkman, O.D. Mass Spectrometry Desk Reference; 2nd ed.; Global View Publishing: Pittsbur-gh, 2006.; [2] Mallet, A.; Down, S. Dictionary of Mass Spectrometry; John Wiley and Sons: Chichester, 2009.;[3] Murray, K.K.; Boyd, R.K.; Eberlin, M.N.; Langley, J.G.; Li, L.; Naito, Y. Standard Definitions of Terms Rela-ting to Mass Spectrometry (IUPAC Recommendations 2006). Pure and Applied Chemistry 2009, under review.;[4] http://www3.interscience.wiley.com; [5] Budzikiewicz, H.; Schäfer, M. Massenspektrometrie - Eine Einfüh-rung; 5th ed.; Wiley-VCH: Weinheim, 2005.Thema: Organische MassenspektrometrieKeywords: Fachbegriffe, Wörterbuch, Deutsch, Englisch, ÜbersetzungKontakt: [email protected]

80

V40

Cyclization and Rearrangement Reactions of a Ions of ProtonatedPeptides

Paizs, Bela (1); Bythell, Benjamin (1); Maitre, Philippe (2)

1: DKFZ, Germany; 2: Université Paris-Sud, France

Einleitung: The structure and reactivity of N-terminal bn fragments of protonated peptides have recently received

significant attention. It was shown that bn (n >= 5) ions [1] can undergo head-to-tail cyclization forming macro-

cyclic isomers which can open up at various amide bonds forming linear structures with ‘scrambled’ sequences.

Numerous examples demonstrated that neglecting this ‘scrambling’ chemistry in peptide sequencing can lead to

erroneous peptide identification [2]. Recent gas-phase IR experiments [3] indicate that macrocyclic bn isomers are

present in the mass spectrometer as stable species. While an fragments are often formed in CID of protonated pep-

tides, their structures received limited attention in the last few years. Here we report detailed IR and computational

data on an fragments formed from protonated oligoglycines.

Experimenteller Teil: The fragmentation of protonated GGG, GGGG, and GGGGG was studied in a quadrupole

ion trap mass spectrometer (Bruker Esquire 3000+). Following ionization, the singly charged precursor ions were

subjected to CID to form an ions which were mass-selected and irradiated with the free electron laser beam

at CLIO. Upon resonant vibrational excitation, dissociation of an ions was observed. The abundances of the

corresponding fragments were recorded as a function of the IR wavelength in order to derive the IR action spectra.

The theoretical IR spectra were determined using harmonic frequencies calculated for various an isomers. The

calculated stick spectra were convoluted assuming a Lorentizan profile with a 20 cm−1 full-width at half-maximum

to allow direct comparison to the experimental spectra.

Ergebnisse und Diskussion: an ions have generally been assumed to exist as immonium derivatives (R1HN+ =

CHR2). Here we show using infrared spectroscopy and DFT calculations that the a2 ion with the GG sequence

undergoes cyclization to form a five-membered ring isomer. The a3 ion with the GGG sequence undergoes

cyclization that is initiated by nucleophilic attack of the carbonyl oxygen of the N-terminal glycine residue on

the carbon center of the C-terminal immonium group forming a seven-membered ring isomer. The barrier to this

reaction is low at 13.6 kcal mol−1 and the resulting cyclic isomer is energetically more favored than the linear

form. The a4 ion with the GGGG sequence undergoes head-to-tail cyclization via nucleophilic attack of the N-

terminal amino group on the carbon center of the C-terminal immonium ion forming a 11-membered macroring

which contains a secondary amine and three amide bonds. Then an intermolecular proton shift transfers the

initially formed -CH2-NH+2 -CH2-NH-CO- moiety to -CH2-NH-CH2-NH+

2 -CO- which can be cleaved at the -CH2-

NH+2 - bond leading to opening of the macrocycle and forming a rearranged linear isomer with the CH2=N-CH2-

moiety at the N- and the -CO-NH2 amide bond at the C-termini, respectively. This rearranged linear structure

is 13 kcal mol−1 more energetically favored than the most favored linear a4 ion structure. Furthermore, the

barriers to the above cyclization and ring-opening reactions are at 9 - 12 kcal mol−1 allowing facile formation

of the rearranged species. The experimental infrared spectrum confirms the presence of this species in the mass

spectrometer showing a highly specific CO stretch vibration of the un-substituted C-terminal amide group at 1750

cm−1. Our results indicate that one needs to consider both cyclization and rearrangement reactions in order to

decipher fine details of the structure and fragmentation pathways of peptide an ions.

Neue Aspekte: Structure and cyclization reactions of an ions studied by IR spectroscopy and theory

Referenzen: [1] Harrison, A. G.; Young, A. B.; Bleiholder, B.; Suhai, S.; Paizs, B. J. Am. Chem. Soc. 2006, 128,

10364.; [2] Bleiholder, C.; Osburn, S.; Williams, T. D.; Suhai, S.; Van Stipdonk, M.; Harrison, A. G.; Paizs, B.

J. Am. Chem. Soc., 2008, 130, 17774.; [3] Erlekam, U.; Bythell, B.J.; Scuderi, D.; Van Stipdonk, M.; Paizs, B.;

Maitre, P. J. Am. Chem. Soc. 2009, 131, 11503.


Keywords: Peptide fragmentation, mechanism, IR spectroscopy, theory


81

V41

High Performance MALDI Imaging

Spengler, Bernhard; Römpp, Andreas; Takats, Zoltan; Günther, Sabine; Schulz, Oliver; Schober, Yvonne

Justus-Liebig-Universität Gießen, Germany

Einleitung: MALDI Imaging mass spectrometry has gained broad interest over the last decade. After a proof ofprinciple in 1994 [1] it has found broad acceptance and applicability over the years, driven by academic develop-ments and more recently by availability of commercial instruments [2]. Today MALDI imaging is a method ofvisualizing biomedical states in tissue in a broad applicational range. While the qualitative usefulness of MALDIImaging was quite obvious from the very beginning, its analytical relevance in terms of identification of imagedsignals, reproducibility and authenticity was still questionable. Focusing on accurate mass based identification ofimaged compounds and improving lateral resolution in biological tissue analysis has resulted in a new level ofquality and reliability in MALDI imaging.Experimenteller Teil: For the first time high spatial resolution of 3 to 10 µm was combined with high mass

accuracy in biological tissue imaging with our setup. FT-MS in the low ppm accuracy range enabled us to identify

analytes directly from tissue during imaging from individual sample spots ("pixels"). Images were generated from

high resolution / high accuracy data with a selectivity (a m/z bin width) of 0.01 u. MALDI imaging was performed

with a home-built atmospheric pressure MALDI imaging source [3] attached to a LTQ Orbitrap mass spectrometer

or a LTQ-FTICR mass spectrometer (Thermo Scientific GmbH, Bremen). Matrix was applied onto tissue samples

using a dedicated pneumatic spray procedure [4].

Ergebnisse und Diskussion: The analytical performance of the new instrumental setup and methodology was

found to be sufficiently high to characterize histological features of biological tissue in a highly detailed and

reliable manner. Structural features of various tissue types could be differentiated on a molecular basis and could

be directly compared to the performance of classical histological staining experiments. Lipids, peptides and drug

compounds were imaged in a number of biological samples at 3 to 10 µm lateral resolution with a mass accuracy

under imaging conditions of better than 2 ppm. The pattern of phospholipid compounds was used to distinguish

between different tissue types. The identity of lipids could be determined by accurate mass analysis directly from

tissue. Additional MS/MS imaging was employed to verify lipid head groups. The results demonstrate a new

quality of MALDI imaging under high accuracy / high resolution conditions.

Neue Aspekte: MALDI imaging mass spectrometry of biological samples with accurate mass analysis at 3 to 10

µm spatial resolution.

Referenzen: [1] B. Spengler, M. Hubert, R. Kaufmann, "MALDI Ion Imaging and Biological Ion Imaging with

a new Scanning UV-Laser Microprobe". Proc. of the 42nd Annual Conference on Mass Spectrometry and Allied

Topics, Chicago, Illinois, May 29 - June 3, 1994, page 1041.; [2] L.A. McDonnell, R.M.A. Heeren, "Imaging Mass

Spectrometry". Mass Spec. Reviews 26 (2007) 606-643.; [3] M. Koestler, D. Kirsch, A. Hester, A. Leisner, S.

Guenther, B. Spengler, Rapid. Comm. Mass Spectrom. 22 (2008) 3275.; [4] W. Bouschen, O. Schulz, D. Eikel, B.

Spengler, Rapid Comm. Mass Spectrom. 24 (2010) 355.

Thema: Imaging

Keywords: Imaging, Massengenauigkeit, Massenauflösung, Ortsauflösung, Selektivität


82

V42

Lucky Survivor zum Letzten - Experimentelle Bestätigung eineslange postulierten Modells

Jaskolla, Thorsten W.; Karas, Michael

Goethe Universität Frankfurt, Germany

Einleitung: Obwohl die Matrix-unterstützte Laserdesorptions-/ionisations-Massenspektrometrie (MALDI-MS)

bereits seit mehr als 20 Jahren genutzt wird, steht eine eindeutige mechanistische Erklärung der Analytionenbil-

dung noch immer aus. Zwei Haupttheorien wurden und werden diskutiert: DasModell der Gasphasen-Protonierung,

das als Ursache der Analyt-Ionisation einen Protonentransfer protonierter Matrizes auf neutrale Analyte annimmt

sowie das Modell des "Lucky-Survivors", das von Ladungstrennungen bereits präformierter Ionen ausgeht. In

diesem Beitrag wird der experimentelle Nachweis der real ablaufenden Prozesse geführt werden.

Experimenteller Teil: Zur Differenzierung zwischen den konkurrierenden Analyt-Ionisationsmodellen wurden

verschiedene (un)deuterierteα-Cyanozimtsäureester als Matrizes für dieMALDI-Massenspektrometrie dargestellt.

Ausgehend von Cyanessigsäure erfolgte Synthese der (un)deuterierten Cyanessigsäureethyl- und tertbutylester

durch Kopplung mit den entsprechenden Alkoholen und Dicyclohexylcarbodiimid gemäß einer Steglich-Veresterung.

In nachfolgender Knoevenagel-Kondensation mit substituierten Benzaldehyden wurden die Cyanessigsäureester

zu den gewünschten para-substituierten α-Cyanozimtsäureestern umgesetzt. Der Deuterierungsgrad wurde mit-

tels ESI überprüft. Die Zimtsäureester unterliegen einer intramolekularen Umlagerung nach Energieeintrag mit-

tels Laser, wodurch die entsprechenden freien (un)deuterierten Zimtsäurecarbonsäuren dargestellt werden. Zur

Analyse wurden Peptide als präformierte Ionen sowie verschiedene kleine Moleküle (z.B. 4-OH-Acetophenon,

Anisaldehyd, Phloroglucin, Salicylamid, Nicotinsäureamid) als neutrale Analyte eingesetzt und die resultierenden

protonierten bzw. deuterierten Analytsignale analysiert.

Ergebnisse und Diskussion: Das Modell der Gasphasen-Ionisation postuliert eine Protonierung des neutralen An-

alyten A durch protonierte Matrix MH+ in der Gasphase. Eine Überprüfung dieser Vorhersage wird durch Einsatzeiner Matrix mit deuterierter acider Funktionalität (z.B. Säurefunktion) möglich, als Ergebnis eines Gasphasen-

Protonentransferprozesses muss der Analyt folglich eindeutig als AD+ detektierbar sein. Im Gegensatz dazu

geht das "Lucky-Survivor"-Modell von in der Ausgangslösung und festen Matrixpräparation präformierten Ionen

[AHnn+ + nX−] aus, das Auftreten von Analytionen ist nach diesem Modell das Ergebnis einer Clusterdesol-

vatisierung mit unvollständiger Neutralisation durch entweder bereits fehlende Anionen oder partieller Anionen-

Neutralisation bedingt durch MH+(oder MD+ im Falle deuterierter Matrizes), was in der eindeutigen Bildung von

AH+resultiert.

Da durch das Lösungsmittel in bei MALDI typischen Dried-Droplet-Präparationen keine stabile Protonierung

mit paralleler Deuterierung von funktionellen Gruppen mit austauschbaren Wasserstoffen möglich ist, lässt sich

dieser Ansatz nur durch Verwendung einer Matrix mit "geschützter" deuterierter Säurefunktion ermöglichen.

Diesem Zweck dienen die dargestellten deuterierten Matrix-Ester, die erst durch Energiezufuhr in der frühen

Ablationsphase in die korrespondierenden deuterierten Säuren (z.B. α-Cyano-4-hydroxyzimtsäure-d1, CHCA-

d1) zerfallen. Eine Analyse des reinen Matrix-Spektrums bestätigt die quantitative Bildung der deuterierten und

deuteronierten Säure CHCA-COOD2+. Ein Vergleich der Peptidsignale (Standard-Peptide im niedrigen femtomol-

Bereich) mit den entsprechenden undeuterierten Matrix-Verbindungen zerfallend zu Matrix-COOH2+ erlaubt die

experimentelle Bestätigung des "Lucky-Survivor"-Modells für präformierte Ionen (basische Analyte wie z.B. Pep-

tide) und ermöglicht die Erkenntnis, dass neutrale Analyte in Abhängigkeit der (deuterierten) Matrix entsprechend

dem Gasphasen-Ionisationsmodell entweder ausschließlich protoniert oder deuteroniert werden. Die hierzu er-

forderlichen Analyt-Mengen erlauben einen groben Effizienzvergleich beider Mechanismen (min. 10.000:1) und

beschränken die Gasphasen-Ionisierung auf einen wenig effizienten Nebenprozess. Weiterhin kann der Einfluss

der Matrix-Protonenaffinität mit dem verifizierten "Lucky-Survivor"-Modell kombiniert sowie der experimentelle

Nachweis von Matrix-Analyt-Wasserstoffaustauschreaktionen in der Gasphase geführt werden.

Neue Aspekte: Die hier vorgestellten Experimente ermöglichen eine abschließende Klärung des in MALDI do-

minierenden Analyt-Ionisationswegs unter Bestätigung des "Lucky-Survivor"-Modells.

83

Referenzen: [1] Karas M.; Glückmann, M.; Schäfer, J., Ionization in Matrix-assisted Laser Desorption Ionization:

Singly Charged Molecular Ions are the Lucky Survivors, J. Mass Spectrom. Special Feature: Perspective 2000,

35, 1-12.; [2] Krüger, R.; Karas, M., Ion Formation in MALDI: The Cluster Ionization Mechanism, Chem. Rev.

2003, 103, 427-439.; [3] Zenobi, R.; Knochenmuss, R., Ion Formation in MALDI Mass Spectrometry, Mass

Spectrometry Reviews 1998, 17, 337-366.; [4] Knochenmuss, R. Ion Formation Mechanisms in UV-MALDI,

Analyst 2006, 110, 12728-12733.; [5] Knochenmuss, R.; Zenobi, R. MALDI Ionization: The Role of In-Plume

Processes, Chem. Rev. 2003, 103, 441-452.

Thema: Organische Massenspektrometrie, Ionen-Molekül-Reaktionen

Keywords: Protonierung, Ionisation, Lucky-Survivor, Gasphase, Präformierung


84

Abstracts der Posterbeiträge

90

P1

Affinity Chromatographic Isolation of Low-abundant EndogenousTranscription Factors - Background Reduction by Enhancing

Chemical Specificity during Elution.

Eickner, Thomas (1); Lorenz, Peter (2); Ding, Gouhoi (3); Li, Yixue (3); Thiesen, Hans-Jürgen (2);

Glocker, Michael O. (1)

1: Proteom-Zentrum Rostock, Germany; 2: Institut für Immunologie, Universität Rostock, Germany; 3: Shanghai

Center for Bioinformatics Technologies, China

Einleitung: Affinity chromatographical isolation of proteins is a well established method. However, a serious

problem of this powerful isolation procedure is the enrichment of non-specific bound proteins and interaction-

mimics. This causes highly complex mixtures of bound molecules. As a consequence, the analysis of the desired

target proteins is quite difficult. In order to improve the background reduction, we focused on the fine-tuning

of the chemical conditions during the elution step. TRIM28 is a multi-interaction key-protein that takes part in

transcriptional repression, protein modification and oligomerization. In its amino-terminus, a so-called "RBCC-

domain", a protein interaction domain, is located. It consists of three zinc finger domains and a coiled-coil domain.

TRIM28 trimerizes and binds to KRAB-domain containing transcription factors [1,2] via that "RBCC-domain".

Experimenteller Teil: TRIM28-interacting proteins were harvested from cells that expressed the RBCC-domain

of TRIM28 combined with a OneStrepTagII and an HA-Tag. RBCC-associated proteins were bound on Streptactin-

columns using a TST2-buffer. After washing, the RBCC-bound proteins were eluted using Phenanthrolin, EDTA,

and Desthiobiotin, respectively. Eluted proteins were subjected to SDS-PAGE analysis. Western Blots were

performed by using antibodies against C2H2 zinc finger domains, TRIM28 and HA-Tag for staining of KRAB-

zinc finger proteins,TRIM28 and the RBCC-construct. Protein bands were analyzed with MALDI-ToF-MS peptide

mass fingerprinting. Identification results were confirmed by mass spectrometric sequencing of selected peptide

ions on an Axima MALDI-QIT-ToF-MSn [3].

Ergebnisse und Diskussion: By destroying the zinc-finger structures of the RING finger and the B-Boxes of

the RBCC-part of TRIM28, we were able to reduce the background of the affinity chromatographic isolation of

KRAB-domain containing proteins. The specific release of KRAB-domain containing proteins was achieved by

using Phenanthrolin, a zinc complexing reagent, for elution. Herewith co-elution of TRIM28 and the RBCC-

construct was prevented, eliminating the two most dominant contaminating proteins.

Note, that not only were we able to reduce the background by this optimized isolation procedure, but we also

achieved a deeper insight into the chemical and structural nature of the TRIM28-KRAB and TRIM28-TRIM28

interaction. We now have for the first time clear evidence that the zinc finger domains of the TRIM28-RBCC

complex are necessary for interaction. These results support the rationing that only the coiled-coil domain but not

the RB-part of TRIM28-RBCC complex is needed for (homo)oligomerization.

Neue Aspekte: New insights for interactions between KRAB-domains and TRIM28, and between TRIM28 and

TRIM28 Background reduction in affinity chromatographical methods.

Referenzen: [1] J. F. Margolin, J. R. Friedman, Meyer, K.-H. Wolfram, H. Vissing, H.-J. Thiesen, F. J. Rauscher

(1994). "Krüppel-associated boxes are potent transcriptional repression domains." Proc Natl Acad Sci U S A 91:4509-4513.; [2] P. Moosman, O. Georgiev, B. LeDouarin, J.-P. Bourquin, W. Schaffner, (1996). "Transcriptionalrepression by RING finger protein TIF1β that interacts with the KRAB repressor domain of KOX1." Nucleic AcidsRes. 24(24): 4859-4867.; [3] C. Koy, S. Mikkat, E. Raptakis, C. Sutton, M. Resch, K. Tanaka, M. O. Glocker(2003). "Matrix-Assisted Laser Desorption/Ionization Quadrupole Ion Trap-Time of Flight Mass SpectrometrySequencing Resolves Structures of Unidentified Peptides Obtained by in-Thema: Proteine und PeptideKeywords: KRAB, TRIM28, Affinity Chromatography, Background ReductionKontakt: [email protected]

91

P2

C3 exoenzyme of Clostridium botulinum induces changes in thenuclear proteome of murine cells

Schröder, Anke; Hülsenbeck, Stefanie; Just, Ingo; Pich, Andreas

Hannover Medical School, Germany

Einleitung: C3 exoenzyme produced by Clostridium botulinum (C3botWT) catalyzes the transfer of ADP-ribose

to Rho proteins leading to their inactivation (1). Rho proteins are small GTPases that are involved in signal

transduction in eukaryotic cells affecting cytoskeleton, cell cycle, and apoptosis. Additionally, C3bot has a neu-

rotrophic effect on murine primary hippocampal neurones during development (2) leading to axonal growth and

ramification. These effects are independent from the enzymatic activity and also induced by an inactive mutant

(C3botE174Q). Recently it had been shown that treatment of neuronal cells with C3bot revealed dramatic changes

in the nuclear proteome. Thus, in this study nuclei were isolated and a proteome analysis was conducted using the

ICPL technique and LC-MALDI (3).

Experimenteller Teil: To analyze C3bot-dependent effects on cellular system the J774A.1 cells were chosen

which efficiently internalize C3bot. Cells were treated with 100 nM C3botWT and C3botE174Q for 24 hours and

uptake of C3bot was checked by detection of ADP-ribosylated RhoA. Nuclei were isolated and extracted proteins

were labelled with different ICPL reagents. After combining the samples proteins were separated using a gradient

SDS-PAGE analysis. The gel lane was manually cut into 24 pieces followed by tryptic digestion of the proteins.

Thereafter, peptides were separated using nano RP-LC and spotted directly onto a MALDI target plate using a

microfraction collector. Mass spectrometric analysis including relative quantitation was performed using MALDI

TOF/TOF MS (Ultraflex I, Bruker) and WARP-LC and Mascot software tools.

Ergebnisse und Diskussion: J774A.1 cells were chosen to elucidate proteome changes induced by C3bot. Several

other cell lines had been tested that did not respond to C3bot. Primary hippocampal cells are not suited for

proteome analysis since these cells are highly heterogeneous and contaminated by a high extend of glia cells.

Uptake of C3bot was controlled by identification of ADP-ribosylated RhoA. Cells were treated either with the

wild type C3bot or with the mutant C3botE174Q and nuclei were isolated. Extracted proteins were labelled

with the ICPL reagent and analyzed by mass spectrometry. With this technique 294 proteins with at least two

peptides have been identified from the nuclear fraction of J774 cells. 49 of these proteins exhibited an altered

concentration as evident from the peaks intensities of the modified peptides. Control experiments revealed that

regulation factors above 1.5 correspond to truly altered protein concentration. Proteins that were found up or down

regulated belong to a heterogenous group of proteins. With an apparent regulation factor greater than 2 the "Farupstream element binding protein" 1 and the "SAM domain and HD domain containing protein 1" were found withthe highest regulation after treatment with 100 nM C3botWT and C3bot E174Q. Several proteins responded only tothe treatment with wild type C3bot and some proteins only to the treatment with the inactive mutant C3botE174Q.Proteins regulated differentially were e. g. "Thyrosine-protein phosphatase non receptor type 6" and "Serinehydroxymethyltransferase".These results provide new insights into the function of C3 exoenzyme. For a statistical analysis the experimentsare currently repeated in our laboratory.Neue Aspekte: The C3 exoenzyme of C3bot effects Rho and so far unknown proteins of cells independent fromits enzymatic activity.Referenzen: [1] Just I, Hofmann F, Genth H, Gerhard R (2001) Bacterial protein toxins inhibiting lowmolecular-mass GTP-binding proteins. Int J Med Microbiol. 291, 142-250; [2] Höltje M, Djalali S, Hofmann F, Münster-Wandowski A, Hendrix S, Boato F, Dreger SC, Große G, Henneberger C, Grantyn R, Just I, Ahnert-Hilter G(2008) A 29-amino acid fragment of Clostridium botulinum C3 protein enhances neuronal growth, connectivity,and; [3] Muetzelburg MV, Hofmann F, Hahner S, Just I, Pich A (2009) Quantitative proteome analysis of humanneuroblastoma cells treated with Clostridium botulinum C3 exoenzyme - application of isotope-coded proteinlabels and LC-MALDI techniques, Manuscript submitThema: Proteine und PeptideKeywords: Rho proteins, bacterial toxins, ADP-ribosyltransferase, nuclear proteomeKontakt: [email protected]

92

P3

LC-MALDI-MS based analysis to investigate effects of

Clostridium difficile Toxin A

Jochim, Nelli; Gerhard, Ralf; Just, Ingo; Pich, Andreas

Medizinische Hochschule Hannover, Germany

Einleitung: Toxin A and B are the causative agents of Clostridium difficile-associated diseases (CDAD) like

diarrhoea and pseudomembranous colitis. The toxins glucosylate and inactivate Rho-GTPases leading to reorgani-

zation of the actin cytoskeleton and cell rounding (1). The inactivation of Rho-GTPases is responsible for intestinal

barrier disfunction resulting in diarrhoea. The molecular mechanisms of inflammation of colitis are unknown, so

far. However, evidence was provided that toxins A and B induce changes in colonic cells, which are independent

of glucosylating activity (2, 3). Thus, we investigated effects of toxin A on colonic cells and compared with ef-

fects caused by a glucosyltransferase deficient mutant of toxin A using ICPL (Isotope coded protein label) and

LC-MALDI-MS techniques (4).

Experimenteller Teil: To show enzyme-independent effects, Caco-2 cells were treated with toxin A wild type

or with a glucosyltransferase deficient toxin A mutant for 5 h. Caco-2-cells were lysed at confluent stage and

nuclear and cytosolic proteins were separated. The proteins were labelled with light, medium or heavy ICPL

reagents, combined, separated by SDS-PAGE. The resulting gel lane was cut into 20 pieces, which were processed

by tryptic digestion and generated peptides were separated by nanoscale RP-LC and directly fractionated onto a

MALDI target. Peptides were analyzed in an Ultraflex I MALDI-TOF/TOF-MS (Bruker Daltonics), quantified

using WARP-LC software (Bruker Daltonics), and identified by the MASCOT search engine.

Ergebnisse und Diskussion: Overall 322 proteins with at least two peptides were identified in two technical

replicates and for 152 proteins regulation factors were calculated. The reproducibility was 50 % or 88 % for

first and second replicate, respectively. Proteins exhibiting a regulation factor greater than 1.67 and less than 0.6

were termed up- or down- regulated. Two proteins, acetyl-coA acetyltransferase and cytoplasmic actin 2, were

up-regulated in Caco-2-cells treated with toxin A mutant in both technical replicates. Four proteins, glucosidase 2

b-subunit, hsp 90 kDa B1, neutral a-glucosidase AB and protein disulfide-isomerase, were up-regulated by factor

about 2 in Caco-2-cells treated with toxin A wild type. Eight proteins were down-regulated in this approach. The

most responsive proteins were ribosomal proteins 40 S and 60 S that were down-regulated by factor 3. Thus,

ICPL-TriplexTM is a novel approach well adapted to the identification and relative quantification of proteins from

different samples. This study shows as yet non-identified target proteins, which respond to treatment with trans-

ferase active and transferase deficient toxin A. Kinetic studies of toxin treatment are currently under investigation.

Neue Aspekte:

• Identification and relative quantification using ICPL-triplex-technique

• System biological analysis of toxin impact on colonic cells

Referenzen: [1] I Just and R Gerhard Rev. Physiol.Biochem. Pharmacol. 2005, 152, 23-47; [2] D He et. al.

Gastroenterology 2002, 122, 1048-1057; [3] Kim et.al. Gastroenterology 2005, 129, 1875-1888; [4] A Schmidt et.

al. Proteomics 2005, 5, 4-15


Keywords: ICPL, Toxicoproteomics


93

P4

Auf Substratsuche von wenig bekannten Proteasen mittels2D-Gelelektrophorese und Massenspektrometrie

Frochaux, Violette (1); Hildebrand, Diana (2); Linscheid, Michael (1); Schlüter, Hartmut (2)

1: Humboldt-Universität zu Berlin, Germany; 2: University Medical Center Hamburg-Eppendorf, Germany

Einleitung: Enzyme welche die Hydrolyse von Peptid-Bindungen katalysieren, werden Proteasen genannt. Pro-teasen sind nicht nur für den Protein-Abbau im Körper verantwortlich sondern erfüllen auch hochspezifische

und präzise Aufgaben, durch die Hydrolyse eines Pro-Peptids können Proteine aktiviert oder deaktiviert wer-

den womit Protease eine essentielle Rolle in Zellfunktionen und Signal-Kaskaden spielen. Eine Störung der

proteolytischen Aktivität der Zelle kann zu schweren Krankheiten führen. Aus diesem Grund sind Proteasen

Zielmoleküle vieler Pharmaka. Um die biologische Rolle einer einzelnen Protease zu verstehen, muss zunächst

sein Substrat-Repertoire aufgeklärt werden. Von der menschlichen Serin-Protease HtrA1 sind bis jetzt nur wenige

Substrate bekannt. HtrA1 ist unter anderem in einigen Tumorarten und in der Plazenta gesunder Schwangerer

überexprimiert, was auf eine Beteiligung bei Zell-Wachstum und Proliferation hindeutet.

Experimenteller Teil: Um das Substrat-Spektrum von HtrA1 in der Plazenta aufzuklären, wurden Proteine aus

der menschlichen Plazenta isoliert und mit der Protease HtrA1 12h in PBS-Puffer bei 37°Cinkubiert. Als Kontroll-Probe wurde die Inkubation ohne Zugabe der Protease durchgeführt. Mittels zweidimensionaler Gelelektrophorese(2D-GE), bei der die Proteine anhand ihrer isoelektrischen Punkte (erste Dimension) und ihrer Größe (zweite Di-mension) aufgetrennt und mit Silberfärbung angefärbt werden, wurde versucht, die Differenzen des Poteinspek-trums beider Proben zu erkennen. Die potentiellen Substrate und ihre Fragmente wurden herausgeschnitten, mitTrypsin im Gel proteolysiert, mit nHPLC getrennt und an einem FT-ICRMassenspektrometer gemessen. Die Iden-tifizierung der Proteine erfolgte mit der Datenbank OMSSA. Um die erhaltenen Ergebnisse zu validieren wurdenweitere Inkubationsexperimente mit HtrA1 und den potentiellen, reinen Substraten durchgeführt.Ergebnisse und Diskussion: Aus den 2-D-GE konnten viele Veränderungen durch die Einwirkung von HtrA1auf die Plazenta-Proteine sichtbar gemacht werden. Vergleicht man die Protein-Spots der mit Protease behan-delten Probe mit der Kontroll-Probe können die möglichen Substrate anhand der reduzierten Mengen oder neuerzeugte Fragment-Spots identifiziert werden. Es wurden sechs neue Kandidaten gefunden: Serum Albumin,α-1-Antitrypsin, Heat Shock 70 kDa Protein (HSP), Actin Cytoplasmic 1, Placental 17-β-Hydroxysteroid De-hydrogenase1 (17 HSD 1) und Peroxiredoxin 1. Es wurden viele Fragmente von Serum Albumin gefunden.Möglicherweise sind im Plazenta-Protein-Gemisch noch weitere Proteasen aktiv, die die durch HtrA1 erzeugteSubstrat-Fragmente noch weiter proteolysieren.Bei den Inkubationsexperimenten mit HtrA1 und den reinen Protein-Substraten wurden die Proteine und ihreFragmente mittels eindimensionaler Gelelektrophorese (1D-GE) aufgetrennt. Überraschenderweise wurde dabeiersichtlich, dass Serum Albumin und α-1-Antitrypsin von HtrA1 nicht geschnitten werden. Dieses steht in Wider-spruch mit den Ergebnissen vom 2D-GE-Experiment. Als positiv-Kontrolle wurde β-Casein mit HtrA1 inkubiertund dabei vollkommen abgebaut. Diese Ergebnisse führen zu der Annahme, dass manche der Kandidat-Substratenicht direkt von HtrA1 gespalten worden sind, sondern indirekt durch die Aktivierung einer anderen Protease.Um diese Theorie zu stützen sollen Inkubations-Reihen mit verschiedenen Protease-Inhibitoren an HtrA1 und denPlacenta-Proteinen durchgeführt werden. Somit könnte man eine Indikation über die Protease erhalten, die vonHtrA1 aktiviert worden ist.Neue Aspekte: Mittels zweidimensionaler Gelelektrophorese und hochauflösender nHPLC-MS Methoden kon-nten sechs neue potentielle Substrate der Protease HtrA1 identifiziert werden.Referenzen: [1] Doucet, A.; Overall, C. M. Mol Aspects Med 2008, 29, 339-358.; [2] Overall, C. M.; Blobel, C.P. Nat Rev Mol Cell Biol 2007, 8, 245-257.; [3] Faccio, L.; Fusco, C.; Chen, A.; Martinotti, S.; Bonventre, J. V.;Zervos, A. S. J Biol Chem 2000, 275, 2581-2588.; [4] Nie, G. Y.; Hampton, A.; Li, Y.; Findlay, J. K.; Salamonsen,L. A. Biochem J 2003, 371, 39-48.Thema: Proteine und PeptideKeywords: Protease, Substrat-Suche, 2D-GE, HtrA1, PlazentaKontakt: [email protected]

94

P5

The Effects of Toxin A from Clostridium difficile on Colon Cells -a proteomic approach based on differential two-dimensional

electrophoresis

Zeiser, Johannes (1); Gerhard, Ralf (1); Braun, Hans-Peter (2); Just, Ingo (1); Pich, Andreas (1)

1: Medizinische Hochschule Hannover, Germany; 2: Leibniz Universität Hannover, Germany

Einleitung: The anaerobe Clostridium difficile evolves to a growing sanitary threat in hospitals. Antibiotic treat-

ment and the resulting suppression of the physiological gut flora enable the bacterium to colonize the intestine.

Here it releases its main virulence factors toxins A (TcdA) and B (TcdB), which are 300 kDa single-chain pro-

teins. After endocytosis of the toxins their glucosyl-transferase domain selectively inactivates low molecular Rho

GTP-binding proteins leading to a reorganization of the cytoskeleton [1]. In the presented work the proteome of

human colonic cells (Caco-2) treated with wild type TcdA and a D285/287N mutant that is deficient in glucosyl-

transferease activity were compared. Resulting changes in the protein pattern of the colonic cells were investigated

by differential two-dimensional electrophoresis (DIGE)(2).

Experimenteller Teil: Confluent Caco-2 cells grown in Dubelcco´s Modified Eagle Medium were treated for 24

hours with either 500 ng/mL of TcdA or 500 ng/mL of the D285/287N mutant. Cells were lysed by sonication in a

urea containing buffer and proteins were extracted. Labeling with the fluorescent dyes (CyDye, GE Healthcare) and

two-dimensional electrophoresis was optimized for Caco-2 protein extracts and performed basically as described

before [2,3] in three biological replicates including label-switching. The gels were scanned with a Typhoon TRIO

(GE Healthcare) laser scanner and the images were analyzed with the Delta2D software (Decodon). Protein spots

showing significant changes (p<0,05) in intensity were cut out from preparative (700 ug protein) gels. Proteins

were identified by LC-ESI-Iontrap-MS/MS (Esquire3000+, Bruker Daltonics) analysis.

Ergebnisse und Diskussion: DIGE analysis was based on 819 spots that could be matched among each of the

nine gel images. Comparing the spot pattern of the D285/287N -mutant treated cells with the control group, no

significant changes in intensity of the spots were found. However comparing the protein spots of the TcdA group

with those of the control revealed seven of higher and one of lower intensity (p<0,05). Differential analysis of

the spot intensities of the TcdA treated group with a combination of both the D285/287N mutant treated and the

control group indicated twelve additional putatively regulated protein species. Protein identification was based on

at least two peptides with an individual MASCOT (www.matrixscience.com) ion score of at least 40 each.

The function of the proteins correlates well to dominant effects of TcdA which include the reorganization of the

cytoskeleton and subsequent rounding of cells [1]. Five proteins are involved in the assembly of the cytoskeleton

(beta-Actin, Keratin, Ezrin, T-complex protein 1 subunit gamma and beta). Ten up regulated proteins are involved

in metabolism and/or oxidative stress response (Catalase, several Dehydrogenases). The latter effect could be due

to the formation of reactive oxygen species [4]. Ubiquitin, involved in protein degradation, was found in lower

concentrations in TcdA treated cells.

To further investigate these findings and elucidate so far unknown cellular targets of the clostridial toxins, stable

isotope labeling with amino acids in cell culture (SILAC)[5] has been established and proteomes of the differen-

tially treated cells are analyzed using high-resolution mass spectrometry.

Neue Aspekte: Application of the DIGE technique provides new insights into changes of the protein pattern of

colonic cells after TcdA treatment.

Referenzen: [1] Ingo Just, Ralf Gerhard, Rev Physiol Biochem Pharmacol. 2004;152:23-47; [2] Ünlü et al.,Electrophoresis (2005) 18 (11): 2071-7; [3] Heinemeyer et al, J. Prot. (2009) 72, 539-544; [4] He et al., Gastroen-terology (2002) 122: 1048-57; [5] Ong et al., Mol. Cell. Proteomics (2002), 1, 376-386Thema: Proteine und PeptideKeywords: Toxin A, Clostridium difficile, DIGEKontakt: [email protected]

95

P6

Data processing workflow for homology-driven proteomics byLC-MS/MS

Thomas, Henrik; Knaust, Andrea; Shevchenko, Andrej

MPI-CBG, Germany

Einleitung: Protein identification in organisms with unsequeced genomes relies on a combination of database

mining approaches. Proteins could be identified by stringent cross-species matching of peptide MS/MS spectra by

conventional software (like, MASCOT). Alternatively, MS/MS spectra could be interpreted de novo and produced

sequenced candidates used for identifying proteins by sequence-similarity searches (MS BLAST). At each stage

spectra, de novo sequence candidates and results of database searches should be correlated and sorted according to

various criteria. Here we describe an automated homology driven proteomics workflow which combined several

data mining routines in user defined manner.

Experimenteller Teil: The entire pool of MS/MS spectra is interpreted de novo by PepNovo software and se-

quence candidates are saved within the mgf file that is further submitted to MASCOT searches [1]. Database search

results are stored in a single spreadsheet and spectra could be automatically selected for subsequent sequence-

similarity searches according to user-defined criteria. De novo candidates could either validate assignments made

by MASCOT or used for independent identifications. To this end, the output of MS BLAST searches is also

converted into the table format that facilitates its correlation to MASCOT results.

Ergebnisse und Diskussion: The presented workflow automates time consuming interactive interpretation of LC-

MS/MS datasets in homology-driven proteomics. Sequence candidates produced by PepNovo and peptide hits

from MASCOT searches are associated and compared. Confident hits identified by MASCOT are automatically

excluded from the list of predictions and are not further analyzed by MS BLAST. At the same time, MS BLAST

hits are back-associated to original spectra files, their de-novo interpretations and identifications by MASCOT or

alternative software. Reliable sequence candidates that were not matched to any proteins by MS BLAST could be

further searched against a raw genome database.

The workflow supports an alternative reductionist strategy by selecting only most confident de-novo predictions

for MS BLAST searches are from very large data sets. To this end, each spectrum is searched against its own

prediction list using X!tandem software and only corroborating predictions are selected. Using a relatively small

non-redundant list of highly reliable candidate sequences facilitates the identification in crude mixtures of unknown

(i.e. not present in a database) proteins.

Spectra filtering applied at several stages of the workflow helps to eliminate background proteins and occasional

contaminants.

Neue Aspekte: Automation of homology-driven proteomics for large screening projects.

Referenzen: [1] Thomas H, Shevchenko A: Simplified validation of borderline hits of database searches:Proteomics

8(20) 4173 - 4177; [2] Junqueira, M; Spirin, V; Balbuena, TS; Thomas, H; Adzhubei, I; Sunyaev, S; Shevchenko,

A, Protein identification pipeline for the homology-driven proteomics. J Proteomics 2008; 71: 346-56.; [3]

Wielsch, N; Thomas, H; Surendranath, V; Waridel, P; Frank, A; Pevzner, P; Shevchenko, A, Rapid validation of

protein identifications with the borderline statistical confidence via de novo sequencing and MS BLAST searches.

J Proteome Res 2006; 5: 2448-56


Keywords: PepNovo, MASCOT, MS-Blast, in-gel


96

P7

Two-dimensional-Liquid Chromatography-Mass Spectrometryemploying Displacement Chromatography for the Analysis of

Proteomes

Schlüter, Hartmut (1); Ahrends, Robert (1); Lichtner, Björn (1); Kohlbacher, Oliver (2); Bertsch, Andreas (2);

Trusch, Maria (1)

1: Universitätsklinikum Hamburg-Eppendorf; 2: Universität Tübingen, Wilhelm Schickard Institute for Computer

Science, Division for Simulation of Biological Systems

Einleitung: For the mass spectrometric identification of proteins via the bottom-up approach a chromatographic

separation guaranteeing a high resolution is mandatory because of the huge number of tryptic peptides. For

this reason a combination of at least two different chromatographies is common. Usually both chromatographic

steps are perfomed in the gradient elution mode. Instead of the gradient we applied the displacement elution

mode in the first chromatographic step (cation-exchange chromatography) of a two-dimensional chromatographic

separation prior to the mass spectrometric analysis of the tryptic peptides. We tested the displacement modus

for its application in proteome analysis because it is one of its major advantages yielding significantly higher

concentrations of analytes eluting from the column in comparison to the gradient mode.

Experimenteller Teil: A tryptic digest of a plasma protein fraction was fractionated with cation-exchange chro-

matography either in the gradient mode or in the displacement mode. Each of the resulting fractions was analysed

with a LC-MS system, consisting of a reversed-phase HPLC-chip module and an ESI ion-trap mass spectrometer

(Agilent Technologies). The mass spectrometric data were used for data base (SwissProt) searches, done with

Mascot, OMSSA and X!Tandem. Only proteins with a probability of at least 0.99 and covered by at least two dis-

tinct peptides were kept for further analysis. For comparing the elution behaviour of the different elution modes,

the charge of each peptide being fully protonated was calculated based on the amino acid sequences determined

by the data base searches.

Ergebnisse und Diskussion: In total nearly 80 proteins were identified via displacement and approximately 60

proteins via gradient chromatography. About 30 proteins were exclusively identified by displacement chromatog-

raphy and abot 10 proteins exclusively by gradient chromatography. Comparison of the elution behaviour of the

peptides demonstrated a higher resolution. The results clearly show the advantages of the displacement chromatog-

raphy [1] over the gradient elution mode for the analysis of proteomes in the bottom-up approach verifying the

advantages of displacement chromatography including the concentrating effect and a higher separation efficacy.

Neue Aspekte: The capability of displacement chromatography towards its application in LC-MS based analysis

of proteomes is demonstrated.

Referenzen: [1] Schlüter H, Jankowski J. Displacement Chromatography. In: Protein Purification. Ed. Kastner

M. Amsterdam: Elsevier Science; 2000. Pages: 505-522


Keywords: two-dimensional liquid chromatography coupled with mass spectrometry, proteomics, bottom-up,

plasma proteins


97

P8

The proteomic analysis of Human Formalin-fixedparaffin-embedded (FFPE) tissue by on-line 2D-LC-MS

Wiegand, Manfred (1); McKenna, Therese (2); Hughes, Chris (2); Langridge, James (2); Nirmalan, Niroshini (3);

Banks, Rosamonde E (3)

1: Waters GmbH, Eschborn, Germany; 2: Waters Corporation, Manchester, UK; 3: CRUK Clinical Center,

St.James Univ.Hospital, Leeds, UK

Einleitung: In this work, we apply multiple LC-MS strategies to the analysis of extracted tryptic peptides from

Formalin-fixed paraffin-embedded (FFPE) tissue archives. FFPE tissue samples are the common way to archive

biopsy material and as such these offer an attractive alternative to fresh tissue samples. In initial experiment we

have looked at five FFPE tissue samples; independently extracted, digested, and analyzed using LC-MS to assess

technical reproducibility and variability in a novel sample preparation procedure. In subsequent experiments 1D

LC-MS has been compared to 2D RP-RP LC-MS approach to assess depth of proteome coverage and analytical

reproducibility.

Experimenteller Teil: Proteins extracted from FFPE tissue slices were digested with trypsin and diluted to 1µg/µLusing aqueous 0.1% formic acid including an ADH internal standard. Samples were analysed by 1D LC-MS,2D-

5step and 2D-10step at 500ng, 2.5µg, and 4.5µg respectively. 2D separations were performed on a 150µm x 5cm

XBridge C18 (5µm) column with a discontinuous step gradient at pH=10 in the first dimension, followed by a

pH=2 separation on a 75µm x 20cm BEH C18(1.7µm) column in the second dimension. In each case triplicate

injections were performed. Column eluent was coupled to the nanoelectrospray source of a Xevo Q-ToF MS, using

the alternating low energy / elevated energy MSE analysis technique. Data was processed, database searched and

compared with PLGS2.4 and a non-redundant Human database.

Ergebnisse und Diskussion: Analysis of the different FFPE tissue extracts showed that the number of proteins

identified was highly consistent across the replicates, using 1D LC-MS. From the identified proteins the overlap

was high with 80% of the proteins common across the samples, showing reproducible qualitative and quantitative

metrics at the protein and peptide level. For the 2D analysis data processing, merging of individual fractions and

searching shows that an increasing number of proteins are identified when the number of first dimension steps is

increased. Approximately 250-300 proteins are identified in each of the pseudo 1D experiments, > 450 proteins

in each of the 2D 5 step experiments and > 600 proteins in each of the 2D 10 step experiments. This increase in

protein identifications can be correlated to the protein loads which increase with the number of steps, the optimum

load of sample onto the first dimension column being approximately 500ng for each first dimension step. When

assessing the data for replication between the experiments using the same number of steps, high reproducibility

between repeat injections of the 2D RP/RP technique is observed at both the peptide and protein level for each

individual step and for each of the steps combined. Absolute quantitation, utilizing the intensity of the top three

peptides from the yeast ADH internal standard, shows that with an increased number of first dimension steps,

there is an increase in the number of low abundant species that are sampled as interferences from higher abundant

species are reduced with the greater separation capability when using more steps.

Neue Aspekte: The usages of 2D-RP/RP chromatography allows for a significant increase in dynamic range of

reproducible protein identifications from complex samples.

Thema: Instrumentelle Entwicklungen, Proteine und Peptide

Keywords: 2D RP/RP on-line LC-MS, Human FFPE tissue, Absolute quantitation, low abundant species


98

P9

LC-MS based Protein Analysis of Microdissected TissuesRevealed Cell-Type Specific Biological Functions in Developing

Barley Grains

Kaspar, Stephanie; Matros, Andrea; Weschke, Winfriede; Mock, Hans-Peter

IPK-Gatersleben, Germany

Einleitung: Laser capture microdissection combined with pressure catapulting (LMPC) enables target cells to be

isolated from complex tissues and allows analysis of specific cell types representing the in vivo state at the time

of sample preparation [1], e.g. by applying "-omics" techniques. As proteomic approaches cannot benefit fromamplification protocols, generally far fewer proteins than transcripts can be detected [2]. However, the assessmentof proteins has gained substantial impact from the advances in mass spectrometric techniques, which made thembetter accessible for qualitative and quantitative analysis. We are interested in the proteome analysis of barleyas a major cereal crop plant. In particular we want to have a closer insight into tissue specific differences in theproteome during barley grain development.Experimenteller Teil: In our study, nucellar projection (NP) and endospermal transfer cells (ETC) were preparedby LMPC from cryo-sections of developing barley grains (8 days after flowering (DAF)). Dissected material wascatapulted into the lid of 0.5-mL PALM Adhesive Caps, respectively. For each cell type three individual prepa-rations were performed. Protein extracts from collected samples were subsequently analyzed by separation on ananoUPLC combined with ESI-Q-TOF mass spectrometry. Data acquisition was performed by a data independentstrategy, called multiplexed LC-MS. For data processing and protein profiling the ExpressionE system solution(Waters) was utilised processing the intensities of molecular ions for quantification and the fragment and molecu-lar ions for identification.Ergebnisse und Diskussion: Typically, between 70 and 75 sections were processed per experiment for dissectionof ETC, which refers to about 4.500.000 µm2 of section area. For dissection of NP, about 40 sections wereprocessed per experiment, which refers also to about 4.500.000 µm2 of section area. Proteins have been directlyextracted from the dissected tissues and digested in solution using trypsin. Complex tryptic peptide mixtureswere subsequently analysed as described above. Initial analysis revealed the need of a desalting step prior toreversed phase separation. Thus, peptide separation was performed on a 180µm x 20mm Symmetry (5µm) C18pre-column coupled to a 100mm x 75µm BEH (1.7µm) C18 column, with a gradient of 3-40% actonitrile over80min. Each experiment was analysed in three technical replicates. Data evaluation showed good reproducibilityacross individual runs. For protein identification mass spectra have been searched against the Uniref90 database forOryza sativa and Hordeum vulgare and the HarvEST database. About 140 proteins were significantly identified,when considering only protein identifications which were found in all three individual experiments, and in at least2 out of 3 of the technical replications. Most of the proteins found were involved in translation, protein synthesis,as well as in protein folding. Absolute protein quantities have been calculated from the data set based on standardpeptides of known amount using PLGS software (Waters). The comparison of results for the different tissue typesindicated a pronounced function in stress defence for the ETC and a dedicated role in transport and mobilisation ofnutrients for the NP and the ETC. All together, qualitative and quantitative protein profiling led to identification ofa number of proteins with tissue specific expression indicating distinct biological functions of different cell-typesin developing barley grains.Neue Aspekte: The multiplexed LC-MS analysis of microdissected tissues revealed cell-type specific biologicalfunctions in developing barley grains at the protein level.Referenzen: [1] Ramsay K. et al., 2006, Molecular Plant Pathology, 7 (5): 429-435.; [2] Hennig L., 2007,TRENDS in Plant Science, 12 (7): 287-293.Thema: Proteine und PeptideKeywords: seed development, barley, microdissection, multiplexed LC-MSKontakt: [email protected]

99

P10

Investigation of proteomic changes in liver affected bynon-alcoholic steatohepatitis

Thomas, Anja (1); Reinders, Jörg (1); Hellerbrand, Claus (2); Oefner, Peter J. (1)

1: Institute of Functional Genomics, University Regensburg, Germany; 2: Department of Internal Medicine I,

University Medical Center Regensburg, Germany

Einleitung: The increasing number of obesity in western society is a major cause for higher incidence of metabolic

aberrations as insulin resistance and non-alcoholic fatty liver disease (NAFLD). Non-alcoholic steatohepatitis

(NASH) is the most severe form of NAFLD, characterized by liver steatosis and inflammation leading to a sig-

nificant deterioration in liver function, possibly culminating in liver cirrhosis. The exact mechanism of NASH

development is still unclear. A potential cause for inflammation is a higher rate of β-oxidation in the liver mito-

chondria induced by elevated levels of plasma free fatty acids. Due to the harmful effect of elevated formation

of reactive oxygen species, damage particularly on the mitochondrial proteome may occur resulting in significant

impairment of cellular respiration.

Experimenteller Teil: Here, an established mouse model system for NASH is used to assess the early changes in

hepatic protein regulation. Wild-type mice are fed with a high-fat diet facilitating the development of adipositas

and NASH. Proteomic changes in different compartments of murine liver cells are investigated, thereby focusing on

mitochondria as one of the key players in disease progression. The analyses of protein-related changes are accom-

plished by means of two-dimensional differential gel electrophoresis (DIGE), followed by protein identification

using liquid chromatography-mass spectrometry. In a complementary approach, changes in protein abundances

are quantified by a shotgun approach using stable-istotope labelling. Furthermore, results will be confirmed using

functional assays.

Ergebnisse und Diskussion: To assess changes in early NASH development, female C57/BL6 mice were fed

with a high fat diet for 2, 4, 6 and 10 days. At each of these time points, livers were perfused for blood removal

and dissected. The grade of steatosis was determined by histological examination. During the course of time, a

microvesicular steatosis with mild inflammatory infiltration was confirmed. The degree of inflammation and fibro-

sis was assessed by quantitative real-time-PCR of relevant marker genes (TNFα and Collagen-1). The expression

analysis shows no development of fibrosis up to this early points in disease progression, whereas an up-regulation

of the pro-inflammatory cytokine TNFα after 10 days of high fat feeding indicates the onset of inflammation of

hepatic tissue. No significant increase in total cholesterol content was observed during the time course. In contrast,

the triglyceride content of liver tissue increased significantly during feeding time, which is in accordance with the

findings of triglyceride inclusions in hepatocytes by histology. Purification techniques for different compartments

of murine liver cells were established. Mitochondria were isolated from whole tissue by differential centrifuga-

tion and ultracentrifugation using a sucrose density gradient. For purity control of mitochondria, immunoblotting

against endoplasmatic reticulum and cytoplasmatic proteins was performed. Furthermore, liver cells were parti-

tioned in a soluble and a membrane fraction. In a first differential analysis, the soluble fraction was examined

for differentially regulated proteins by DIGE. After 10 days, an upregulation of various proteins involved in lipid

metabolism could be shown. Also, some proteins participating in primary energy metabolism were shown to be

regulated in early disease progression. For several proteins, a graduated regulation over the experimental course

was observed.

Neue Aspekte: The molecular basis of NASH emergence and progression are to be elucidated for deeper insights

into pathogenesis.

Referenzen: [1] Prokisch et al.: Integrative analysis of the mitochondrial proteome in yeast. PLoS Biol 2004,

2, 795-804; [2] Douette et al.: Steatosis-Induced Proteomic Changes in Liver Mitochondria Evidenced by Two-

Dimensional Differential In-Gel Electrophoresis. J Prot Res 2005, 4, 2024-2031; [3] Pessayre et al.: Nonalcoholic

Steatosis and Steatohepatitis; V. Mitochondrial dysfunction in steatohepatitis. Am J Gastrointest Liver Physiol

2002, 282, G193-G199


Keywords: Non-alcoholic steatohepatitis, proteomics, mitochondrial dysfunction


100

P11

Application of Protein-SIP and decimal place calculation of 13Cincorporation

Jehmlich, Nico (1,4); Fetzer, Ingo (2); Seifert, Jana (1); Taubert, Martin (1); Maier, Stefan K. (1);

Vogt, Carsten (3); Richnow, Hans-Herrmann (3); Schmidt, Frank (1,4); von Bergen, Martin (1)

1: Department of Proteomics, Helmholtz Centre for Environmental Research - UFZ, Leipzig, Germany;

2: Department of Environmental Microbiology Department of Isotope Biogeochemistry, Helmholtz Centre for

Environmental Research - UFZ, Leipzig, Germany; 3: Department of Isotope Biogeochemistry, Helmholtz Centre

for Environmental Research - UFZ, Leipzig, Germany; 4: Interfaculty Institute for Genetics and Functional

Genomics, University of Greifswald, Germany

Einleitung: Different strategies based on labeling proteins or peptides with heavy isotopes have become a powerful

tool in MS-based proteomics. By the metabolic incorporation of heavy isotope labeled substrates we recently

introduced an approach that enables monitoring the carbon flux in microbial consortia [1,2]. Thereby we were

able to elucidate mechanisms in the degradation of environmental pollutants. Here we present a novel method to

calculate the incorporation level of 13C into proteins using the half decimal place rule (HDPR) on tryptic peptides

[3]. This calculation depends only on accurate peptide masses and not on known sequence information.

Experimenteller Teil: The limits for 0 and 100 atom % 13C incorporation have been defined by a representative in

silico data set of 89,773 theoretical tryptic peptide masses from Mycobacterium tuberculosis H37Rv which were

plotted against their decimal places. In our study, P. putida ML2 grew in the presence of [13C6]-benzene (solely

energy and carbon source) and as a control in the presence of [12C6]-benzene. Afterwards the proteins were

extracted, subjected to 1-D gel electrophoresis, tryptic in-gel digested, and peptides were analyzed by nano-LC

LTQ Orbitrap-MS. The difference of the isotopic pattern between 12C/13C peptides allowed the calculation of the13C incorporation within various labeling contents using the highest intensity 13C peak by decimal place slope.

Ergebnisse und Diskussion: In the present study, the applicability of the half decimal place rule approach is

demonstrated by using Pseudomonas putida ML2 proteins labeled uniformly via the consumption of [13C6]-

benzene present in the medium in concentrations of 0 atom %, 10 atom %, 25 atom %, 50 atom % and 100

atom %. The incorporation of 13C was calculated on the basis of several labeled peptides derived from benzene

1,2-dioxygenase. The accuracy of the calculated incorporation level depended on the number of peptide masses

included - 100 peptide masses were required to reduce the deviation below 4 atom %. This accuracy was compara-

ble with calculation of incorporation based on the isotope envelope. Current studies have shown that this technique

is suitable to elucidate the carbon flow in mixed bacterial communities and identify pathways and key players of

degradation even when no sequence information is available. This technique can be applied in several fields rang-

ing from screening schemes for microorganisms with defined functions (e.g. proteolytic activities) to degradation

of environmental harmful xenobiotics.

Neue Aspekte: Assessing metabolic activity of microbial consortia by Protein-SIP and half decimal place rule

Referenzen: [1] Jehmlich N, Schmidt F, von Bergen M, Richnow HH, Vogt C. ISME Journal (2008) 2,1122-1133;

[2] Jehmlich N, Schmidt F, Hartwich, M, von Bergen M, Richnow HH, Vogt C. Rapid Commun. Mass Spectro

(2008) 22,2889-2897; [3] Fetzer I, Jehmlich N, Vogt C, Richnow HH, Seifert J, Harms H, von Bergen M, Schmidt

F Bioinformatics (2009) submitted; [4] Jehmlich N, Fetzer I, Mattow J, Vogt C, Harms H, Thiede B, Richnow HH,

von Bergen M, Schmidt F. Molecular and Cellular Proteomics (2009) in press


Keywords: half decimal place rule, Protein-SIP, microbial consortia, degradation of xenobiotics


101

P12

Speeding-up snails: relieved and successful data mining routinefor homology-driven proteomics in evolutionary distant organisms

Knaust, Andrea (1); Jin, Sun (2); Shevchenko, Anna (1); Shevchenko, Andrej (1)

1: MPI-CBG, Germany; 2: Hong Kong Baptist University

Einleitung: Mass spectrometry-driven proteomic studies in organisms with unsequenced genomes still remain a

challenging task. They rely on cross-species matching and therefore very much depend on availability of protein

and genomic information from phylogenetically close species [1]. In addition, it is extremely laborious procedure

which usually requires a lot of manual work. Here we describe identification of proteins in perivitelline fluid of

apple snail Pomacea Canaliculata, a serious plague to rice agriculture in south-east Asia. We applied systemized

and automated data mining routine and variable sample preparation procedure to facilitate proteomics in the

organism with no related sequencing data and increase its success rate.

Experimenteller Teil: After separation on a 2D gel, proteins from perivitelline fluid were in-gel digested and ex-

tracted peptide mixtures were analyzed by LC MS/MS on an Orbitrap LTQ mass spectrometer (Thermo Scientific)

coupled with Ultima 3000 HPLC (Dionex) [2]. A multistage automated data mining routine was applied for anal-

ysis of the acquired dataset. The pipeline comprised unspecific background filtering (EagleEye), stringent spectra

matching (MASCOT, X!Tandem), spectrum-sequence matching evaluation (ProteinProphet), de-novo sequence

prediction (PepNovo) and sequence similarity searching in various databases (MS BLAST)[3,4]. In addition, bor-

derline matches were manually inspected. Results generated in each step were cross-correlated and connected to

original MS/MS spectra. In duplicated experiment we improve the recovery of long and hydrophobic peptides.

Ergebnisse und Diskussion: The automated pipeline which connected multiple laborious data mining steps

enabled the analysis of large and complex LC MS/MS data in straightforward, transparent and handy manner.

Altogether, we gave identity to 2/3 of analyzed protein spots. A half of protein identifications was achieved

via stringent spectra matching, whereas another was obtained at the end of the data mining routine based on

combination of de-novo prediction and similarity searching results. The cross-species matches were made to

protein sequences from various organisms from mammals to worm. Modifying sample preparation procedure, we

gained additional peptides and so confirmed a number of borderline hits. Despite complex analysis, we matched

less than 10% of the set of meaningful tandem mass spectra and there are still several data mining approaches to

be tested. For example, to improve matching of spectra acquired from multiply charged precursors, sequence tag

generation followed by error-tolerant searches can be included in the pipeline.

Neue Aspekte: Facilitated homology-driven proteomic routine

Referenzen: [1] Shevchenko A, Valcu CM, Junqueira M. Tools for exploring the proteomosphere. J Proteomics.

2009 Mar 6;72(2):137-44.; [2] Junqueira et al, J proteom res Aug;7(8):3382-95; [3] Junqueira, M; Spirin, V; Bal-

buena, TS; Thomas, H; Adzhubei, I; Sunyaev, S; Shevchenko, A, Protein identification pipeline for the homology-

driven proteomics. J Proteomics 2008; 71: 346-56.; [4] Keller, A., Nesvizhskii, A. I., Kolker, E., and Aebersold,

R. (2002) Anal.Chem.74, 5383 -5392


Keywords: homology-driven proteomics, ms data mining automation, unsequenced genomes


102

P13

Mascot Delta Score: a simple approach to evaluating theperformance of large-scale phosphoproteomics data

Savitski, Mikhail (1); Lemeer, Simone M. (2); Boesche, Markus (1); Bantscheff, Marcus (1); Kuster, Bernhard (2)

1: TU Muenchen, Germany; 2: Cellzome AG, Germany

Einleitung: High-throughput mass spectrometry has identified thousands of phosphorylation sites on human

proteins. Despite substantial experimental and computational improvements, the assignment of a phosphorylation

site to a precise amino acid can still be difficult and time consuming because tandem MS spectra are often not

straightforward to interpret and there are no reference standards for any of the detected putative phosphopeptides.

Using a library of synthetic phosphopeptides we evaluated the performance of different fragmentation modes for

their ability to call the site of phosphorylation correctly and introduce a simple score that discriminates between

alternative phosphorylation sites.

Experimenteller Teil: Standard Fmoc solid phase parallel peptide synthesis (2 umol scale, Intavis, GER) was

utilised to generate several hundred phosphopeptides with precisely known modification sites. LC-MS/MS of

individual and pools of phosphopeptides was performed on three mass spectrometers: a) an LTQ (for CID, MSA,

MS3), b) a LTQ Orbitrap XL (for HCD, ETD,) and c) a QTOF Micro (for CID). All systems were coupled to a

nanoLC using 75 um i.d. reversed phased columns. Tandem MS data was searched against the IPI database using

Mascot. The Mascot Delta Score was computed by subtracting the Mascot peptide ion score of the first wrong

phosphorylation site assignment from the ion score of the correct site assignment.

Ergebnisse und Diskussion: Using a set of 2173 tandem mass spectra from 182 synthetic p-peptides with pre-

cisly known PTM-sites, we developed a simple scoring scheme for the validation of p-site assignments. The

MD-Score simply reflects the difference of Mascot ion scores between the best and the second best p-site match

for an otherwise identical peptide sequence. Simply put, ion score differences of >10 enables to call the cor-

rect phosphorylation site with >95% confidence. Importantly, this confidence can be calculated for any putative

phosphorylation site which will therefore become an important quality criterion for future phosphoproteomics ap-

plications. Comparison of the MD-Score and the customised Ascore algorithm revealed that both perform very

well for S/T phospho-site validation, whereas the MD-Score outperforms the Ascore for pY-site assignments. At

0.05 FDR, MD-score assigns 324 correct pY sites while Ascore only assigns 82. Differences in MD scores were

observed for different fragmentation techniques. Against common notion, ETD was not necessarily the best frag-

mentation technique for unambiguous phosphorylation site assignment. Owing to its simplicity, the MD score can

easily adopted in any proteomics laboratory without the need for sophisticated software tools.

Neue Aspekte: Development of a new scoring scheme for phosphorylation site assignment


Keywords: phosphorylation, proteomics, tandem MS, bioinformatics


103

P14

Ultra-high-performance nanoLC-MS/MS Analysis of ComplexProteomic Samples

Liedtke, Steffen

Dionex GmbH, Germany

Einleitung: Determination of the proteome and identification of biomarkers is required to monitor dynamic

changes in living organisms and predict the onset of an illness. One popular method to tackle contemporary

proteomic samples is called shotgun proteomics, in which proteins are digested, the resulting peptides are sep-

arated by high-performance liquid chromatography (HPLC), and identification is performed with tandem mass-

spectrometry. Digestion of proteins typically leads to a very large number of peptides. For example digestion of

a cell lysate easily generates 500,000 peptides. The separation of these highly complex peptide samples is one of

the major challenges in analytical chemistry.

Experimenteller Teil: The main strategy to improve the efficiency of packed columns is either to increase column

length or by decreasing the size of the stationary phase particles. However, to operate these columns effectively

the LC conditions need to be adjusted accordingly. Naturally, the on-line coupling to MS systems has to be taken

into account in the optimization process.

Ergebnisse und Diskussion: Here, we report on the performance of nanoLC columns operating at ultra-high

pressure. The effects of column parameters (particle size and column length) and LC conditions (gradient time,

flow rate, column temperature) were investigated with reversed-phase (RP) gradient nanoLC. High-resolution

LC-MS separations of complex proteomic peptide samples are demonstrated by combining long columns with

2 µm particles and long gradients. The effects of LC parameters on performance and the influence on peptideidentification are discussed.Neue Aspekte: Ultra-high performance nanoLC; combined optimization of stationary phase, column length andLC conditions.Thema: Instrumentelle Entwicklungen, Proteine und PeptideKeywords: Ultra-high performance nanoLC, proteomicsKontakt: [email protected]

104

P15

Small is beautiful - Improving the identification rate of lowmolecular weight proteins

Müller, Stephan (1); Kohajda, Tibor (2); von Bergen, Martin (1,2); Kalkhof, Stefan (1)

1: Department of Proteomics, Helmholtz Centre for Environmental Research - UFZ, Leipzig; 2: Department of

Metabolomics, Helmholtz Centre for Environmental Research - UFZ, Leipzig

Einleitung: Low molecular weight (LMW) proteins represents about 1/3 of eukaryotic proteome including growth

factors, cytokines, toxic proteins as well as proteins involved in signal transduction, transcription regulation or cell

communication. However, small proteins are underrepresented in many proteome studies. Small proteins often get

lost with standard methods like 2D gel electrophoresis or gel-free LC-MS/MS analysis, because most procedures

are not adjusted to them. Beside loss effects, small proteins also tend to appear in low copy number. Additionally,

MS identification is hampered by the low number of peptides generated from small proteins by proteolysis.

Experimenteller Teil: In our study, we improved a workflow for mass spectrometry based identification of small

proteins with E.coli as model organism. Therefore, we used different lysis buffers for protein extraction. Depletion

of proteins bigger than 50kDa was performed by filtration. Proteins in the permeate were precipitated with TCA.

Additionally, separation of proteins was done on 20% Tricine-SDS-Gels with a high resolving power in the LMW

range. The unstained gel was cut into several slices and in-gel digestion was performed with two complementary

proteases (Trypsin, AspN) to increase the number of protein identification. Furthermore, a gel-free approach was

used. For the identification different of LMW proteins by online nano-HPLC/nano-ESI-MS/MS with an Orbitrap

LTQ XL mass spectrometer different MS/MS-methods were compared.

Ergebnisse und Diskussion: The LC-MS/MS analysis of in-gel digests resulted in 454 different proteins beyond

25 kDa, when searching with Mascot against the Swiss-Prot database with E.coli as taxonomy. The usage of

two different extraction methods and two different proteases appreciably improved the identification rate of small

proteins. 61% of these proteins could be identified in both lysis systems, whereas the remaining proteins were only

matched with TFA (18%) or urea lysis (23%). The usage of two different proteases provided a reconfirmation of

50% of the identified proteins and thus increased protein verification. The other proteins were exclusively found

with Trypsin (35%) or AspN (15%) digestion. 38% of the Ecoli proteome consists of small proteins lower than 25

kDa. We have been able to identify 27% of the E.coli proteome below 25 kDa covering a broad pI and mass range

down to 4.3 kDa.

Neue Aspekte: Optimized extraction, separation and LC-MS-analysis protocols for low molecular weight proteins

Referenzen: [1] Klein et al. The Low Molecular Weight Proteome of Halobacterium salinarum. Journal of

Proteome Research 6, 1510 - 1518 (2007).; [2] Schägger. Tricine-SDS-PAGE. Nature Protocols 1 (1), 16 - 22(2006)Thema: Proteine und PeptideKeywords: Low molecular weight proteins, LC-MS, optimized sample preparationKontakt: [email protected]

105

P16

Strategies towards a complete organellar proteome.

Sickmann, Albert (1); Meisinger, Chris (2); Pfanner, Nikolaus (2); Wortelkamp, Steffi (1); Zahedi, Rene (1)

1: Leibniz Institut für Analytische Wissenschaften - ISAS, Germany; 2: Institut für Biochemie, UniversitätFreiburg, Germany

Einleitung: Amajor topic in proteomics is the coverage of qualitative and quantitative results in a given sample.Wedeveloped strategies to overcome general limitations like sample complexity and undersampling and applied themto the yeast mitochondrial proteome. Till today we generated the the most complete organelle proteome withis currently a rich source of numerous other studies to deliver detailed functional analysisofknown and novelmitochondrail proteins.Experimenteller Teil: The experients were done with different HPLC and mass spectrometry based techniques.We mainly used capillary and nano HPLC for prefactionation and trace analysis of low abundand proteins. Thesorting of selected peptides was done semi automatic with microbore HPLC techniques followed by chemical andenzymatic modifications. MS detection was done with high resolution mass spectrometry (global and unbiasedanalysis) and SRM/MRM experiments (dedicated analysis). Data analysis ad interpretation was done with thecurrent version of mascot server as well as the open source algorithms MS-LIMS and OPEN-MS.Ergebnisse und Diskussion: We produced the following data sets which will be presented:- complete proteome (with different strategies)- posttranslational modifications ( e.g. phosphorylation, proteolytic cleavage, acetylation)- the proteome of the outer membrane- the N-proteome (clevage products)- analysis of protein complexes (unbiased and dedicated)Neue Aspekte: Increased coverage of proteomes, subfractionation, reduction of measurement time, maturation ofproteins.Referenzen: [1] Voegtle, Wortelkamp et. al, Cell, 2009; [2] Sickmann et al. PNAS, 2003; [3] Zahedi et al. MCB,2005; [4] Reinders et al. MCP, 2007; [5] Zahedi et al. Proteomics, 2006Thema: Proteine und PeptideKeywords: COFRADIC, bioinformatics, undersampling, dynamic range, membrane proteinsKontakt: [email protected]

106

P17

Overcoming Undersampling in Proteomic Experiments with theHelp of a New Hybrid Linear Trap-Orbitrap Mass Spectrometer

Damoc, Eugen (1); Denisov, Eduard (1); Scheffler, Kai (2); Griep-Raming, Jens (1); Moehring, Thomas (1)

1: ThermoFisher Scientific, Bremen, Germany; 2: ThermoFisher Scientific, Dreieich, Germany

Einleitung: Enormous improvements in mass spectrometry technology over the last few years have allowed for

ever deepening analysis of complex proteomes. However, many low abundance proteins remain undetected in

current large-scale proteomic analyses due to the limits on the rate at which MS/MS spectra can be acquired. To

solve this under-sampling problem, we developed a new hybrid linear ion trap-orbitrap mass spectrometer that

provides faster MS/MS scanning in the ion trap (up to 10 peptide sequences per second), thereby increasing the

depth of analysis of complex protein mixtures. In the present work we evaluated the ability of the newly developed

hybrid instrument to identify more proteins, with increased sequence coverage and confidence.

Experimenteller Teil: Several technological improvements have been implemented: (a) a progressive stacked-

ring ion guide (SRIG) with five-fold increased ion transmission; (b) a dual-cell differentially pumped ion trap with

a higher pressure region for improved ion trapping, isolation, and CID efficiencies, and a lower pressure region

for improved resolution and/or scan speed; (c) predictive AGC for increased scan speed; (d) an HCD-collision

cell with axial field for improved performance; (e) improved vacuum in the Orbitrap chamber for improved intact

protein analysis. To assess improvements in proteome coverage, nano-LC-MS/MS analyses of C. Elegans and E.

Coli protein digests were performed using various LC gradients. The raw data files from duplicate analyses were

searched with Mascot using Proteome Discoverer software.

Ergebnisse und Diskussion: To assess the extent of under-sampling for a real-life sample, nanoLC MS/MS

analysis of 1µg C. elegans protein digest was carried out using two different LC gradients (2-40% acetonitrile in 75

minutes, and 2-40% acetonitrile in 150 minutes). Extensive data analysis allowed us to estimate the extent of under-

sampling for this proteome. Various contributors to under-sampling are reviewed. Using the new instrument, a two-

fold increase in the number of MS/MS spectra acquired per unit time was observed. This provided identification

of a higher number of proteins and peptides in the 75’ run than in the 150’ run using the LTQ Orbitrap XL.

With the novel hybrid linear ion trap-orbitrap mass spectrometer, we were able to identify 41.5% more proteins

and 63% more peptides (75’ run, at 0.25% FDR) or 34% more proteins and 50.5% more peptides (150’ run, 0.25%

FDR) than in the corresponding runs with the LTQ Orbitrap XL. Similar results were obtained analyzing a very

complex E. Coli protein digest. For the analysis of this sample three different dissociation methods have been used

(CID, ETD and HCD). All these results clearly indicate that the newly developed instrument can identify more

proteins with increased sequence coverage and confidence.

Neue Aspekte: The problem of under-sampling in proteomics is effectively addressed by a new linear ion trap-

Orbitrap mass spectrometer.


Keywords: novel hybrid mass spectrometer, linear trap-orbitrap, overcoming undersampling, proteomics


107

P18

Quantitative Protein Expression Analysis Of Murine Hepa CellsExposed To Carcinogenic Contaminant Benzo(a)pyrene with

DIGE and SILAC

Kalkhof, Stefan (1); Dautel, Franziska (1); Michaelson, Jacob (2); Trump, Saskia (3); Beyer, Andreas (2);

Lehmann, Irina (3); von Bergen, Martin (1)

1: Helmholtz Centre for Environmental Research, Department of Proteomics; 2: Biotechnology Centre TU

Dresden; 3: Helmholtz Centre for Environmental Research, Department of Enviromental Immunology

Einleitung: In this part of a large-scale systems biology project presented, the cellular responses of murine Hepa-

cells to the exposition of the carcinogenic polyaromatic contaminant Benzo(a)pyrene (B(a)P) on the protein scale

are monitored and analyzed to generate a predictive mathematical model. Polycyclic aromatic hydrocarbons

(PAHs) constitute a substance class of widely dispread environmental contaminants, which are side products

of everyday life processes like smoking, barbecuing or car exhaustion. In this study time- and concentration-

dependent effects on the proteome were analyzed with MS using DIGE and SILAC to identify and differentiate

acute and chronic response mechanisms. To further sustain the extracted cellular response mechanism the datasets

were compared with a comprehensive transcriptome dataset obtained from the same cells.

Experimenteller Teil: The cytotoxicity of B(a)P to Hepa cells was tested with Alamar Blue-, a Neutral Red- and

a lactate dehydrogenase-assay Hepa1c1c7-cells are exposed with 5 µM (toxic) and 50 nM (subtoxic) of B(a)P

and DMSO as a negative control for 0, 2, 4, 12, 24 and 48 h. Changes in the protein expression following

B(a)P-exposure are monitored using two-dimensional difference gel electrophoresis (2D-DIGE) and SILAC. Due

to an independent analysis using multiple testing and ANOVA and further analysis BaP-induced time curve sof

differential protein expression were created. Cellulare reponse mechanismn were extracted by a human protein

reference database network analysis.

Ergebnisse und Diskussion: Applying the DIGE-technology, we detected 1227 spots of which 120 were differ-

entially regulated over time and 95 were identified. 77 proteins respond before 2 hours of BaP-exposure, thus

indicating an immediate response to BaP on the protein level. BaP alters the expression of proteins involved in

oxidative stress, protein degradation, cytoskeleton remodelling, growth signaling pathways, cell cycle control and

others. The analysis of the SILAC samples was performed in a gel-based and a LC-based approach. This datasets

offers time- and concentration-dependent quantitative information about more than 1.000 proteins which are used

currently for the confirmation and extension of the DIGE results.

Neue Aspekte: Time- and concentration dependent proteomic study with DIGE and SILAC cellular response to

BaP, correlation proteomics genomics


Keywords: SILAC, DIGE, Proteomics


108

P19

LC-MS based secretome analysis of various Clostridium difficilestrains

Boetzkes, Alexander; Felkel, Katharina Wiebke; Zeiser, Johannes; Just, Ingo; Pich, Andreas

Institut für Toxikologie, Medizinische Hochschule Hannover, Germany

Einleitung: C. difficile is an anaerobic gram-positive spore forming pathogen causing antibiotic associated gas-

trointestinal infections from mild C. difficile associated diarrhea (CDAD) to severe pseudomembranous colitis

(PMC) [1,2]. Since 2003 new hypervirulent C. difficile strains (PCR ribotype 027) spread across North America

and Europe accompanied by dramatically increasing morbidity and mortality rates [1]. The secretomes of three

C. difficile strains were compared: R20291 (ribotype 027, isolated 2006), CD 196 (ribotype 027, isolated 1985),

and CD 630 (ribotype 012, isolated 1982) as a reference strain [2]. The aim was to identify putative protein candi-

dates in the secretome of C. difficile, which might play a role as so far unknown pathogenicity factors and explain

hypervirulence of ribotype 027 strains.

Experimenteller Teil: C. difficile strains were cultivated in modified defined growth medium [3] and brain heart

infusion broth under anaerobic conditions at 37°C. Samples were withdrawn during stationary growth phase. Aftertwo steps of centrifugation at 2.500 g, proteins of the culture supernatants were precipitated by chloroform-methanol. Proteins were separated by one-dimensional SDS-PAGE and lanes were cut into 10 pieces followedby tryptic in-gel digestion. Peptide analysis was performed by RP-HPLC (1100 series, Agilent) coupled online toan Esquire 3000+ ion trap mass spectrometer (Bruker Daltonics). MS data were searched against strain specificprotein databases using Mascot [2]. To identify signal peptides, sequences of all matched proteins were processedwith Signal P 3.0 [4].Ergebnisse und Diskussion: C. difficile was grown in brain heart infusion broth and in defined medium withnearly similar growth rates but different final optical densities. As evident from SDS-PAGE, protein pattern variedonly slightly in different growth phases and after growth in complex or defined medium. LC-MS analysis revealedabout 90 different proteins in the supernatant of C. difficile. Most of the identified proteins originate from thecytoplasm and might be present due to cell lyses. However, only a subset of cytoplasmic proteins was identified,indicating that protein release was somehow specific. 21 proteins in R20291, 25 in CD 196 and 26 in CD 630contained a signal sequence and should be secreted by C. difficile strains. Proteins known to be secreted weremainly S-layer proteins, substrate binding proteins of ABC - transporters, cell wall hydrolases, pilin and unknownhypothetical proteins. The major pathogenenic factors toxin A and toxin B [5] were only identified after growthin brain heart infusion medium. Only in the hypervirulent ribotype 027 strains the ADP-ribosyltransferase bindingcomponent protein was identified which is a part of the binary toxin CDT which is an additional pathogenic factorexpressed by C. difficile strains. The analysis of the secretome of C. difficile strains revealed several differentproteins for each strain that might contribute to different pathogenicity, too. Further analysis will focus on theverification of the results. LC-MS-analysis is currently repeated and immunological techniques will be carried outto target and quantify different secreted proteins like e.g. toxin A and toxin B and CDT.Neue Aspekte: For the first time an LC-MS based comparison of the secretome of C. difficile strains has beendescribed.Referenzen: [1] Kelly CP, LaMont JT. Clostridium difficile-more difficult than ever. N. Engl. J. Med. 2008;359: 1932-1940; [2] Stabler RA, He M, Dawson L, Martin M, Valiente E, Corton C, et al. Comparative genomeand phenotypic analysis of Clostridium difficile 027 strains provides insight into the evolution of a hypervirulentbacterium. Genome Biol. 2009; 10: 102; [3] Karlsson S, Burman LG, Akerlund T. Suppression of toxin productionin Clostridium difficile VPI 10463 by amino acids. Microbiology 1999; 145: 1683-1693; [4] Bendtsen JD, NielsenH, von Heijne G, Brunak S. Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 2004; 340:783-795;[5] Just I, Gerhard R. Large clostridial cytotoxins. Rev. Physiol. Biochem. Pharmacol. 2004; 152: 23-47Thema: Proteine und PeptideKeywords: Clostridum difficile, LC-MS, bacterial toxins, secretomeKontakt: [email protected]

109

P20

Elastaseverdaus und MALDI MS von TMTzero derivatisiertenProteomen

Bäumlisberger, Dominic; Arrey, Tabiwang N.; Rohmer, Marion; Rietschel, Benjamin; Beckhaus, Tobias; Meyer,

Björn; Karas, Michael

Goethe-Universität Frankfurt am Main, Germany

Einleitung: Die Anwendbarkeit des Enzyms Elastase mitr einer deutlich geringeren Schnittspezifität für die Anal-yse von cytosolischen Proben und Membranproteomen konnte in der Vergangenheit bereits gezeigt werden. MitMALDI als Ionisationsmethode werden aber überwiegend nur leicht saure und basische Peptide identifiziert, diezusätzlich eine gewisse Mindestmasse besitzen müssen, da der Massenbereich unter 700 aufgrund des Auftretensvon Matrixclustern kaum brauchbare MS/MS Daten liefert.Bei der Derivatisierung mit TMTzero (Thermo Scientific, Bremen), das normalerweise für die Methodenopti-mierung von Quantifizierungsexperimenten verwendet wird, erhalten die Peptide einen Massezuwachs und dieBasizität des N-Terminus und der Lysinreste wird erhöht. In einem systematischen Vergleich von Elastase ver-dauten Membran- und Cytosolproteom mit und ohne TMTzero-Modifikation sollte deshalb untersucht werden, obdas Detektionsspektrum von MALDI optimiert werden kann.Experimenteller Teil: Membranen und Cytosol von Corynebacterium glutamicum wurden mit Elastase in einemTEAB-Puffer verdaut. Ein Teil wurde jeweils mit dem Derivatisierungsreagenz TMTzero umgesetzt, wodurchdie N-Termini der Peptide und die Lysin-Seitenketten modifiziert wurden. Die Kontrollen wurden nicht weiterbehandelt. Die Proben wurden dann mittels nLC getrennt und MALDI-MS/MS analysiert.Ergebnisse und Diskussion: In der Kontrolle und der TMTzero behandelten Probe konnte ungefähr die gleicheAnzahl an Peptiden signifikant identifiziert werden, aber nur ungefähr ein Fünftel der Peptide kam in beidenProben vor. Im Gegensatz dazu fand sich die Mehrzahl der identifizierten Proteine in beiden Proben wieder. Dadie Kontrollen und derivatisierten Proben aber aus dem identischen Verdau stammten, konnte dieses Ergebnis nichtauf unterschiedliche Bedingungen bei der Proteolyse zurückgeführt werden. In der behandelten Probe wurden vorallem kleine bis mittelgroße Peptide gefunden, da das TMTzero-Reagenz einen Massezuwachs von ungefähr 224Da bewirkt. Deshalb konnten auch sehr kleine Peptide unterhalb 700 Da detektiert werden, die normalerweiseunterhalb des erfassbaren MALDI-Massebereich liegen. Größere Peptide werden besser in der nicht modifiziertenKontrollprobe erfasst. Außerdem wurden in der derivatisierten Probe verstärkt stark saure Peptide identifiziert.Ein Großteil der Peptide war zusätzlich im pH-neutralen Bereich zu finden. Die meisten Peptide der Kontrollelagen dagegen im leicht sauren oder im basischen Bereich. Die Evaluierung der identifizierten Peptide nach ihrerHydrophobizität mittels des GRAVY-Scores zeigte, dass in der derivatisierten Probe wesentlich mehr hydrophobePeptide nachweisbar waren.Durch die Kombination der komplementären Datensätze konnte die Sequenzabdeckung der identifizierten Pro-teine in der Membran- und Cytosolprobe signifikant erhöht werden. Zudem waren für einige Proteine nur durchderivatisierte Peptide nachweisbar.Neue Aspekte: Derivatisierung der Peptidaminseitengruppen von Elastase verdauten Proteomen mittel TMTzero

verbessert die Identifikation von hydrophoben und stark sauren Peptiden in MALDI.Thema: Proteine und PeptideKeywords: MALDI, Elastase, TMTKontakt: [email protected]

110

P21

Towards 100 % sequence coverage in protein QC: In-depthsequence characterization of monoclonal antibodies

Czentnar, Zoltan; Asperger, Arndt; Suckau, Detlev; Hufnagel, Peter; Schweiger-Hufnagel, Ulrike;

Hebeler, Romano; Witt, Matthias


Einleitung: Quality control of biotherapeutic proteins requires access to the full protein sequence to account

for every single aminoacid and its modification status. This is typically not achieved in a single LC-MS/MS

analysis, but requires combined analytical strategies. However, this complicates the downstream data analysis

due to distribution of relevant information in multiple datasets. A new bioinformatics platform, ProteinScape, is

described here, which facilitates in-depth protein characterization tasks at an unparalleled level of efficiency.

Experimenteller Teil: A monoclonal antibody (mAb) was figested by either trypsin, chymotrypsin, LysC orGluC

with or without prior SDS-PAGE chain separation, followed by LC-MALDI-TOF/TOF. ProteinScape 2.1 was used

to compile a single result peptide list across all experiments.

Ergebnisse und Diskussion: LC-MALDI analysis of these digests yielded individual sequence coverages between

64 and 88 %. A significant increase in sequence coverage was achieved when compiling the four individual digest

analyses into one overall sequencing result using the ProteinExtractor algorithm implemented in ProteinScape.

ProteinExtractor compilation of the LC-MALDI data yielded 100 % coverage for the light chain (LC), and 98 %

for the heavy chain (HC). Additional electrospray measurements provided additional information on HC-[177-

184] which was not accounted for by MALDI. Combinastion of ESI and MALDI data increased the sequence

coverage for the HC to 99 %. In addition to the aminoacid sequence, the HC´s N-terminal modification status was

characterized. Evaluation of the respective MS/MS spectra confirmed N-terminal conversion of Gln to pyroGlu

based on consistent ESI and MALDI data.

Neue Aspekte: Protein QC Bioinformatics allows to combine multiple analysis in a single report. This is needed

for full protein coverage


Keywords: Bioinformatics, Protein, QC, LC-MALDI, ESI


111

P22

Comparison of label-free protein quantification approaches

Wu, Zhixiang; Kuster, Bernhard

TU Muenchen, Germany

Einleitung: Mass spectrometry-based proteomic quantification technologies are promising methods for the dis-

covery of protein biomarkers, which might be used as a guideline for physicians to choose the optimal treatment

for individual patients.Current MS-based methods can be divided into two main categories: stable isotopic labeling

and label-free quantification, both of which have their own strengths, limitation and requirements. As what con-

cerns biomarker discovery within large numbers of clinical samples, label-free quantification has the conceptual

advantage that it allows comparing many samples in any combination. Here we evaluate two label-free methods

(MS intensity and spectrum counting) for their ability to analyze kinase protein expression profiles of 34 cancer

cell lines.

Experimenteller Teil: Human kinases were affinity purified from 34 cancer cell lines using kinobeads, an affinity

resin displaying broad spectrum kinase inhibitors. Affinity purifications were digested into peptides and analysed

by LC-MS/MS on a LTQ-Orbitrap XL mass spectrometer. Raw LC-MS/MS data was converted into Mascot

Generic Format files (MGF) either by Mascot Distiller or Progenesis LCMS. Protein identification was performed

byMascot. TheMascot output dat files were imported into Progenesis LCMS or Scaffold for quantification. emPAI

values were extracted from the corresponding dat files by a custom Perl script.

Ergebnisse und Diskussion: When comparing MS intensity and spectrum counting for their ability to quantify

proteins between a large data set comprising 34 highly similar experiments and a total of approx. 750,000 tan-

dem mass spectra, several observations could be made: 1. Missing values: any statistical comparison between

experiments requires that all data points are present in all experiments. Quantification by MS intensity (Progenesis

LCMS) clearly outperformed spectrum counting (Scaffold). Only 0.5 % of all intensity values (661 out of 133042)

were missed in the Progenesis data while 52.2% (26751 out of 51272) of all spectrum counting values were zero. 2.

Quantitative trends: With few exceptions, the quantification data of both methods was consistent. This means that

a protein showing high (low) abundance by MS intensity was also highly (low) abundant by spectrum counting.

Owing to the few missing data points, many more proteins could be quantified by MS intensity across the 34 ex-

periments compared to spectrum counting. 3. Protein abundance: It has been well established, that the correlation

of MS intensity and protein quantity is linear over many orders of magnitude. For spectrum counting, there also

is a linear range but the correlation is skewed at both ends of the protein abundance scale. For high abundance

proteins, e.g. EGFR, the MS intensity ratio between two samples was 0.22 while it was only 0.12 for spectrum

counting. This indicates saturation of the spectrum counting approach leading to the underestimation of the true

abundance. At the low abundance end, e.g. illustrated by PKN1 (max spectrum count of 2), the spectrum counting

data becomes binary (present, absent) thus leading either to under- or overestimation of ratios. MS intensity for

this protein was detected in all 34 samples and revealed that its abundance was not even the highest in the sample

where the highest spectrum count was observed.

Neue Aspekte: Performance comparison of label free quantification in affinity proteomics


Keywords: quantitative proteomics, label-free quantitation, affinity proteomics


112

P23

Identification of novel microRNA-155 target proteins in thecancer cell line HEK293T using quantitative proteomics

Warnken, Uwe (1); Lößner, Christopher (1); Meier, Jan (2); Pscherer, Armin (2); Lichter, Peter (2);

Schnölzer, Martina (1)

1: German Cancer Research Center, Functional Proteome Analysis, Germany; 2: German Cancer Research

Center, Division of Molecular Genetics, Germany

Einleitung: MicroRNAs (miRNAs) are endogenous ∼ 22 nucleotides long RNAs pairing to sites within mes-

senger RNAs (mRNAs). miRNAs deregulations have a strong impact in various diseases and especially in cancer.

Although miRNAs exert their way of action favourably by inhibition of translation only little is known about exper-

imentally validated miRNA targets so far. In B cell lymphomas miRNA-155 is accumulated 30-fold compared to

normal circulating B cells (1). To decipher changes caused by miRNA-155on the protein level weapplied relative

quantitative proteomics using SILAC (stable isotope labeling by amino acids in cell culture) followed byESI-MS

analysis to miRNA-155 transfected cancer cell line HEK293T.

Experimenteller Teil: In order to overexpress miR-155,the miR-155 sequence was cloned intoa pCMX-PL1

vector. HEK293T cells were cultured in SILAC D-MEM mediumand were tranfected with either 0,45 µg of

pCMX empty vector or pCMX-miR-155. 100 µg protein of each forward and reverse labelled cell lysate were

separated on a 4-12% SDS-PAGE gel (NuPAGE Novex Bis-Tris Gel; Invitrogen). Lanes were cut to get 26 slices.

Following tryptic digestion samples were applied to nanoLC-ESI-MS analysis using a LC-Packings Ultimate

nanoLC (Dionex) and a LTQ-Orbitrap XL (Thermo) mass spectrometer. MaxQuant (Cox and Mann) software was

used for protein quantification and Mascot (Matrix Science) database algorithm was used for protein identification.

Differentially regulated proteins were validated by Western blot and qRT-PCR analysis.

Ergebnisse und Diskussion: Using the SILAC approach, we could identify a total of 3148 protein groups with a

false discovery rate (FDR) of less than 1%, using a target decoy database and at least one unique peptide. Out of

the initial protein groups, 2434 protein groups could be quantifiedanalyzing at least three independenttransfected

HEK293T cultures. Eighty-eight protein groupsexhibited changes in regulation with a FDR of less than 1%

and a variability of less than 50%, where of 42 protein groups were upregulated and 46 downregulated. These

results demonstrate that, employing the mass spectrometry based SILAC relative quantification technique, miR-

155 regulated target proteinscan be significantly detected in HEK293T cells, even with fairly subtle regulations.

Neue Aspekte: Identification and quantification of miRNA target proteine by relative quantification using SILAC

labellingandmass spectrometry analysis.

Referenzen: [1] Eis PS, Tam W, Sun L, Chadburn A, Li Z, Gomez MF, Lund E, Dahlberg JE. Accumulation of

miR-155 and BIC RNA in human B cell lymphomas. Proc Natl Acad Sci USA. 2005 Mar 8;102(10):3627-32.


Keywords: SILAC, miR-155, HEK293T,


113

P24

Introducing MeCAT with iodine reactivity for cysteine labeling ofproteins and peptides

Schwarz, Gunnar; Weber, Ralf; Linscheid, Michael W.

Humboldt-Universität zu Berlin, Germany

Einleitung: In modern life science, the focus of proteomics nowadays turns from sole identification of proteins

to their quantification. Since the development of stable isotope labeling techniques including ICAT,[1] iTRAQ[2]

and SILAC[3] mass spectrometry has been shown to be capable of providing both qualitative and quantitative

information. Furthermore, with the combination of high capacity separation techniques even complex mixtures

can be analyzed. MeCAT (Metal Coded Affinity Tag)[4] uses chelate complexes of lanthanides for relative and

absolute quantification with elemental mass spectrometry, while molecular mass spectrometry is used for sequence

elucidation of peptides and proteins by tandem mass spectrometry.

Experimenteller Teil: Peptides and proteins are labeled with the MeCAT-reagent, which contains a iodine residue

as a cysteine-reactive group for quantitative labeling and an elemental tag harboring a lanthanide ion for quantifi-

cation. Complex mixtures of MeCAT labeled peptides, such as protein digests, are chromatographically separated

before identification by ESI-MSn. Quantification is done by HPLC-ICP/MS with external calibration. Using dif-

ferent metal labels, it is also possible to obtain relative information about the amount of peptides or proteins, while

the advantage of MeCAT in ICP/MS is the opportunity for absolute quantification.

Ergebnisse und Diskussion: The structures of labeled peptides were investigated using CIDMS/MS. For complex

peptide mixtures, reversed phase HPLC-ESI/MS coupling was applied to distinguish between labeled and unla-

beled peptides. Identification of labeled peptides was achieved using CID experiments, since MeCAT-labeled pep-

tides form b- and y-series fragments. Using external calibration with lanthanide standards, it is possible to quantify

peptides using reversed phase HPLC-ICP/MS. This was demonstrated on a peptide mixture from b-lactoglobulin

trypsin digests. In order to avoid disadvantages of quantification on the peptide level, such as sample loss or in-

complete digestion of proteins, labeling at the protein level was also applied and optimized. Compared with the

maleinimide functionalized MeCAT reagent, which also labels cysteins, the reaction with iodine is faster and a

smaller excess of MeCAT reagent is needed for quantitative labeling.

Neue Aspekte: New MeCAT reagent with iodine reactivity for cysteine labeling.

Referenzen: [1] Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R, Nat. Biotechnol. 1999, 17, 994;

[2] Hunt T, Huang Y, Ross P, Pillai S, Purkayastha S, Pappin D, Mol. Cell. Proteomics 2004, 3, S286.; [3] Ong SE,

Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M, Mol. Cell. Proteomics 2002, 1, 376.;

[4] A metal-coded affinity tag approach to quantitative proteomics. Ahrends R, Pieper S, Kühn A, Weisshoff H,

Hamester M, Lindemann T, Scheler C, Lehmann K, Taubner K, Linscheid MW. Mol Cell Proteomics. 2007 Nov;

6(11):1907-16.

Thema: Element-Massenspektrometrie, Proteine und Peptide

Keywords: quantitative proteomics, metal labeling, fragmentation


114

P25

Strategies for protein quantitation in human serum by QTRAPLC mass spectrometry

Wielsch, Natalie (1); Ceglarek, Uta (1); Dojahn, Jörg (2); Glückmann, Matthias (2); Leichtle, Alexander (1);Kortz, Linda (1); Thiery, Joachim (1); Fiedler, Georg Martin (1)

1: Universitätsklinikum Leipzig AöR, Institut für Laboratoriumsmedizin, Klinische Chemie und MolekulareDiagnostik; 2: Applied Biosystems, Darmstadt

Einleitung: Quantitative proteome analysis in human body fluids by multiple reaction monitoring (MRM) coupledwith stable isotope dilution mass spectrometry has become important for clinical diagnostics due to its potentialof multiplexed testing. However, verification of protein biomarkers in clinical routine requires a high degreeof robustness and the potential for high throughput. In this study we compare two analytical strategies for thequantitation of C-reactive protein (CRP) in human serum samples: 1) nanoflow-HPLC and 2) UPLC combinedwith QTRAP instrument.Experimenteller Teil: Serum samples containing CRP at clinically relevant concentrations were selected andsubjected to analysis by the use of nanoHPLC (75 µm i.d.) and analytical UPLC (2.1x100mm) coupled to QTRAP5500. To evaluate the influence of the biological matrix (u.a.high abundant proteins) on the protein quantitation,samples were optionally subjected to depletion (Albumin and IgG). Serum samples were denaturated, trypticallydigested and desalted prior to LC-MS/MS analysis. CRP peptide ESDTSYVSLK was quantified using externalcalibration by MRM and newly patented MRM3 approach.Ergebnisse und Diskussion: Our results demonstrated applicability of the robust and high throughput compatibleUPLC coupled to the novel QTRAP 5500 system for the quantitation of CRP at clinically relevant concentrations(µg/ml range) without depletion. By fastLC-MS/MS 350 amol/ul of the CRP peptide standard were detetctedwith SN 13.6 for MRM and 175 amol/ul were still detected with S/N 1.3 using MS3. Although nanoLC-MS/MSanalysis showed higher sensitivity for quantification of peptides, the limited robustness of nanoflow-HPLC restrictsits application in clinical proteomics.The amounts of CRP quantified by MRM3 were close correlated to those determined by ELISA tests. The demon-strated MRM3 approach showed significant improvement in the specificity and sensitivity of peptide detection,opening new prospects in clinical proteomics.Neue Aspekte: New MRM cubed approach significantly improves the specificity and sensitivity of peptide detec-tion.Referenzen: [1] Fortin, T. et al. MCP 2009, 8.; [2] Fortin, T. et al. Anal Chem. 2009.Thema: Instrumentelle Entwicklungen, Proteine und PeptideKeywords: protein biomarkers, targeted proteomics, multiple reaction monitoring (MRM), multiple reactionmonitoring cubed (MRM3)Kontakt: [email protected]

115

P26

Quantitative Analysis of Cellular Levels of ErythropoietinReceptor by LC-

Böhm, Martin Erich (1); Zinn, Nico (1); Rodriguez, Agustin (2); Klingmüller, Ursula (2); Lehmann, Wolf Dieter

(1)

1: Molecular Structure Analysis, DKFZ Heidelberg, Germany; 2: Systems Biology of Signal Transduction,

DKFZ Heidelberg, Germany

Einleitung: Anemia is a frequent implication in the etiopathology of different types of cancer and their treatment.

Anemia generally impairs patients and leads to a higher mortality. Anemia can be treated by Erythropoietin

(Epo) because Epo stimulates red blood cell formation and survival. However, recent data indicate a tumor-

promoting effect for Epo-receptor (EpoR) expressing tumor cells [1]. In characterizing the Epo-sensitivity of cells,

quantification of the EpoR levels is a first important step. In this study we implemented a sensitive method to

quantify EpoR in eukaryotic cells by LC-MS/MS.

Experimenteller Teil: Lysates of different cell lines were immunoprecipitated and purified by 1D-PAGE. Gel

bands were excised and in-gel digested according to standard protocols. To quantify the receptor, we added

absolutely quantified isotopically labelled EpoR-peptides (PASTA peptides) to the digest. The samples were

analysed with a nanoAcquity UPLC (Waters) interfaced to a LTQ-Orbitrap XL (Thermo) using either a top6 DDA

method or a predefined precursor ion mass list.

Ergebnisse und Diskussion: Method development was started using recombinant proteins representing the intra-

cellular or extracellular part of EpoR, respectively. Tryptic digestion revealed a set of proteotypic EpoR peptides,

which were used to establish an inclusion mass list. This list was employed for detection of EpoR in 13 cell lines

with elevated sensitivity compared to the standard mode of data-directed analysis. Using an inclusion list-only

acquisition of LC-MS/MS data, the semiquantitative data evaluation for EpoR derived peptides (spectral counts)

showed a good agreement with expected EpoR levels. To improve the quantification, synthetic labeled and quan-

tified peptide standards (PASTA peptides [2]) were prepared and added to the digests of gel bands prepared from

the corresponding immunoprecipitate fractions. For coverage of the intracellular part of EpoR carrying the pTyr

sites, the sole use of trypsin results in a poor sequence coverage, so that for this purpose a combination of several

proteases such as Asp-N, Glu-C, and trypsin was found to be essential.

Neue Aspekte: Qualitative and quantitative LC-MS/MS-analysis of EpoR in tumor cell lines.

Referenzen: [1] Wright JR, Ung YC, Julian JA, Pritchard KI, Whelan TJ, Smith C, Szechtman B, Roa W, Mulroy

L, Rudinskas L, Gagnon B, Okawara GS, Levine MN. J CLIN ONCOL 2007, 25, 1027-1032.; [2] Zinn N, Hahn

B, Pipkorn R, Schwarzer D, Lehmann WD. J Proteome Res 2009, 8, 4870-4875.

Thema: Isotopen-Massenspektrometrie, Proteine und Peptide

Keywords: Erythropoietin-receptor, quantitative proteomics, isotope dillution, tumor cells


116

P27

Understanding the degradation of elastin and its precursor

Heinz, Andrea (1); Jung, Michael C. (1); Taddese, Samuel (1); Ihling, Christian (1); Weiss, Anthony S. (2);

Neubert, Reinhard H. H. (1); Schmelzer, Christian E. H. (1)

1: Martin-Luther-Universität Halle-Wittenberg, Germany; 2: School of Molecular and Microbial Biosciences,

University of Sydney, Sydney, Australia

Einleitung: Elastin provides elasticity and resilience to various organs such as arterial blood vessels, lungs, and

the skin. Degradation of elastic fibers upon repeated exposure of elastin to the attack of proteases may result in

the development of pathological conditions such as aneurysm, atherosclerosis, and loss of skin elasticity. Among

other factors bioactive peptides containing the GxxPG motif released upon degradation of elastin and its precursor

tropoelastin are known to be associated with the formation of the abovementioned diseases and conditions. It is,

hence, of particular importance to investigate the susceptibility of tropoelastin and elastin to the degradation by

biologically relevant enzymes such as matrix metalloproteinase MMP-12 and the serine protease human leukocyte

elastase (HLE).

Experimenteller Teil: Human tropoelastin lacking domains encoded by exons 22, 24A, and 26A (isoform 2) and

human skin elastin (Elastin Products Company, Owensville, MO, USA) were digested using recombinantly pro-

duced MMP-12 (Biomol, Plymouth Meeting, PA, USA) and HLE (Sigma, Steinheim, Germany). The generated

peptides were analyzed by on-line nano-LC-nanoESI-qTOF and off-line nano-LC/MALDI-TOF/TOF mass spec-

trometry and identified by means of a combined de novo and database sequencing approach using PEAKS and

Mascot.

Ergebnisse und Diskussion: The aim of the study was to investigate the degradation of the natural substrate

tropoelastin by the elastinolytic matrix metalloproteinases MMP-12 and the serine protease HLE and compare

the cleavage site specificities of the enzymes. Furthermore, the ability of the two proteases to release bioactive

peptides was studied. It was found that MMP-12 and HLE rapidly and substantially degrade tropoelastin while

upon degradation of elastin significantly fewer peptides were released which is due to the extensive cross-linking

of elastin as compared to monomeric tropoelastin. With respect to the cleavage site specificities, the study revealed

that the two enzymes similarly accept the hydrophobic and/or aliphatic amino acids Pro, Ala, and Val as well as

the charged amino acid Lys in S1’ while S1 of both MMP-12 and HLE mainly accommodates the hydrophobic

and/or aliphatic amino acids Gly, Ala, and Val [1, 2]. An analysis of the peptides generated during degradation of

human skin elastin showed that both MMP-12 and HLE release bioactive GxxPG sequences which underlines the

biological relevance of these two enzymes.

Neue Aspekte: Investigation of the release of bioactive motifs by matrix metalloproteinases and serine proteases

from the natural substrates tropoelastin and elastin

Referenzen: [1] S. Taddese, A.S. Weiss, G. Jahreis, R.H.H. Neubert, C.E.H. Schmelzer, In vitro degradation of

human tropoelastin by MMP-12 and the generation of matrikines from domain 24, Matrix Biol 28 (2009), 84-91;

[2] A. Heinz, M.C. Jung, L. Duca, W. Sippl, S. Taddese, C. Ihling, A. Rusciani, G. Jahreis, A. Weiss, R.H.H.

Neubert, C.E.H. Schmelzer, Degradation of tropoelastin by matrix metalloproteinases: cleavage site specificities

and release of matrikines, submitted


Keywords: GxxPG, matrikines, macrophage elastase, human neutrophil elastase, cleavage site specificities


117

P28

Mass Spectrometric Analysis of Protein Methyltransferase Action

Knut, Kölbel (1); Ihling, Christian (1); Uwe, Kühn (2); Andrea, Sinz (1); Elmar, Wahle (2)

1: Institut für Pharmazie, MLU Halle, Germany; 2: Institut für Biochemie und Biotechnologie, MLU Halle,

Germany

Einleitung: Asymmetric dimethylation of arginine residues is a common posttranslational modification of proteins

carried out by type I protein arginine methyltransferases, including PRMT1. We report that the consecutive

transfer of two methyl groups to a single arginine side chain by PRMT1 occurs in a distributive manner, i. e.

with intermittent release of the monomethylated intermediate. The oligomeric state of PRMTs together with the

clustering of methylated arginine residues in most proteins carrying this type of modification suggests that multiple

methyl transfers to a single polypeptide chain might proceed in a processive manner by cooperation of multiple

active sites. However, we present evidence that the reaction is distributive even with substrates containing multiple

methyl-accepting arginines, such as the nuclear poly(A)-binding protein (PABPN1).

Experimenteller Teil: The methylation assays were conducted with 12 µM PABPN1 and 0.3 µM PRMT1.

Reactions were started by the addition of S-adenosylmethionine (SAM) to a final concentration of 238 µM . After

different time intervals, 20 µl aliquots were taken, the reaction was stopped by addition of the methylation inhibitor

sinefungin to a final concentration of 0.6 mM. The samples were digested with endoproteinase Lys C. Proteolysis

products were analyzed by nanoLC / nano-ESI-MS using reversed phase chromatography on an UltiMate Nano-

LC-system coupled to an LTQ-Orbitrap with nano-electrospray ionization source.

Ergebnisse und Diskussion: Endoproteinase Lys C cleaves off the entire C-terminal domain of PABPN1 con-

taining all 13 methyl-accepting arginines as a single peptide. LC/MS of the digestion mixture allowed the direct

analysis of the methylation state of this domain after different time intervals of incubation of PABPN1 with SAM

and PRMT1. Several differentially methylated species were observed; products containing up to four methyl

groups were sufficient in intenstiy to allow quantification. Monomethylated products were the dominant product

species over the first 20 minutes of the time course, encompassing approximately 16 methylation events per en-

zyme molecule. Over the first 30 minutes, the distribution of the various methylated species was in reasonable,

though not in perfect agreement with a random distribution, supporting a distributive action of PRMT1 on its sub-

strate PABPN1. Only at later time points, when a significant fraction of the substrate had been methylated at least

once, the experimentally determined distribution differed from the prediction. We conclude that the consecutive

transfer of two methyl groups to a single arginine side chain by PRMT1 occurs in a distributive manner.

Neue Aspekte: Nano-HPLC/nano-ESI-LTQ-Orbitrap-MS is used for analyzing protein methyltransferase action


Keywords: protein, orbitrap


118

P29

The role of arginine in chemical cross-linking with NHS esters

Mädler, Stefanie; Gschwind, Sabrina; Zenobi, Renato

ETH Zürich, Switzerland

Einleitung: N-hydroxy succinimide (NHS) esters are often used as specific and efficient immobilizing, labeling

or cross-linking agents for the detection, characterization and quantification of interactions between biomolecules.

Apart from acylation of the ǫ-amino group of lysines and the α-amino group of N-termini, occasional side reactions

with hydroxyl side chains have been observed, although hydroxyl groups are much less nucleophilic.1,2 The

presence of His increases the reactivity of hydroxyl groups towards NHS labeling agents significantly for certain

sequences.3 However, additional catalytic effects are likely to occur. In order to clarify whether Arg has an

enhancing effect on the acylation of hydroxyl groups by homobifunctional cross-linking agents in aqueous solution,

we carried out systematic experiments with model peptides.

Experimenteller Teil: Peptides containing Ser, Thr or Tyr in close proximity to Arg and control samples without

Arg were investigated. In order to be able to disregard other possible sequence-specific effects in comparative ex-

periments, Arg residues were covalently blocked using 2,3-butanedione and phenylboronic acid.4 Systematic cross-

linking experiments were carried out with the NHS esters disuccinimidyl suberate (DSS) or bis(sulfosuccinimidyl)

suberate (BS3). Samples were analyzed by MALDI-TOF-MS (UltraFlex II MALDI-TOF-MS, Bruker Daltonics

GmbH, Bremen, Germany) comparing relative reaction yields of intramolecular crosslinks with covalently pro-

tected and unprotected Arg.

Ergebnisse und Diskussion: Reactions of Ser, Thr and Tyr occurred predominately as intramolecular linkages

connecting the N-termini and the hydroxyl groups for all investigated peptides. Interestingly, all peptides hav-

ing high Type-1 yields with unprotected Arg showed a lower reactivity of the hydroxyl groups when Arg was

covalently blocked. In control experiments with an Arg-free peptide DPAFNSWG-NH2, no changes occurred,

thus confirming the direct effect of Arg blockage. As catalytic mechanism, the formation of hydrogen bonds be-

tween the guanidinium hydrogens and the oxygen of the attacked carbonyl group of the cross-linker should be

considered to facilitate the nucleophilic attack of the hydroxyl groups by lowering the activation barrier of the

nucleophilic substitution. In summary, Ser, Thr and Tyr are of significant importance as reactive sites for chemical

cross-linking and can be used as additional constraints for structure elucidation studies. The guanidinium group

of Arg was demonstrated to contribute to the reactivity of hydroxyl groups towards NHS esters and to catalyze

the nucleophilic substitution.This finding can explain the enhanced reactivity of Ser, Thr or Tyr in Arg-containing

peptides.5

Neue Aspekte: Elucidation of a reactivity enhancing effect for hydroxyl acylation with homobifunctional NHS

esters

Referenzen: [1] S. Kalkhof, A. Sinz, Chances and pitfalls of chemical cross-linking with amine-reactive N-

hydroxysuccinimide esters, Anal. Bioanal. Chem. 392 (2008) 305-312.; [2] S. Madler, C. Bich, D. Touboul, R.

Zenobi, Chemical cross-linking with NHS esters: a systematic study on amino acid reactivities, J. Mass Spectrom.

44 (2009) 694-706.; [3] S. Madler, C. Bich, D. Touboul, R. Zenobi, Chemical cross-linking with NHS esters: a

systematic study on amino acid reactivities, J. Mass Spectrom. 44 (2009) 694-706.; [4] A. Leitner, W. Lindner,

Probing of arginine residues in peptides and proteins using selective tagging and electrospray ionization mass

spectrometry, J. Mass Spectrom. 38 (2003) 891-899.; [5] S. Madler, S. Gschwind, R. Zenobi, Role of arginine in

chemical cross-linking with NHS esters, Analytical Biochemistry, doi:10.1016/j.ab.2009.11.020


Keywords: Chemical cross-linking, NHS esters, reactivity of hydroxyl amino acids, MALDI-MS, arginine catal-

ysis


119

P30

Acid-labile histidine phosphorylation becomes accessible byfunctional proteomics

Hohenester, Ulli Martin; Ludwig, Katrin; Krieglstein, Josef; König, Simone


Einleitung: In functional proteomics mass spectrometric (MS) techniques have been widely-used for the detectionof phosphohydroxyamino acids such as phosphoserine, phosphothreonine or phosphotyrosine. Phosphohistidine,which is 10 to 100 times more abundant in nature than pY and is also of importance for protein signalling andregulation, was hardly investigated using bioanalytical methodology. One reason for this was the acid lability ofHis-phosphorylation. Another reason were initial difficulties using MS for phosphorylation site analysis, becausepHis seemed to lose the phosphate group even more readily that pS, pT or pY. In this work, several His-containingpeptides have been chemically phosphorylated and analyzed by low-pH reversed-phase chromatography and MSin order to evaluate the analytical possibilities in a dedicated effort.Experimenteller Teil: Peptides (AHPF, CheA peptide IFRAAHSIKGG, Gβ-peptide LMTYSHDNIIC, angiotensinI and II, renin, bombesin) were treated with alkaline potassium phosphoramidate solution overnight. The reactionmixtures were diluted with aqueous formic acid for manual nanospray MS/MS and LC-MS/MS. These exper-iments were performed using the Esquire3000 ion trap (Bruker Daltonics, Bremen, Germany) with a modifiedoff-line nanospray source and Q-TOF Premier coupled to Acquity nanoUPLC (Waters Corp., Manchester, UK).RP-chromatography was carried out on C18 material with a water/acetonitrile gradient.Ergebnisse und Diskussion: Phosphorylation with potassium phosphoramidate was highly selective and mainlygenerated 3N-pHis with up to 90% yield. 1N-pHis and 1,3N-dipHis were also formed at much lower quantities. Allthree forms could be separated by RP-LC running at low pH. The phosphorylation yield was directly proportionalto peptide solubility and bombesin, the Gβ-peptide and renin were not modified under the described conditions.Phosphorylated peptides were diluted at least 1:100 with 0.1% formic acid could be used for MS without fur-ther purification. Storing them overnight at room temperature did not influence their degree of phosphorylation.Synthesized peptides were stable about several hours at room temperature and fragment spectra from low energycollision-induced dissociation could be generated. Peptide size, charge, sequence as well as the experimental pa-rameters influenced the number and intensity of detected backbone fragments which retained the phosphate group.Moreover, the His phosphoimmonium ion was detected which is a helpful marker for the presence of pHis.Neue Aspekte: Phosphohistidine identification with low pH reversed-phase LC-MS/MReferenzen: [1] Anal Bioanal Chem 2010, DOI 10.1007/s00216-009-3372-xThema: Proteine und PeptideKeywords: Phosphohistidine, Protein phosphorylation, Fragmentation, ChromatographyKontakt: [email protected]

120

P31

Peptide backbone conformation affects the substrate preferenceof protein arginine methyltransferase I

Kölbel, Knut (01); Ihling, Christian (01); Stichel, Jan (02); Beck-Sickinger, Annette G. (02); Sinz, Andrea (01);

Wahle, Elmar (01)

1: Martin-Luther-Universität Halle, Germany 01; 2: Universität Leipzig 02

Einleitung: Asymmetric dimethylation of arginine residues is a eukaryotic posttranslational modification thatoccurs frequently in DNA- and RNA-binding proteins. One of them, the nuclear poly(A) binding protein 1(PABPN1), contains thirteen arginine residues in its C-terminal domain, all of which are quantitavely dimethylatedin vivo. In higher eukaryotes, several protein arginine methyl transferases (PRMTs) exist, among them PRMT1,which is mainly responsible for the modification of PABPN1 [1]. In vitro methylation of PABPN1 and PABPN1-derived peptides proceeds distributively, but depends strictly on the length of the substrate (poly-) peptide chain[2]. An inverse turn harboring a crucial proline residue was shown to present the initial methylation site withinPABPN1.Experimenteller Teil: Recombinant (i.e. unmethylated) His-tagged PABPN1 and PABPN1-P288A were pro-duced in E. coli and purified by affinity as well as ion exchange chromatography. The purified proteins wereincubated with PRMT1 and S-adenosylmethionine (SAM) for different times followed by chymotryptic diges-tion. Information about methylation sites was derived from MALDI-TOF-MS (Ultraflex III, Bruker Daltonik)and from nano-HPLC/MALDI-TOF/TOF-MS/MS experiments. Substrate peptides were synthesized and purifiedusing Fmoc-chemistry and HPLC. After incubation with PRMT1 and SAM, their methylation patterns were ana-lyzed by MALDI-TOF-MS and MS/MS. Steady-state kinetics were determined by incubation of PAPBN1 peptideswith PRMT1 and 14C-SAM, subsequent SDS-PAGE, and phosphoimaging. The secondary structure of selectedpeptides was deduced from far UV CD spectra.Ergebnisse und Diskussion: Only two fragments of wildtype PABPN1, both covering the region N285-V292/Y293,were found to be methylated to a significant amount. By MALDI-TOF/TOF-MS/MS, R289 was identified to bemethylated. In a peptide resembling the respective region of PABPN1 the arginine residue R10, corresponding toR289, formed the exclusive methylation site in a similar manner. The specificity for both arginines, R289 and R10,was abolished if the preceding proline residue (P288 and P9, respectively) was exchanged to glycine or alanine.Proline is frequently found in i+1 position of type I or type II β-turns. Therefore, three pentapeptides of the se-quence D-Ala-PRGN were investigated, which exhibit free, protected, and connected (cyclic peptide) termini. Byfar UV CD and steady state kinetics, a striking correlation was observed between the turn structure content of thepeptides and their ability to be methylated.Neue Aspekte: PRMT substrates have been regarded as essentially unstructured. Here we present evidence ofcertain secondary structures being preferred substrates.Referenzen: [1] Fronz K, Otto S, Kölbel K, Kühn U, Friedrich H, Schierhorn A, Beck-Sickinger AG, Ostareck-

Lederer A, Wahle E. 2008: Promiscuous modification of the nuclear poly(A)-binding protein by multiple protein-

arginine methyltransferases does not affect the aggrega; [2] Kölbel K, Ihling C, Bellmann-Sickert K, Neundorf I,

Beck-Sickinger AG, Sinz A, Kühn U, Wahle E. 2009: Type I Arginine Methyltransferases PRMT1 and PRMT-3

Act Distributively. J Biol Chem. 284 8274-82


Keywords: arginine methylation, PABPN1, inverse turn conformation


121

P32

Non database IgG heavy chain C regions: de novo sequencing andelucidation of N-glycosylation

Neue, Kristina; Mormann, Michael; Peter Katalinic, Jasna; Pohlentz, Gottfried


Einleitung: For mass spectrometric analysis of N-glycosylation the glycans are frequently cleaved enzymatically- mostly with PNGase F - prior to proteolytic digest of the deglycosylated protein. Oligosaccharides and peptidesare analyzed separately yielding the overall glycan pattern and the previously occupied glycosylation sites [1, 2].Recently, zwitter-ionic hydrophilic interaction liquid chromatography on ZIC®-HILIC is used for separating gly-

copeptides taking advantage of their hydrophilicity. The HPLC method is usually laborious and time-consuming.

A number of papers describe a combination tryptic digest with ZIC®-HILIC SPE separation. However, either

a deglycosylation step follows because the relatively high mass of glycopeptides causes problems with matrix-

assisted laser desorption/ionization (MALDI) MS analysis [3] or a further separation, e.g. by use of LC-MS/MS,

is made [4].

Experimenteller Teil: Bovine and caprine IgG preparations were digested with trypsin, chymotrypsin, and/or

thermolysin in an ammonium hydrogen carbonate buffer over night. Nanoelectrospray mass spectrometry experi-

ments were carried out using a quadrupole time-of-flight (Q-TOF) mass spectrometer in the positive ion mode. A

Z-spray atmospheric pressure ionization (API) source was used with the source temperature set to 80°Cand a des-

olvation gas (N2) flow rate of 75 L/h. For low energy CID peptide precursor ions were selected in the quadrupole

analyzer and fragmented in the collision cell by use of a collision gas (Ar) pressure of 2.5 x 10−5 mbar and col-

lision energies of 20 to 40 eV (Elab). Separation of glycopeptides by use of ZIC®-HILIC SPE was performed

according to manufacturer’s instructions.

Ergebnisse und Diskussion: Immunoglobulins gamma (IgGs) have an important role as antibodies in the mam-

malian circulation. They are involved in the humoral immune response and therefore also recombinantly pro-

duced for therapeutic purposes. They are soluble serum glycoproteins with a high variety in the sequence of the

antigen-binding region and have a conserved N-glycosylation site in the constant region of their heavy chain.

Since the databases provide sequence data of IgG heavy chain C regions only from man, rat, mouse, Guinea pig,

rabbit, and horse the present study aimed de novo sequencing of constant regions derived from commercially avail-

able bovine and caprine IgG preparations. Direct nanoESI Q-Tof MS/MS analysis of corresponding digests with

trypsin, chymotrypsin, and/or thermolysin yielded high sequence coverages of the heavy chain C regions as well

as of the light chains. Moreover, the nanoESI Q-Tof MS analysis and the collision-induced fragmentation of N-

glycopeptides separated from proteolytic digests by use of ZIC®-HILIC tips enabled the direct elucidation of the

glycopeptides structures without further purification or separation.Our results clearly demonstrate that separation

of N-glycopeptides with ZIC®-HILIC is a powerful and fast method for the exploration of the highly heterogenous

N-glycosylation of IgGs.

Neue Aspekte: Amino acid sequences and N-glycan structures were elucidated without laborious separation

procedures.

Referenzen: [1] Raju, T.S. et al. (2000) Glycobiology, 10, 477-486.; [2] Yu, Y. Q. et al. (2005) Rapid Commun.

Mass Spectrom., 19, 2331-2336.; [3] Thysen-Andersen, M. et al. (2006) American Biotechnology Laboratory, 24,

14-17.; [4] Hägglund, P. et al. (2004) Journal of Proteome Research, 3, 556-566.

Thema: Kohlenhydrate, Proteine und Peptide

Keywords: IgG, de-novo sequencing, N-glycopeptides, HILIC, mass spectrometry


122

P33

Disulfide Bonding Patterns of Laminin Beta 1 and Gamma 1N-Terminal Constructs Analyzed by ESI-LTQ-Orbitrap-MS

Witte, Konstanze (1); Keller, Manuel (2); Ihling, Christian (1); Paulsson, Mats (2); Sinz, Andrea (1)

1: Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin Luther University

Halle-Wittenberg, Wolfgang-Langenbeck-Strasse 4, D-06120 Halle(Saale), Germany; 2: Center for Biochemistry

and Center for Molecular Medicine, Faculty of Medicine, University of Cologne, Joseph-Stelzmann-Strasse 52,

D-50931 Cologne, Germany

Einleitung: Laminins constitute a family of heterotrimeric glycoproteins, which are the main non-collagenous

components of the basement membrane. Basement membranes are multifunctional thin extracellular layers, sep-

arating endothelial cells from connecting tissues. Laminins form large polymeric networks, acting as scaffold for

basement membrane formation. We analyze disulfide pattern of laminin beta 1 and gamma 1 N-terminal regions.

Both of them consist of a globular N-terminal (LN) domain and four cysteine-rich epidermal growth factor-like

(LE) domains [1]. Using mass spectrometric methods we investigated the disulfide bonding patterns of these

constructs based on homology to laminin gamma 1 LE 7-9.

Experimenteller Teil: For assigning disulfide bonding patterns two different strategies were applied. Both are

based on a complete alkylation of the free cysteines with iodoacetamide as a first step to prevent disulfide shuffling.

In the first strategy, alkylation was followed by enzymatic digestion with different proteases. In the second strategy,

disulfides were reduced after alkylation of free SH-groups, before the newly created cysteines were alkylated with

an alternative reagent and enzymatic digestion is performed. Peptide mixtures were analyzed by nano-HPLC/ESI-

LTQ-Orbitrap-MS. We chose beta lactoglobulin, a 18-kDa protein containing five cysteines and two disulfide

bonds, as a model protein to optimize both strategies. Different enzymes (trypsin, chymotrypsin, GluC, LysN, and

mixtures) at different pH values were evaluated.

Ergebnisse und Diskussion: We performed proteolytic digests at pH 7.5 and pH 4.0 under different conditions.

Experiments at pH 4 did not lead to any expedient results. We were able to identify the correct disulfide bonding

patterns and the free cysteine for the model protein beta lactoglobulin when digestion was performed at pH 7.5.

Preliminary experiments with the laminin beta 1 construct indicated a linear disulfide bonding pattern, which

differs from the predicted structures based on homology to laminin gamma 1 LE 7-9. Experiments with the

laminin gamma 1 construct are currently in progress. Even though iodoacetamide yields a complete alkylation of

free cysteines, disulfide shuffling cannot be completely excluded as the pH value for digestion is 7.5 and disulfide

shuffling is preferred at neutral to slightly alkaline pH. Therefore, we are planning to conduct digestions of the

laminin constructs at pH 6 using endoproteinase LysN. Recent studies show that LysN retains a 50% proteolytic

activity at pH 6. A linear disulfide bonding pattern in laminin LE domains is expected to have a great impact

on future structural and functional studies of laminins and will shed light onto the mechanisms underlying the

interactions of basement membrane proteins.

Neue Aspekte: Disulfide analysis indicated different disulfide bonding patterns in laminin LN domains in contrast

to predicted patterns.

Referenzen: [1] Aumailley M, Smyth N, The role of laminins in basement membrane function, J Anat 193, 1-21

(1998)


Keywords: laminin, disulfide bonds, disulfide shuffling


123

P34

Applications of SAW - ESI MS to structural and bioaffinitycharacterization of β - amyloid peptides

Muresan, Adina Roxana (1,2); Slamnoiu, Stefan (1); Vlad, Camelia (1); Zamfir, Alina (2);

Przybylski, Michael (1)

1: Universitaet Konstanz, Germany; 2: Universitaet Aurel Vlaicu, Arad, Romania

Einleitung: PyroGlu3 Aβ(3-40) peptide accumulates in the brain of AD patients and has a high pathogenic effect

because early aggregates alter membrane permeability and thereby form pores in the membrane of neurons and also

the cyclisation of the N-terminal glutamate may lead to partial resistance to extracytoplasmic aminopeptidases.1

The aim of the study is to characterize the interaction between PyroGlu3 Aβ(3-40) and a commercial monoclonal

Aß(1-16) antibody.

Experimenteller Teil: The affinity - MS method of the online coupling of SAW biosensor and ESI-ion trap MS

was applied on antigen-antibody complexes related to Alzheimer’s disease and the results compared with those

obtained by other immuno-analytical methods (ELISA, Dot Blot). The SAW biosensor is suitable to analyze

samples in solution, being highly sensitive to mass loading and viscosity changes. All the reactions involved in

the immobilization of the first partner on a gold chip and the second partner in conducted through the microfluidic

cell of the biosensors. In the first experiment anti-Aβ(1-16)antibody was immobilized than were added different

dilutions of Glu3Aβ(3-40) and in a second experiment the reverse system. The same systems investigated by Dot

Blot and ELISA experiments.

Ergebnisse und Diskussion: The affinity interactions between the antibodies and Aβ-peptides were first investi-

gated by Dot Blot and ELISA experiments and further using the online coupling of SAW biosensor and ESI ion

trap MS. Were investigated the differences between the bioaffinities of pyro-Aβ(3-40) and Aβ(3-40) to the anti-Aβ(1-16) antibody. The antibodies were covalently immobilized on the surface of the chip used by the SAW sensor

and the interactions with the antigens (peptides) present in solution were determined. Further, the affinity bound

antigens were eluted with a newly developed online interface under acidic conditions and analyzed by ESI mass

spectrometry. We determined the dissociation constant using the SAW biosensor at an average of 10.79 nM in the

case of the affinity binding of pyroGlu3-Aβ(3-40) to anti-Aß (1-16) antibody .

Neue Aspekte: Affinity of Aβ peptides to antibody was proved with SAW, ELISA and DotBlot, for pyro Aβpeptide kD is 10.79nM .

Referenzen: [1] Piccini A., Russo, C. Gliozzo A., Relini A., Vitali A., Tabaton M., (2005) β-Amyloid is different

in normal aging and in Alzheimer disease, J. Bio. Chem. 280, 34186-34192; [2] Thomas M.A. Gronewold, Antje

Baumgartner, Eckhard Quandt, and Michael Famulok, (2006), Discrimination of single mutations in cancer-related

gene fragments with a surface acoustic wave sensor, Anal. Chem., vol. 78, No.14, 4865


Keywords: amyloid β (Aβ) / anti-Aβ antibodies / pyro Aβ peptides / affinity-mass spectrometry


124

P35

A Novel Cleavable Cross-Linker for Protein Structure Analysis -Constant Neutral Losses Simplify Identification of Cross-Linking

Products

Müller, Mathias (1); Dreiocker, Frank (2); Ihling, Christian (1); Schäfer, Mathias (2); Sinz, Andrea (1)

1: MLU Halle-Wittenberg, Germany; 2: Universität zu Köln, Germany

Einleitung: A novel thiourea cross-linker is described, which allows distinguishing different cross-linking prod-

ucts based on their characteristic constant neutral losses (CNLs) during collision-induced dissociation (CID)

MS/MS experiments. The development of this cross-linker is part of our ongoing efforts to synthesize novel

reagents, which create characteristic fragment ions during tandem mass spectrometry allowing a simplified analy-

sis of cross-linked products [1].

Experimenteller Teil: For probing reactivity and fragmentation mechanisms, a model peptide (Munc 13-1 [2]) as

well as a model protein (PPARα [3]) were cross-linked. Proteolytic cleavage was performed with trypsin and GluC

in solution. Peptide mixtures were analyzed both by offline Nano-HPLC/MALDI-TOF/TOF mass spectrometry

(Ultraflex III, Bruker Daltonik) and Nano-HPLC/Nano-ESI-LTQ-Orbitrap mass spectrometry (LTQ Orbitrap XL,

ThermoFisher Scientific). Characteristic CNLs were added into a neutral loss list for MS/MS experiments.

Ergebnisse und Diskussion: This novel dissociative cross-linker is applicable both to ESI and MALDI mass

spectrometry and as such, possesses a versatile applicability for chemical cross-linking studies. The characteristic

CNLs observed for different cross-linked species allow a facilitated analysis of cross-linking products, underlining

the potential of the novel reagent for protein conformational studies and for mapping protein-protein interfaces [4].

Neue Aspekte: Cleavable cross-linkers simplify identification of cross-linking types.

Referenzen: [1] F Dreiocker et al., J Mass Spectrom 2009; [2] K Dimova et al., Biochemistry 2009; [3] MQ

Müller et al., J Med Chem 2009; [4] MQ Müller et al., submitted


Keywords: cross-linking, constant neutral loss, fragmentation


125

P36

Phytochelatin identification by nanoelectrospray ionization massspectrometry

Bräutigam, Anja (1); Schaumlöffel, Dirk (2); Krauß, Gerd-Joachim (1); Wesenberg, Dirk (1)

1: Martin-Luther-Universität Halle-Wittenberg, Institut für Biochemie und Biotechnologie, Ökologische undPflanzen-Biochemie, Kurt-Mothes-Straße 3, 06120 Halle (Saale), Germany; 2: CNRS-Université de Pau,

Laboratoire de Chimie Analytique Bio-Inorganique et Environnement, MR 5254 IPREM, Hélioparc, 2, av. Pr.Angot, 64053 Pau, France

Einleitung: Phytochelatins (PCs) are peptides with the general structure (γ-GluCys)n-Gly, but also different iso-forms exist. Cadmium and other metals can be bound by their thiol groups, thus they are part of metal detoxi-fication machinery in many organisms. PCs were already described to play a role in metal detoxification in thegreen alga Chlamydomonas reinhardtii but were not clearly identified. The focus was to identify PC synthesized byC.reinhardtii exposed to Cd, with special emphasis made on low sample consumption. Nanoelectrospray ionizationtime-of-flight tandem mass spectrometry (ESI-QTOF MS/MS) was chosen for precise PC identification. [1]Experimenteller Teil: Chlamydomonas was maintained in medium containing 70µM CdCl2. 100mg biomasswere suspended in 300 µL 0.1 N HCl. Extracts were cleaned up via HPLC. Mobile phase A was TFA in water (pH2) and mobile phase B acetonitrile (1 mLmin−1). Detection was carried out via postcolumn derivatization (300µMDTNB in 50 mM KH2PO4). PC containing eluates were lyophilized and resuspended in 10 mM CH3COONH4.The ESI-QTOF MS/MS system was a QSTAR-XL (Applied Biosystems, Foster City, USA) equipped with ananospray source. Sample flow was between 300 and 500 nL min−1. The tip diameter of the spraying needlewas 10 µM . Mass spectra were acquired in the positive ion mode in the range of m/z 300-2,000.Ergebnisse und Diskussion: A high percentage of oxidized PCs was observable in the ESI-MS spectra. E.g.the oxidation of CysPC3, whose corresponding oxidized ions were observed between m/z 871 and 874, occurs.CysPC3 can form intramolecular S-S bonds (m/z 871) and intermolecular S-S bonds (m/z 1,741 and 1,745). Notethat oxidized PCs identified in this study by tandem MS are in a similar molecular mass range as canonic PCs withn>4. This can lead generally to artefacts in identification only by chromatography.In order to reduce the oxidizedthiols before ESI-MS analysis TCEP was used for further experiments. The masses (m/z) of cadmium inducedcanonical PC2−6 of the isoforms CysPC2−5, CysPC2−5desGly, PC2−5desGly, CysPC2−4Glu, PC2−3Ala, and ofPC2Glu could be detected. Among these compounds, PC2−5, CysPC2−5, PC2desGly, PC2Glu, and PC2Ala wereunambiguously identified by MS and MS/MS. Only small deviations between theoretical and experimental masseswere observed (±30 ppm). Their sequences were clearly confirmed by fragmentation in MS/MS.In addition, ithad to be verified that the identified iso-PCs were synthesized in vivo and not formed by in source fragmentationof canonical PCs. E.g. a b7 fragment of CysPC3 was detected in mass spectrum, and its identity was confirmedby MS/MS. Unwanted fragmentation can occur in the orifice skimmer region of the MS caused by the curtain gasflow as well as the orifice and focusing ring voltages. To investigate the fragmentation, the intensity ratio of the b7

ion to its parent ion was measured. A variation of the three parameters within the instrumental limits showed lowinfluence on the percentage of fragmentation. At optimized operating conditions there was 11% fragmentation.Because the ion intensity of the CysPCn is much higher than the potential precursor PCn+1, the part of fragmentions contributing to the ion intensity is negligible.Neue Aspekte: It was shown that ESI-QTOF MS/MS is the method of choice for PC identification. The dogma,of PC11 was doubted.Referenzen: [1] BRÄUTIGAM, A., SCHAUMLÖFFEL, D., KRAUSS, G. J. and WESENBERG, D. (2009).Analytical approach for characterization of cadmium-induced thiol peptides-a case study using Chlamydomonasreinhardtii. Anal Bioanal Chem 395, 1737-1747.Thema: Proteine und Peptide, Umweltanalytik und AerosoleKeywords: ESI-MS/MS, Phytochelatins, thiols, Chlamydomonas, TCEPKontakt: [email protected]

126

P37

Synthesis and structural characterization of a proteolyticParkinson-related fragment of a-Synuclein, a-Syn(71-140)

Lindner, Kathrin; Vlad, Camelia; Przybylsi, Michael

Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, Department of Chemistry, University of

Konstanz, 78457 Konstanz, Germany

Einleitung: The Parkinson-related protein α-Synuclein is an intrinsically unstructured protein that binds to mem-

branes, forms fibrils and inclusions generally known as Lewy bodies in the substantia nigra of dopamineric neurons

[1]. The 140 amino-acid α-Syn consists of three main domains, (i), an N-terminal region (1-60) containing several

imperfect KTKEGV repeats; (ii), a hydrophobic non-amyloid component of Alzheimer‘s disease (NAC) region

(61-95); and (iii), a highly negatively charged C-terminal region (96-140). However, how α-Syn is involved in the

pathogenesis of neurodegenerative disease is not clearly understood at present [2, 3]. Recently, we have shown

by ion mobility mass spectrometric analysis the formation of a specific intermediate proteolytic fragment α-Syn

(71-140).

Experimenteller Teil: The α-Synuclein fragment a-Syn (71-140) was synthesized by solid phase peptide synthe-

sis (SPPS) on a semiautomatic peptide synthesizer using fluorenylmethoxycarbonyl (Fmoc) with MBHA resins

and PyBop activation. Each coupling consisted of (i) removal of the Nα-Fmoc protection by 2% Piperidin/2%

DBU/96% DMF, (ii) 2x60 min coupling and (iii) acetylation of the N-terminus was carried out with acetic anhy-

drid, NMM and DMF for 30 min. The peptide was cleaved by 2,5 % triethylsilan (TES), 2,5 % water and 95 %

TFA. The purification of crude α-Syn (71-140) was performed on a Vydac C8 RP-HPLC column, and the resulting

peptide analyzed using MALDI-TOF and ESI mass spectrometry.

Ergebnisse und Diskussion: The proteolytic Parkinson-related fragment α-Syn (71-140) was successfully ob-

tained out by manual and automated chemical synthesis using the Fmoc-strategy. It has been reported that a 12-

amino acid stretch (71VTGVTAVAQKTV82) in the middle of the hydrophobic domain of human α-Synuclein is

necessary for its fibrillization [2]. Therefore, aggregation studies of this domain will be performed using thioflavin

T (ThT) fluorescence and mass spectrometry.

Neue Aspekte: Synthesis of a proteolytic Parkinson-related fragmentα-Synuclein (71-140) using Fmoc-chemistry.

Referenzen: [1] M.G. Spillantini, M.L. Schmidt, V.M. Lee, J.Q. Trojanowski, R. Jakes, M. Goedert, Alpha-

synuclein in Lewy bodies, Nature 388 (1997) 839-840.; [2] B.I. Giasson, I.V. Murray, J.Q. Trojanowski, V.M.

Lee, A hydrophobic stretch of 12 amino acid residues in the middle of alpha-synuclein is essential for filament

assembly, J Biol Chem 276 (2001) 2380-2386.; [3] M.G. Spillantini, R.A. Crowther, R. Jakes, M. Hasegawa, M.

Goedert, alpha-Synuclein in filamentous inclusions of Lewy bodies from Parkinson’s disease and dementia with

lewy bodies, Proc Natl Acad Sci U S A 95 (1998) 6469-6473


Keywords: alpha Synuclein, SPPS, structural characterization


127

P38

Affinity binding study of Aβ-autoantibody CDR peptide toAβ-(12-40) peptide

GALUSCA, MIRELA (1,2); COZMA, CLAUDIA (1); ZAMFIR, ALINA (2,3); PRZYBYLSKI, MICHAEL (1)

1: Department of Chemistry, Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, University

of Konstanz, 78457 Konstanz, Germany; 2: Department of Chemical and Biological Sciences, "Aurel Vlaicu"University of Arad, Romania; 3: Mass Spectrometry Laboratory, National Institute for Research and

Development in Electrochemistry and Condensed Matter, 300224, Timisoara, Romania

Einleitung: Production and aggregation of the amyloid-beta (Aβ) peptide plays a key role in Alzheimer’s disease(AD). Abeta antibodies have been shown to reduce amyloid plaques[2]. The amino acid sequence of Abetaautoantibodies was determined in our laboratory, partial sequences of the CDRs (Complementary DeterminingRegion) have been established [1].The aim of the study was to characterization of the bioaffinities of the isolatedsynthetic CDR peptides and the core containing epitope fragments.Experimenteller Teil: In order to analyze the sequence and affinity against the Abeta(12-40), a peptide containinga CDR region was synthesized using solid phase peptide synthesis (SPPS) and Fmoc strategy. The sequence of thepeptide was 1VTITC(x)RESQGIRNYLAWYQQKG22 containing the CDR (underlined) and flanking regions ofthe antibody sequence to mimic the 3D structure of the epitope. The peptide purified by RP-HPLC on a C4 andcolumn was characterized by mass spectrometry.Ergebnisse und Diskussion: The affinity interaction was checked by affinity-MS. Affinity column was a UltraLinkIodoacetyl matrix covalently bound with Cys-Aβ(12-40). After incubation with CDR peptide in physiologicalconditions, and elution was performed using 0.1%TFA. The supernatant, the last milliliter of the washing solutionand the elutate were analyzed by mass spectrometry. The obtained results showed clearly that the synthetic CDRpeptide 1 presents affinity towards Aβ(12-40).Neue Aspekte: An Abeta autoantibodies CDR peptide was investigated by affinity-MS against Abeta(12-40)Referenzen: [1] M. Przybylski et al., EPA and US Patent applications; Universities of Konstanz and Bonn, 2007;[2] R C Dodel et al., J.Neurol. Neurosurg. Psychiatry 2004;75;1472-1474Thema: Proteine und PeptideKeywords: amyloid beta, Aβ-antibodies, CDR peptide, affinity-MSKontakt: [email protected]

128

P39

High yield expression of mature, bioactive TNF-alpha bySUMO-fusion system

Hoffmann, Andreas (1); Müller, Mathias Q. (2); Gloser, Manja (3); Sinz, Andrea (2); Rudolph, Rainer (4);

Pfeifer, Sven (1)

1: NWG Künstliche Bindeproteine, Institut für Biochemie und Biotechnologie, Technische Biochemie,

Martin-Luther-Universität Halle-Wittenberg, Heinrich-Damerow-Str. 4, 06120 Halle, Germany; 2:Institutsbereich Pharmazeutische Chemie und Bioanalytik, Institut für Pharmazie, Martin-Luther-Universität

Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120 Halle, Germany; 3: Scil Proteins GmbH,Heinrich-Damerow-Str. 1, 06120 Halle, Germany; 4: Institut für Biochemie und Biotechnologie, Technische

Biochemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle, Germany

Einleitung: Tumor necrosis factor (TNF-α) inhibitors, used for the treatment of common inflammatory diseases,currently belong to the most important biotechnologically produced pharmaceuticals [1]. So far four TNF-α an-tagonists have been approved by regulatory authorities for defined subsets of applications. Furthermore, numerousapproaches are being taken to develop new protein-based pharmaceuticals and to broaden their application areasin the treatment of TNF-α-related diseases. Both the fundamental understanding of disease-related TNF-α activ-ity and the subsequent development of corresponding drug candidates demand the availability of large amountsof TNF-α as a bioactive protein. We have therefore established a protocol for the rapid high-level synthesis ofrecombinant human TNF-α in Escherichia coli shake-flask cultures and the subsequent purification of the matureprotein.Experimenteller Teil: The protein was produced using the advantages of autoinduction medium ZYM-5052 [2]and SUMO-fusion technology [3] to produce protein with an authentic N-terminus in high yield. Two immobilizedmetal ion-affinity chromatography steps with a protease cleavage step in between and subsequent size exclusionchromatography were utilized to purify the protein. The protein was characterized by SDS-PAGE, MALDI-TOFmass spectrometry, nano-HPLC/MALDI-TOF/TOF mass spectrometry and CD-spectroscopy. Furthermore, itsbiological activity was analyzed by an L929 cell assay.Ergebnisse und Diskussion: Using our production and purification protocols we attained two-fold higher proteinyields compared to existing methods [4]. The protein from the last chromatography step was obtained as a trimer,the actual biologically active form of TNF- α. Purity of the purified protein was at least 95% as estimated by SDS-PAGE. The identity of the protein and the formation of an intra-domain disulfide bond were confirmed by MALDI-TOF mass spectrometry. Recombinant mature TNF-α was correctly folded as assessed by CD spectroscopy. Usingan L929 cell assay the biological activity was shown to be in a range comparable to commercially available TNF-α. Although TNF-α is produced in the human body as a secreted protein and forms a homotrimer with one intra-chain disulfide bridge per monomer, we were able to produce a structurally and functionally intact protein bymeans of cytosolic expression in E. coli. The production of mature, bioactive TNF-α obtained with our protocolsis sufficient for experiments that demand abundant protein amounts, e.g. in a drug development process.Neue Aspekte: Combining recent expression improvement technologies to produce biologically active, trimericTNF-α at improved yields in the cytosol of E. coli.Referenzen: [1] 1 B. Huggett, J. Hodgson, R. Lahteenmaki, Public biotech 2008 - the numbers, Nat. Biotechnol.27 (2009) 710-721.; [2] 2 F.W. Studier, Protein production by auto-induction in high-density shaking cultures,Protein Expression Purif. 41 (2005) 207-234.; [3] 3 M.P. Malakhov, M.R. Mattern, O.A. Malakhova, M. Drinker,S.D. Weeks, T.R. Butt, SUMO fusions and SUMO-specific protease for efficient expression and purification ofproteins, J. Struct. Funct. Genomics 5 (2004) 75-86.; [4] 4 F. Curnis, A. Corti, Production and Characterization ofRecombinant Human and Murine TNF, Methods Mol. Med. 98 (2004) 9-22.Thema: Proteine und PeptideKeywords: tumor necrosis factor alpha, SUMO-fusion, cytosolic expressionKontakt: [email protected]

129

P40

Synthesis and characterization of partial ubiquitin contained alysine linked to Gly-Gly and affinity binding studies of lysine 63specific linkage polyubiquitin antibody using mass spectrometry

Cardenas, Vanessa; Jung, Ji Eun; Przybylski, Michael

University Konstanz, Germany

Einleitung: Ubiquity is a highly conserved 76 amino acid protein and is present in virtually all types of cells

as a monomer or as an isopeptide-linked polymer called polyubiquitin chains. Depending on the modification

of proteins by covalent attachment of ubiquitin has different biochemical and biological functions. This chain

formation is carried out by the covalent ligation of the ǫ-amino group of one of the each lysine ubiquitin sequence

to a glycin residue in the C terminus of a single ubiquitin moiety [1]. Recent evidences indicates that only the

function of K48 and K63 ubiquitin chains are well characterized, while very little is known about the function of

the other types of ubiquitin chains[2, 3].

Experimenteller Teil: In order to investigate the specificity of the K63specific linkage antibody, the Gly-Gly

linked to the e-amino group of lysine residues of partial ubiquitin peptide containing one of each K6, K11, K33,

K48 and K63 were synthetized by solid phase peptide synthesis and solid phase fragment condensation. The

reactions were monitored by analytical HPLC, and MALDI- and ESI-mass spectrometry (MS) [4].

Ergebnisse und Diskussion: The affinity studies between the synthetic partial ubiquitin and the K63 specific

linkage ubiquitin antibody were performed by Dot blot experiments and affinity chromatography, where the K63

linkage ubiquitin antibody was immobilized on sepharose column. After washing steps, the peptide bound to the

antibody was eluted and the unbound fractions were collected and analyzed by MALDI-TOF mass spectrometry.

The use of mass spectrometry in combination with affinity method is a suitable tool for the investigation of the

antibody affinity to different peptides.

Neue Aspekte: The use of mass-spectrometry in combination with affinity-method is a suitable-tool for the

investigation of the antibody-affinity to different peptides

Referenzen: [1] Pickart C.M and Fuschman D (2004) Polyubiquitin chains:polymeric protein signals Chemical

biology, 8, 610-616.; [2] Pickart, C.M. (2001) Mechanisms underlying ubiquitylation. Annu. Rev. Biochem.,

70, 503-533.; [3] Eddins M.J. Pickart C.M. (2004) Ubiquitin: structures, functions, mechanisms Biochimica et

Biophysica acta, 1695, 55-72.; [4] J.E Jung, H.P Wollscheid, M. Manea, M. Scheffner and M. Przybylski (2009)

Functional ubiquitin conjugates with lysine-ǫ-amino-specific linkage by thioether ligation of cysteinyl-ubiquitin

peptide building blocks. Bioconjugate Chemistry, 20, 1152-1162.


Keywords: Ubiquitin, K63, Affinity, ESI-MS


130

P41

Identifizierung disulfid-verbrückter Peptide mittels LC-MALDIgenerierten Fragmentionen Chromatogrammen

Merkel, Dietrich; Glueckmann, Matthias; Waidelich, Dietmar; Lenz, Christof

AB Sciex, Germany

Einleitung: Die intakte dreidimensionale Struktur von Proteinen ist essentiell für deren biologische Funktion.

Eine entscheidende Rolle für die Stabilität der Tertiärstruktur spielen hierbei die intramolekularen Disulfidbrücken.Die Struktur von Proteinen ist aber nicht immer unveränderlich, viele wichtige Prozesse wie z.B. Protein-ProteinInteraktion oder Enzym-Substrat Binding gehen oft einher mitÄnderungen der Tertiärstruktur, die durch das Lösenund Verknüpfen neuer Disulfidbrücken unterstützt wird. Aus diesem Grund ist die Identifizierung und Charakter-isierung von Disulfidbrücken ein sehr wichtiges Tool bei der Untersuchung dieser Prozesse mittels Massenspek-trometrie [1]. Wir zeigen hier einen Weg wie man nach LC-MALDI Analyse komplexer Peptidgemische ohneReduktion und Alkylierung vor tryptischem Verdau über Fragmentionen Chromatogramme intramolekulare Disul-fidverknüpfungen charakterisieren kann.Experimenteller Teil: Drei verschiedene Proteine (Lysozyme C, beta-Lactoglobulin und BSA) wurden unternicht-reduzierenden Bedingungen mit Trypsin verdaut. Die resultierenden Peptidmischungen wurden durch RPnano-LC mit einem kurzen 30 Minuten Gradienten getrennt. Das Eluat der Säule wurde nach Zumischung vonMALDI Matrix (α-Cyano 4-Hydroxy Zimtsäure) in Fraktionen von 20 Sekunden auf ein MALDI Target gespottet.Die getrennten Peptide wurden schließlich mit einem AB Sciex TOFTOFTM 5800 im MS mode und nach automa-tischer Precursor Selektion mittels MSMS analysiert. Ein theoretischer Trypsin-Verdau lieferte die molekularenMassen der Cystein-haltigen Peptide. Für diese Massen wurden in der PeakExplorerTM Software FragmentionenChromatogramme erstellt, die bezüglich Cysteinverbrückungen charakteristische Fragmentionen aufweisen.Ergebnisse und Diskussion: MALDI CID MSMS Spektren von disulfid-verknüpften Peptiden zeigen ein charak-teristisches Fragmentmuster. Typischerweise erkennt man zwei Peakgruppen, in deren Zentrum man jeweils dieMH+ Masse einer der beiden verknüpften Peptide sowie ein Ionensignal minus zwei Dalton detektiert, welchesdurch partielle Reduktion des Schwefels entsteht. Dies partielle Reduktion wurde bereits bei ISD Fragmenten vonCystein-verbrückten Peptiden detektiert [2]. Die PeakExplorerTM Software bietet die Möglichkeit FragmentionenChromatogramme zu erstellen. Aufgrund der charakteristischen Fragmentierung von Disulfid-verbrückten Pepti-den lässt sich durch die systematische Erstellung von Fragmentionen Chromatogrammen im MSMS Modus allercystein-haltiger Peptide und dem Vergleich der Retention der entsprechenden Signale sehr einfach die miteinanderin der Proteinstruktur verknüpften Sequenzen feststellen, wie anhand der untersuchten Proteine gezeigt werdenkonnte. Diese Methode würde ebenfalls bei einer intermolekularen Cystein-Verknüpfung funktionieren.Neue Aspekte: LC-MALDI MSMS Analyse disulfid-verbrückter Peptide Charakterisierung mittels Fragmentio-nen ChromatogrammenReferenzen: [1] Jones MD, Patterson SD, Lu HS. Anal Chem 1998; 70:136-143; [2] Quinton L, Demeure K.,Dobson R., Gilles N., Vale´rie Gabelica and De Pauw E. Journal of Proteome Research 2007, 6, 3216-3223Thema: Proteine und PeptideKeywords: Disufidbruecken, Fragmentionen Chromatogramme, MALDI, ProteinstrukturKontakt: [email protected]

131

P42

On the Way towards the Determination of Neoepitope-SpecificAntibody Signatures

Koy, Cornelia (1); Linnebacher, Michael (2); Konus, Metin (1); Lorenz, Peter (3); Klar, Ernst (2);

Thiesen, Hans-Jürgen (3); Glocker, Michael O. (1)

1: Proteome Center Rostock, University Rostock, Germany; 2: Division of Molecular Oncology and Immune

Therapy, University Clinical Center Rostock, Germany; 3: Institute of Immunology, Medical Faculty, University

of Rostock, Germany

Einleitung: Colorectal carcinoma is the second most frequent cancer type in Germany. Incidence is about 57,000

per year. The 5-year mortality rate is 50% and adjuvant immuno-therapeutic concepts are needed that support

the operative, chemo- and radiotherapy. So-called microsatellite instabilities (MSI) are observed in the majority

of patients with hereditary nonpolyposis colorectal cancer (HNPCC) [1,2]. Accordingly, proteins with abnormal

sequences are produced by cancer cells that serve as "neoantigens" which may be targets for the immune system[1,3]. Our concept aimes at the mass spectrometric characterization of specific antibody signatures in patients uponaffinity enrichment of antibody pools using "neoepitope peptides". These signatures shall be brought into clinicalcontext as a measure for the individual immune status against cancer-derived target structures.Experimenteller Teil: Tumor-associated epitopes in sera of colorectal carcinoma patients were identified usingpeptide chip screening methods with patient sera using a specifically designed "colon cancer" chip [4]. To selec-tively characterize "neoepitope-specific antibody signatures" biotinylated neoepitope peptides (ca. 15 amino acidslong) and a non-specific control peptide were synthesized and immobilized on streptavidin beads. Enriched anti-body pools from patient sera were subjected to 2-D gel electrophoresis and mass spectrometric peptide mapping.Bioinformatic gel image analysis determines whether antibodies enriched with "neoepitope" peptides possess char-acteristic profiles. In order to reduce background proteins, pre-enrichment of the antibody pool from patient serawas developed using protein A/G columns. The antibody pool was then subjected to functional analysis (WesternBlot) before generating individual neoepitope-specific antibody signatures.Ergebnisse und Diskussion: Fifty colorectal carcinoma sera and 20 control sera have been supplied for peptidechip analyses. With 323 independent measurements sera have been tested for the presence of colorectal carcinomaspecific signals on peptide chips. To develop a procedure for determining "neoepitope-specific antibody signatures"from sera of patients with colorectal carcinoma, several experimental steps had to be established. First, magneticstreptavidin beads were coated with biotinylated neoepitope peptides. For this purpose, three peptides (includinga non-specific control peptide) were selected from the peptide chip screening experiments. Synthesized peptideswere characterized by MS and MS/MS measurements. Then patient sera were loaded. After washing, eluatesfrom the beads were analyzed by 2-DE (pH 3-10 NL) and showed that antibody pools could be obtained yieldingin individual spot patterns on the gels. In order to reduce and/or remove background proteins, a pre-enrichmentprocedure was developed by which highly purified antibody pools were obtained. Hereto, plasma from patientswas loaded onto protein A/G columns individually, and antibodies were eluated either with glycine buffer or withacetic acid to generate individual antibody pools. Antibody amounts were typically in the range of 40% of thestarting material. These antibody eluates were also subjected to 2-DE and showed only spots for immunoglobulins(IgM, IgG, IgA). Serum albumin and other highly abundant plasma proteins were almost completely removed.Functional analysis of the eluted antibodies is performed by Western blot analysis using known antigens. Next, theso generated individual antibody pools are applied for capturing antibodies that are directed against neoepitopes,yielding in neoepitope-specific antibody signatures. These antibody signatures are now brought into clinicalcontext for assessing their diagnostic and prospective value, hopefully leading to better therapy opportunities forpatients suffering from colorectal carcinoma.Neue Aspekte: Characterization of individual neoepitope-specific antibody signatures as a measure for the indi-vidual immune status against cancer-derived target structures.Referenzen: [1] Schwitalle, Y., Kloor, M., Eiermann, S., Linnebacher, M., Kienle, P., Knaebel, H. P., Tariver-dian, M., Benner, A., von Knebel Doeberitz, M. Gastroenterology, 2008, 134 (4): 988-997.; [2] Thibodeau, S. N.,French, A. J., Cunningham, J. M., Tester, D., Burgart, L. J., Roche, P. C., McDonnell, S. K., Schaid, D. J., Vockley,C.W., Michels, V. V., Farr, G. H. Jr., O’Connell, M. J. Cancer Res. 1998, 58(8):1713-1718.; [3] Linnebacher,M., Wienck, A., Boeck, I., Klar, E., J. Biomed. Biotechnol. in press.; [4] Lorenz, P., Kreutzer, M., Zerweck, J.,Schutkowski, M., Thiesen, H.-J. in: Methods in Molecular Biology. Epitope Mapping Protocols, Vol. 524, Ed. byReineke, U., Schutkowski, M., Humana Press, Totowa, New Jersey, USA, p. 247 (2009).

132


Keywords: Neoepitope screening, antibody profiling, colorectal carcinoma, affinity chromatography, mass spec-

trometry


133

P43

Intein mediated cyclization of the C-terminal domain of humanγ-B-crystallin

Kilka, Susann (1); Müller, Mathias Q. (1); Sinz, Andrea (1); Weiwad, Matthias (2); Rudolph, Rainer (1); Pfeifer,

Sven (1)

1: Martin-Luther-Universität Halle/Wittenberg; 2: Max-Planck Forschungsstelle Enzymologie der ProteinfaltungHalle

Einleitung: Amide bond formation between the N- and C-termini of proteins resulting in covalent cyclizationrepresents a tool to increase protein stability, which is applicable to proteins with termini close in space [1]. Withthe help of a synthetic trans-splicing Synechocystis sp. PCC6803 DnaB split mini-intein the C-terminal domainof human γ-B-crystallin, a structural protein of the eye lens [2], was used as a model system for cyclization [3].Due to the association of the two flanking intein domains and subsequent intramolecular splicing the N-terminusof the target protein is joined to its own C-terminus. This leads to a stabilization of the γ-B-crystallin domain andimproves its applicability as a scaffold for the generation of artificial binding proteins.Experimenteller Teil: Protein overexpression was performed in BL21 cells using 1 mM IPTG. To purify linearand circular proteins the supernatant of a 60 % ammonium sulfate precipitation was loaded to a HiTrap ButylHP 1 ml column. Protein containing elution fractions were added to a HiLoad 16/60 Superdex 75 pg column (GEHealthcare). The identity of linear and circular proteins was determined by MALDI-TOF-MS in linear and positiveionization mode on an Ultraflex III instrument (Bruker Daltonik) using sinapinic acid as matrix. Tryptic peptidemixtures were analyzed by a MALDI-TOF/TOF mass spectrometer (Bruker Daltonik). Thermal denaturation wasmeasured using far UV CD spectroscopy at 215 nm from 20°Cto 95°Cand additional using the SyproOrange assaywhich was established in our group.Ergebnisse und Diskussion: The circular as well as the linear C-terminal domain of human γ-B-crystallin wereproduced in vivo in Escherichia coli using the synthetic Synechocystis sp. PCC6803 DnaB split mini-intein [1].Linear and circular proteins were constructed to be identical in amino acid sequence. Using linear mode MALDI-TOF mass spectrometry both intact domains were compared differing in molecular mass by only that of a singlewater molecule. However, the spectrum of the circular sample showed an impurity which was analyzed by trypticdigestion followed by MALDI-TOF/TOF mass spectrometry to be a linear byproduct of the cyclization reaction.This method showed clearly the separated N- and C-terminus of the linear variant whereas these sequences arelocalized at one peptide of the circular variant. While both variants exhibit a comparable secondary structurecomposed of mainly beta sheets the thermal stability of the circular variant was improved significantly compared tothe linear protein displayed by CD spectroscopy and SyproOrange stability assay. The circular C-terminal domainof human γ-B-crystallin is around 10 °Cmore stable than the linear variant. Stabilization of artificial bindingproteins or their targets have many potential applications in protein engineering and research, which includes thegeneration of affinity matrices or the coupling to surfaces of microchips.Neue Aspekte: Stabilizing scaffold proteins for the generation of artificial binding proteins by in vivo cyclization.Referenzen: [1] Williams N.-K., Liepinsh E., Watt S. J., Prosselkov P., Matthews J. M., Attard P., Beck J. L.,Dixon N. E., Otting G.; Stabilization of native protein fold by intein-mediated covalent cyclization; Journal ofMolecular Biology; 2005; 346; 1095-1108; [2] Ebersbach H., Fiedler E., Scheuermann T., Fiedler M., StubbsM. T., Reimann C., Proetzel G., Rudolph R., Fiedler U.; Affilin-Novel Binding Molecules Based on Human γ-BCrystallin, an All β-Sheet Protein; Journal of Molecular Biology; 2007; 372; 172-185; [3] Wu H., Xu M.-Q., LiuX.-Q.; Protein trans-splicing and functional mini-inteins of a cyanobacterial dnaB intein; Biochimica et BiophysicaActa; 1998; 1387; 422-432Thema: Proteine und PeptideKeywords: cyclization, DnaB split mini-intein, human γB crystallinKontakt: [email protected]

134

P44

Charakterisierung der antimikorbiellen Wirkung von Glycyrrhizinauf Bacillus subtilis

Urbanek, Dorota; Meyer, Björn; Karas, Michael

Goethe Universität, Germany

Einleitung: Die Zahl der Infektionskrankheiten verursacht durch multiresistente Erreger nimmt stetig zu. Dieschnelle Ausbildung von Resistenzen sowie der Mangel an neuen wirksamen Antibiotika limitieren die Behand-lungsmöglichkeiten dieser Krankheiten. Die Ansätze dieser Entwicklung entgegenzuwirken sind weitläufig. DurchdieWiderbesinnung auf pflanzliche Substanzquellen mit ihrem reichen Fundus an Sekundärstoffen ist dieMöglichkeitgegeben, neue antibiotische Substanzen zu finden oder die antimikrobielle Wirkung bekannter oder traditionellangewandter Substanzen zu verstehen. Die Süssholzpflanze (Glycyrrhiza glabra) verfügt über zahlreiche bioaktiveSekundärstoffe verschiedener Stoffklassen [1]. Für das in der Wurzel vorkommende pentazyklische TriterpenoidGlycyrrhizin und dessen Aglykon sollte in dieser Studie die antimikrobille Wirkung näher charakterisiert werden.Experimenteller Teil: Die Hemmung der Proteinbiosynthese durch Glycyrrhizin und dessen Aglykon wurdedurch einen in-vitro Transkritpions/Translations-Assay basierend auf dem RTS 100 E. coli HY Kit (Roche) un-tersucht. Desweiteren wurde die Wirksamkeit der Substanz gegen den gebräuchlichen Testkeim Bacillus sub-tlis 168 überprüft sowie die minimale Hemmkonzentration (MIC) und die subletale Konzentration ermittelt. DieDarstellung der Effekte auf Proteinebene erfolgte mit Hilfe von DIGE. Zu diesem Zweck wurde Bacillus subtilis168 in glycyrrhizin-haltigem und -freiem Minimalmedium angezogen und in der exponentiellen Wachstumsphasegeerntet. Nach Zellaufschluss und Extraktion der löslichen Proteinfraktion nach Büttner et al. [2] erfolgte dieMarkierung der Probe und Auftrennung mittels 2D-Gelelektrophorese. Signifikant regulierte Proteinspots wurdenausgeschnitten und mittels MALDI-Massenspektrometrie identifiziert.Ergebnisse und Diskussion: Die relative Assay-Inhibition [RAI %] des Glycyrrhizinaglykons im in-vitro Tran-skriptions /Translations-Assay gegen einen Streptomycinstandard betrug nahezu 80%. Glycyrrhizin hatte einenschwächeren Effekt, besitzt aber eine bessere Wasserlöslichkeit und wurde deshalb für das DIGE-Experiment ver-

wendet. Hierbei wurden mehrere regulierte Proteine identifiziert, welche für unterschiedliche metabolische Funk-

tionen der Zelle notwendig sind. Desweiteren konnte ein Protein identifiziert werden, welches bereits bei der Inku-

bation von Bacillus subtilis 168 mit Standardantibiotika als hochreguliertes Markerprotein verifiziert wurde [3].

Das Chaperon GroEL wurde schon bei den Aminoglykosidantibiotika Streptomycin, Gentamycin oder Kanamycin

als regulierte Proteinspezies detektiert. Alle drei Aminoglykoside hemmen die Proteinbiosynthese, indem sie sich

an die ribosomale 30S Untereinheit anlagern. Dies ist zusammen mit den Assay-Ergebnissen ein deutlicher Hin-

weise auf die Wirkungsweise von Glycyrrhizin und dessen Aglykon an dieser Stelle.

Neue Aspekte: Hemmender Effekt von Glycyrrhizin und dessen Aglykon auf die Proteinbiosynthese von Bacillus

subtilis . Identifizierung von Markerproteinen mittels DIGE.

Referenzen: [1] Asl et al, Phytotherapy Research 2008, 22: 709-724; [2] Büttner et al, Elektrophoresis 2001, 22:

2908 - 2935; [3] Bandow et al, Antimicrob Agents Chemother. 2003, 47: 948-955


Keywords: DIGE, Antibiotika, Proteinbiosynthese, Glycyrrhizin, Bacillus subtilis


135

P45

Automated enrichment and analysis of phosphopeptides fromENL via a biphasic pre-column with LC MALDI MS/MS

N. Arrey, Tabiwang (1); Baeumlisberger, Dominic (1); Baltruschat, Sabrina (1); Meyer, Bjoern (1); Marschalek,

Ralf (2); Karas, Micheal (1)

1: Cluster of Excellence "Macromolecular Complexes", Institute of Pharmaceutical Chemistry,Goethe-University, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany; 2: Institut of Pharmaceutical

Biology, Goethe-University, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany

Einleitung: Protein phosphorylation is a posttranslational modification, which reversibly regulates many pro-cesses in cells. A fundamentalunderstanding at the molecularlevel of these biological processes requires a char-acterization of the phosphorylation sites in proteins. We introduce a high throughput method for the generalstudy of phosphorylation and non-phosphorylation in protein without column switching. The phosphorylationsites inleukemia-related protein ENL were analyzed. ENL belongs to a protein-network that controls RNA-POLtranscription. Through the interaction with PTEFb, it is possible for the ENL complex to influence this networkand to regulate transcriptional processes. Here we present the first data on phosphorylation sites of ENL.Experimenteller Teil: After transfection in IP buffer (150 mM NaCl, 20 mM HEPES, pH 7.5, 1% Triton X-100, 1mMNa3VO4, 10 mMNaF, 1 mM PMSF, 1x Protease Inhibitor Cocktail V, EDTA free (Calbiochem), 1 x 109 293Tcells were lysed. Prior to lysis, 5 µM MG132 (Calbiochem) was added and the proteins were then purified usingSuperFlow Streptactin columns (IBA GmbH). After centrifugation the supernatant was incubated with Avidin,loaded onto the Gravity Flow Step-Tactin Superflow mini-column (IBA GmbH). Fractions containing ENL werereduced, alkylated and digested with trypsin. Digested sample were loaded on an in-house packed biphasic pre-column (TiO2-C18-beads). Peptides were separated on an in-house C-18 column using the Proxeon EASY-nLCTM

system coupled to a SunCollect MALDI spotter (SunChrom).Ergebnisse und Diskussion: With the use of the biphasic pre-column, we were able to separate and analyze bothnon-phosphorylated and phosphorylated peptides from ENL in a 2 step gradient without any column switching.During loading, the phosphopeptides were trapped on the TiO2 beads and the non-phosphorylated peptides onthe C-18 beads. During the first gradient, the non phosphorylated peptides were eluted from the C-18 materialof the biphasic pre-column, separated and spotted on a MALDI target. Prior to the second gradient, the trappedphosphorylated peptides were eluted with 18 ul 250 mM ammonium bicarbonate (pH 10.5), injected via sampleloop, onto the C18 beads of the biphasic pre-column and separated. Thirteen phosphopeptides covering 9 differentphosphorylation sites where identified. Six out of 9 phosphorylations sites predicted by Mascot and 3 novelphosphorylation sites were identified.Neue Aspekte: The LC-MALDI MS/MS characterization of phosphorylation sites in ENL using a biphasic pre-column.Thema: Proteine und PeptideKeywords: ENL-eleven-nineteen-leukemia protein ENL, P-TEFb Positive Transcription Elongation Factor bKontakt: [email protected]

136

P46

Effect of different ATM-Mutations upon the cellular responseafter ionizing radiation

Schirmer, Sophie; Assmann, Nadine; Reinders, Yvonne; Oefner, Peter J.

Institute of Functional Genomics, University Regensburg, Germany

Einleitung: Every year approximately 57.000 women are diagnosed with breast cancer in Germany and about

5-10% of them are inherited [1]. Beside mutations of BRCA1 and BRCA2 also ATM mutations may lead to an

increased breast cancer risk. ATM (Ataxia Telangiectasia Mutated) encodes for a 3056 AA serine-kinase which is

activated by phosphorylation in response to DNA damage such as double strand breaks, e.g. caused by ionizing

radiation. The activated ATM protein in turn phosphorylates target proteins involved in DNA repair and cell cycle

control. Missense mutations in the carboxyterminal domains of ATM (FAT, kinase and FATC domain) bear an

increased risk for breast cancer while heterozygous ATM protein truncating and splice junction variants, confer

only a marginal risk [2, 3].

Experimenteller Teil: The aim of our study is the analysis of the effect of homozygous nonsense and heterozygous

missense mutations on the cellular response after ionizing radiation (IR). At first, immunoblotting is used to

monitor the regulation of ATM and phosphorylated ATM (Ser1981), respectively with and without IR. To estimate

the effects of ATM activity after irradiation immunoblotting of the downstream targets p53 and phosphorylated p53

is accomplished. Afterwards the nuclear proteome of the irradiated and non-irradiated cells is compared using a

differential 2D-PAGE approach (DIGE) and regulated spots are identified using nano-LC-QTOF-MS/MS. Finally,

mRNA-expression levels will be analyzed on a Human Gene ST Array to complement the proteomics data.

Ergebnisse und Diskussion: EBV-transfected cell lines of breast cancer patients (with heterozygous 7271T>G

mutation), A-T patients (bearing homozygous mutations, but without breast cancer indisposition) and control

patients are subjected to gamma-irradiation with 3 Gy within 30 seconds. Further on, the cell cultures are incubated

for 6 hours and harvested afterwards. RNA is extracted from a small aliquot of the samples and total cell lysate

of a further aliquot is subjected to immunoblotting against ATM and its downstream targets. The immunoblot

analyses show a decrease in the amount of ATM and phosphorylated ATM protein in the heterozygous as well as

in the homozygous cell lines in comparison to the control cell lines. Furthermore, in the homozygous cell lines

a shift of the ATM band to lower molecular weights is observed. This indicates the presence of a truncated form

of the ATM protein in these cells. However, the majority of each sample is used for subcellular fractionation

into nuclei and residual cellular lysate (RCL) for the 2D-DIGE analysis. Therefore, a two-step protocol has been

established consisting of a protein precipitation and a subsequent phenol-extraction to further purify the nuclear

and RCL proteome. Purity control is accomplished by immunoblotting against several marker proteins of different

compartments such as phosphoglycerate kinase (cytsosol), lamin A (nucleus) and calreticulin (endoplasmatic

reticulum). The high-resolution 2D-DIGE analysis of the nuclear proteome carried out on 24x20 cm gels (pH

3-10, 12,5% PA-gel) is currently ongoing. The image acquisition is done on a VersaDoc MP4000 and the gels will

be analyzed with the Progensis SameSpots software for differential analysis.

Neue Aspekte: Analysis of the Impact of ATM mutations on irradiation-induced DNA-damage for a comprehen-

sive view on breast cancer carcinogenesis.

Referenzen: [1] Honrado E. et al., Mod Pathol. 2005 Oct;18(10):1305-20.; [2] Tavtigian SV. et al., Am J Hum

Genet. 2009 Oct;85(4):427-46.; [3] Thorstenson YR. et al., Cancer Res 2003, 63:3325-33.


Keywords: ATM, Breast Cancer, Proteomics, DIGE


137

P47

Targeted Phosphorylation Site Analysis by MRM

Haaf, Erik; Lanner, Ulrike; Lamer, Stephanie; Schlosser, Andreas

Zentrum für Biosystemanalyse (ZBSA), Uni Freiburg, Germany

Einleitung: Phosphorylation site analysis by data-dependent LC-MS/MSmethods often leads to ambiguous results

concerning the exact position of the phosphorylation site, especially if the potential sites are very close to each

other. This is most often due to low quality (e.g. intense neutral loss of phosphoric acid) or to low intensity

of the phosphopeptide MS/MS spectra. Another challenge when analyzing phosphorylation sites is the detection

of multi-phosphorylated peptides. One major reason for that is the selective loss of these multi-phosphorylated

peptides during sample preparation and HPLC separation [1]. In addition, bad fragmentation behavior (multiply

neutral losses) and formation of complex mixtures (e.g. a peptide with 5 phosphorylation sites results in 32 different

peptide species) hamper the analysis of multi-phosphorylated peptides.

Experimenteller Teil: LC-MS/MS analyses were performed on an Agilent 6460 triple quadrupole mass spectrom-

eter and on an Agilent 6520 Q-Tof mass spectrometer (Agilent Technologies, Santa Clara, USA). Both instruments

were coupled to an 1200 Agilent nanoflow system via a HPLC-Chip cube ESI interface. Peptides were separated

on a HPLC-Chip with an analytical column of 75 µm i.d. and 40 mm length and a 40-nL trap column, both packed

with Zorbax 300SB C-18 (5 µm particle size). Peptides were eluted with a linear acetonitrile gradient with 2

%/min at a flow rate of 300 nL/min.

Ergebnisse und Diskussion: We have developed a MRM-based strategy for the targeted analysis of phosphory-

lation sites. This strategy is applicable for phosphorylation site pinpointing within identified phosphopeptides, as

well as for the directed searching of multi-phosphorylated peptides. For phosphorylation site pinpointing fragment

ions are selected that allow distinguishing between the different possible phosphorylation sites. Proline-directed

fragment ions between potential phosphorylation sites are preferred if available. By focusing on the relevant frag-

ment ions this MRM-based strategy is much more sensitive than any data-dependent MS/MS approach and thus

often allows reliable phosphorylation site pinpointing even with very low amount of phosphopeptide. The same

strategy allows searching for higher phosphorylated species of already detected phosphopeptides. The neutral loss

of one molecule of phosphoric acid from the precursor is often the dominating fragmentation reaction for Ser/Thr-

phosphorylated peptides, especially for multi-phosphorylated peptides. Thus, this neutral loss-fragment is a good

choice for a MRM transition. Scanning for higher phosphorylated species is then simply done by shifting the

MRM transition (precursor and fragment m/z) by one (or more) phosphate(s) (80 Da/z).

Neue Aspekte: New strategy that allows the targeted detailed analysis of phosphorylation sites using MRM

Referenzen: [1] [1] Winter et al. J. Proteome Res., 2009, 8: 418-424. Citrate Boosts the Performance of

Phosphopeptide Analysis by UPLC-ESI-MS/MS.


Keywords: MRM, phosphorylation sites, phosphopeptides


138

P48

Isolation und massenspektrometrische Charakterisierung vonhumanem Hautelastin

Jung, Michael (1); Heinz, Andrea (1); Kiesow, Andreas (2); Wohlrab, Johannes (3); Neubert, Reinhard (1);

Schmelzer, Christian (1)

1: Martin-Luther-Universität Halle-Wittenberg, Halle (Saale); 2: Fraunhofer-Institut für Werkstoffmechanik,Halle (Saale); 3: Universitätsklinikum der Medizinischen Fakultät der MLU Halle-Wittenberg, Halle (Saale)

Einleitung: Elastin ist gemeinsam mit anderen Strukturproteinen Bestandteil der extrazellulären Matrix (EZM)und kommt in den elastischen Fasern vieler Organe vor, unter anderem in der Lunge, der Haut und den Blut-gefäßen. Verringerungen der Elastizität des Körpergewebes, die mit dem Älterwerden einhergehen oder infolgeschwerwiegender Erkrankungen auftreten können, sind irreversibel. Um degenerative Veränderungen des Elastinsnachvollziehen und später geeignete Therapien entwickeln zu können, ist ein genaues Verständnis der auf morphol-ogischer und molekularer Ebene stattfindenden Prozesse erforderlich. Da intaktes Elastin jedoch in Wasser undorganischen Lösungsmitteln unlöslich ist, ist es für Untersuchungen nur schwer zugänglich. Zielstellung dieser Ar-beit war es daher, Methoden zu erarbeiten, die es erlauben, Elastinfasern unbeschadet aus einzelnen Hautbiopsienzu isolieren und diese mittels Elektronenmikroskopie und Massenspektrometrie zu charakterisieren.Experimenteller Teil: Stanzbiopsien von Humanhaut wurden mit organischen Lösungsmitteln, reduktiven undchaotropen Substanzen sowie Trypsin derart behandelt, dass andere Matrixbestandteile, insbesondere Kollagen,das ebenfalls Strukturprotein der EZM ist und häufig als Verunreinigung bei der Elastingewinnung auftritt, ent-fernt werden konnten (modifiziert nach [1]). Die Beschaffenheit der verbliebenen Elastinfasern wurde mittelsSEM untersucht. Nach Hydrolyse des Elastins unter Verwendung von Elastasen wurden die Peptide an einemNano-LC-Nano-ESI-qTOF-System vermessen, Tandem-MS-Experimenten unterzogen und unter Verwendung vondatenbank- und de-novo-basierter Sequenzierung identifiziert.Ergebnisse und Diskussion: Im Vergleich zu den in der Vergangenheit verwendeten, teilweise recht destruk-tiven Verfahren, die zur Aufreinigung von Elastin aus Geweben eingesetzt werden, konnten mit den in dieserArbeit beschriebenen Methoden intakte Fasern isoliert werden. Da das Elastin weitestgehend unbeschadet isoliertwurde, stellt es ein sehr interessantes Substrat für enzymologische Studien zum Spaltverhalten und zur Substrat-spezifität von Elastasen dar. Anhand von SEM-Aufnahmen wird deutlich, dass Elastin in der Haut ein dichtes,netzartiges Fasergeflecht ausbildet. Die optische Bestimmung der Faserintegrität und -qualität soll zur Charakter-isierung von morphologischen Veränderungen im Elastingewebe herangezogen werden. In der Natur tritt Elastinin mehreren Isoformen auf, die posttranskriptional aus Spleißvarianten der prä-mRNA resultieren. Das Vorhan-densein bzw. das Ausspleißen von bestimmten Exons geht mit Strukturveränderungen des elastischen Gewebeseinher und scheint mit bestimmten pathophysiologischen Geschehen assoziiert zu sein. Ausgehend von einzelnenHautbiopsien konnte in dieser Arbeitdie Abwesenheitder durch Exon 26A kodierten Aminosäuren auf Proteinebeneindeutig bestimmt werden. Diese in vielerlei Hinsicht außergewöhnliche Domäne tritt insbesondere im Elastinlichtgeschädigter Haut auf [2]. Des Weiteren spielen bei der Charakterisierung der molekularen Veränderungenelastischer Fasern posttranslationale Modifikationen eine große Rolle. Während der Biosynthese des Elastins wer-den Prolinseitengruppen partiell hydroxyliert. Dabei ist der Hydroxylierungsgrad vermutlich abhängig von denphysiologischen Bedingungen des Gewebes. Mit Hilfe von isoliertem Elastin lassen sich solche Veränderungenin der Primärstruktur, die im Zusammenhang mit verschiedenen Krankheiten sowie Alterungsprozessen stehen,auf biochemischer Ebene untersuchen und mittels massenspektrometrischer Verfahren bestimmen. Die Charakter-isierung von nativem Elastin kann zum Verständnis pathologischer Vorgänge entscheidend beitragen und die Suchenach Therapiemöglichkeiten erleichtern.Neue Aspekte: Die Isolation von nativem Elastin aus Humanhaut bietet neue Möglichkeiten der Charakterisierungelastischer Fasern unter Verwendung massenspektrometrischer und elektronenmikroskopischer Methoden.Referenzen: [1] Daamen, W. F., Hafmans, T., Veerkamp, J. H. und van Kuppevelt, T. H. (2005) Isolation of intactelastin fibers devoid of microfibrils. Tissue Eng 11 (7-8): 1168-1176.; [2] Chen, Z., Shin, M. H., Moon, Y. J., Lee,S. R., Kim, Y. K., Seo, J. E., Kim, J. E., Kim, K. H. und Chung, J. H. (2009) Modulation of elastin exon 26AmRNA and protein expression in human skin in vivo. Exp Dermatol 18 (4): 378-386.Thema: Proteine und PeptideKeywords: Elastin, Gewebeextraktion, Elastasen, qTOF-MS, Nano-ESIKontakt: [email protected]

139

P49

Structure, reaction intermediates and topographicalcharacterization of ß-amyloid oligomerisation revealed by ion

mobility mass spectrometry and electron paramagnetic resonancespectroscopy

Iurascu, Marius Ionut (1); Cozma, Claudia (1); Desor, Michael (2); Drescher, Malte (1); Przybylski, Michael (1)

1: Universitaet Konstanz, Germany; 2: Waters GMBH

Einleitung: An increasingly proportion of elderly population is affected by progressive neurodegenerative disor-

ders like Alzheimer’s disease (AD). The major characteristic of AD is the accumulation of high molecular weight

aggregates and plaques in the brain, which comprise the ß-amyloid polypeptide, beta-amyloid (Aß), as a major

component. Aß is a 39-43 aa peptide that is formed proteolytically from the large ß-amyloid transmembrane

precursor protein (APP) and spontaneously aggregates to Aß-tangles and Aß-fibrils. Recently, the formation of

Aß-oligomers has attracted particular interest, since oligomers have been suggested to be a key neurotoxic species.

Experimenteller Teil: For structural studies of Aß-oligomers by (i) mass spectrometric proteome analysis and (ii)

the characterization of aggregates using electron paramagnetic resonance (EPR) spectroscopy, Aß(1-40) and Cys-

Aß(1-40) were synthesized using solid phase peptide synthesis, Fmoc chemistry, and the crude peptides purified by

HPLC [1]. On the N-terminal cysteine a 3-(2-iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy radical spin

label derivative (IPSL) was attached by S-alkylation using overnight incubation [2]. The peptide derivatives with

and without spinlabel were characterized by electrospray (ESI) mass spectrometry. After five days of incubation

at 37 oC [3], oligomers and fibrils were produced and separated by centrifugation. Further characterization was

performed by Tris-Tricine PAGE, and by EPR spectroscopy.

Ergebnisse und Diskussion: The ESI mass spectrometric analysis ascertained the successful synthesis of the

peptide containing the spin label derivative. The soluble oligomers characterized by ion mobility mass spectrom-

etry (IM-MS) using a Waters-Synapt system revealed the presence of at least two conformational forms, and the

Aß-Met36 -sulfon oxidation product. The EPR spectra of the spin-labeled Cys-Aß(1-40) oligomers were matched

with spectra simulations using a multi-component simulation strategy, resulting in complete agreement with the

gel electrophoresis results. In the freshly prepared Aß(1-40) solution, oligomerisation was found to start after

only a few hours, while the insoluble fibril suspension consist of large components and the supernatant contained

predominantly the monomer. ESI-MS and EPR spectroscopy represent a new combined approach for composition

determination of ß-amyloid oligomerisation fractions, and the molecular dimension of aggregates.

Neue Aspekte: ESI-MS and EPR-spectroscopy represent a new approach for composition determination of ß-

amyloid oligomerisation fractions, and the molecular dimension of aggregates.

Referenzen: [1] Iurascu M.I., Cozma C., Tomczyk N., Rontree J., Desor M., Drescher M., Przybylski M., Anal

Bioanal Chem 2009, 8:2509-19; [2] Gettins P., Beth A.H., Cunningham L.W., Biochemistry. 1988, 27(8):2905-11;

[3] Harry LeVine et al., Biochemistry 2005, 44,15937-15943


Keywords: Ion Mobility MS, EPR, ß-Amyloid, aggregates


140

P50

Characterization of Neutral and Acidic Glycopeptides byZIC-HILIC Enrichment and Mass Spectrometry

Meyer-Posner, Franz (1); Wohlgemuth, Jessica (2); Andrecht, Sven (2); Schneider, Andrea (1); Resemann, Anja

(1)

1: Bruker Daltonik GmbH, Bremen, Germany, Germany; 2: Merck KGaA, Darmstadt, Germany

Einleitung: Glycosylation is the most abundant protein posttranslational modification and is involved in many

relevant biological processes and crucial to the understanding of many diseases. In depth analysis of glycosylation

sites is difficult, however, as glycopeptides exhibit a significant micro heterogeneity at glycosylation sites. In

addition, ion suppression effects require selective methods for glycopeptide enrichment. Mass spectrometric

analysis of the two distinct glycopeptide moieties is challenging because both the peptide as well as the glycan

moiety have to be elucidated for a full structural assignment.

Experimenteller Teil: We used Fetuin, Alpha-1-Acidglycoprotein and Asialo-Fetuin to equally representing sia-

lylated and non-sialylated glycosylic structures. In addition, monoclonal antibodies were analyzed as a dedicated

example for pharmaceutical QC. Samples were digested with trypsin and glycopeptides were enriched using a

dedicated ZIC glycocapture resin in combination with an optimized buffer system (EMD Chemicals Inc.). Subse-

quently, the glycopeptides were analyzed using ESI ion trap MS for glycoprofiling and MALDI-TOF/TOF-MS for

in depth characterization of the glycopeptides.

Ergebnisse und Diskussion: In MALDI-TOF/TOF instruments, N-linked glycopeptides undergo an indicative

cross-ring fragmentation of the GlcNAc that links to the Asn. Indicative fragments allow obtaining the peptide

mass safely as well as the glycan composition. Subsequent Mascot searches of the glycopeptide MS/MS spectra

provided for the sequence of the glycopeptide and the localization of the glycosylation site. Searches in glycan

databases based on the same glycopeptide MS/MS spectra allowed to complete the characterization of N-linked

glycopeptides. For the first time, MALDI-TOF/TOF-MS analysis of multi-sialylated glycopeptides was shown

after linear positive ion mode detection of the respective precursor ions.In comparison to direct MS analysis of

glycoprotein digests, the enriched samples allowed to detect more glycopeptides and permitted the acquisition of

MS/MS spectra of higher quality. Additional separation techniques prior to MS analysis such as RP-HPLC may

allow even greater insights into the complexity of protein glycosylation.

Neue Aspekte: Targeted characterization of glycopeptides by combined extraction and peptide and glycan identi-

fication

Thema: Kohlenhydrate, Proteine und Peptide

Keywords: MALDI-TOF, glycopeptides, HILIC, enrichment


141

P51

Massenspektrometrischer Nachweis von Kollagen undGlykosaminoglykanen als Hauptbestandteile der extrazellulären

Matrix in Knorpel und artifiziellen Knorpelkonstrukten

Nimptsch, Ariane; Nimptsch, Kathrin; Süß, Rosmarie; Schiller, Jürgen

Universität Leipzig - Medizinische Fakultät, Institut für Medizinische Physik und Biophysik, Deutschland

Einleitung: Kollagene und Glykosaminoglykane (GAG), wie Chondroitinsulfat (CS) und Hyaluronan (HA) sinddie Hauptbestandteile der extrazellulären Matrix (EZM) von Knorpel, Knochen und Haut. Kollagene und GAGbesitzen wichtige, zugleich aber sehr unterschiedliche Funktionen, so dass die mengenmäßige Zusammensetzungfür die einwandfreie Funktion eines Gewebes entscheidend ist. Ihr qualitativer und quantitativer Nachweis ist de-shalb gerade bei biotechnologisch generierten Geweben von höchster Wichtigkeit [1]. Obwohl bereits Arbeitenzur massenspektrometrischen Charakterisierung der nativen Kollagene vorgelegt wurden [2], ist eine quantita-tive Auswertung dieser Daten kaum möglich. Eine Charakterisierung der intakten Polysaccharide ist aufgrunddes Polyelektrolytcharakters unmöglich [3]. Ziel der Arbeit war deshalb zu untersuchen, inwieweit die durch(möglichst einfachen) enzymatischen Verdau erhaltenen Abbauprodukte mittels MALDI-TOF MS detektiert undquantifiziert werden können.Experimenteller Teil: Neben kommerziell verfügbaren Oligopeptiden und von CS- bzw. HA-abgeleiteten Disac-

chariden, kam natives Kollagen (Typ I aus Achillessehne) sowie nasaler Rinderknorpel zur Anwendung. Der Ver-

dau des Kollagens erfolgte mit Clostridiopeptidase A, wobei für alle weiteren Auswertungen von den entstandenen

Produkten ausschließlich das Tripeptid Gly-Pro-Hyp betrachtet wurde. Die erhaltenen Lösungen wurden direktnach Zusatz eines geeigneten Tripeptid-Standards (Arg-Gly-Asp; m/z 347.2) mittels MALDI-TOF MS (Bruker

Autoflex I Massenspektrometer, Reflektormodus, Positivionendetektion) charakterisiert. Zur Bestimmung des CS-

Anteils wurde die Probe mit Chondroitinase ABC verdaut und das resultierende Disaccharid anschließend mittels

Negativionen MALDI-TOFMS charakterisiert. Die Positivionendetektion erfolgte mit 2,5-Dihydroxybenzoesäure(DHB) als Matrix, während für die Negativionendetektion 9-AA (9-Aminoacridin) Anwendung fand.Ergebnisse und Diskussion: Im Vergleich mit anderen Proteasen, wie z.B. Trypsin, ist nur ClostridiopeptidaseA ("Kollagenase") in der Lage, native, wasserunlösliche Kollagene zu hydrolysieren. Für gewöhnlich bevorzugtman jedoch den tryptischen Verdau (nach vorangegangener thermischer Denaturierung des Kollagens), um spez-ifischere Proteinfragmente zu erhalten, die auch eine Unterscheidung der einzelnen Kollagentypen ermöglichen.Obwohl Clostridiopeptidase A das Kollagen nur unspezifisch spaltet, kann das beim Verdau entstandene Gemischvon Tripeptiden als Maß für den Kollagengehalt verwendet werden. Wir konzentrierten uns dabei auf den MALDI-TOF massenspektrometrischen Nachweis des Tripeptids Gly-Pro-Hyp, da dieses Peptid aufgrund seiner Zusam-mensetzung als besonders kollagentypisch angesehen werden kann. Ein Nachteil bei der Anwendung der MALDI-TOF MS liegt in den relativ geringen Molekulargewichten der relevanten Tripeptide, wodurch es leicht zu Über-lagerungen zwischen Analyt- und Matrixionen kommt. Dies ist ein Grund, warum ausschließlich DHB (2,5-Dihydroxybenzoesäure) als Matrix für die Positivionendetektion verwendet wurde, da DHB im relevanten Massen-bereich einen weit geringeren Hintergrund als Zimtsäurederivate ergibt. Initial wurden Gemische synthetischer,kollagensequenztypischer Tripeptide untersucht, wobei das Signal des Tripeptids Gly-Pro-Hyp (m/z 286,1 für dasH+ Addukt) als besonders geeignet erachtet wurde. Die quantitative Auswertung einer Verdünnungsreihe diesesPeptids ergab eindeutig eine Korrelation, wenn man die Intensität relativ zur Intensität eines Standardpeptids (Arg-Gly-Asp; m/z 347,2) auftrug. Diese Methode kann somit zum quantitativen Kollagennachweis verwendet werden.Um Überlappungen unterschiedlicher Addukte reduzieren zu können, erfolgte die zusätzliche Aufnahme von Neg-ativionenspektren, wobei 9-Aminoacridin (9-AA) als Matrix Anwendung fand. Obwohl die Zuordnung bei denNegativionenspektren einfacher ist, ist die Detektion leider weniger sensitiv und daher zur Quantifizierung gerin-gerer Kollagenmengen ungeeignet. Negativionendetektion ist jedoch das Verfahren der Wahl zum Nachweis vonCS-Abbauprodukten. Wir zeigen, dass hier sogar auf einen (bislang nicht verfügbaren) Standard verzichtet werdenkann und allein das Signal-Rausch-(S/N)-Verhältnis für quantitative Untersuchungen ausreichend ist [4].Neue Aspekte: Dies sind nach unserem Wissen die ersten Arbeiten zur quantitativen, massenspektrometrischenAnalyse von Kollagen, im Anschluß an einen Kollagenaseverdau.Referenzen: [1] R. Schulz et al., J. Nanosci. Nanotechnol. 6 (2006) 2368-2381.; [2] K. Dreisewerd et al., Anal.Chem. 76 (2004) 3482-3491.; [3] J. Schiller et al., Curr. Org. Chem. 10 (2006) 1771-1789.; [4] A. Nimptsch etal., Anal. Chim. Acta 635 (2009) 175-182.

142

Thema: Kohlenhydrate, Proteine und PeptideKeywords: Kollagen, Clostridiopeptidase A, Chondroitinsulfat, Chondroitinase ABC, MALDI-TOF MS.Kontakt: [email protected]

143

P52

Highly Accurate Phosphorylation Degree Determination byMetamorphic Pairs of Stable Isotope Labeled Standards

Hahn, B. (1); D’Alessandro, L. (2); Raia, V. (2); Depner, S. (3); Müller, M. (3); Zinn, N. (1); Klingmüller, U. (2);

Lehmann, W.-D. (1)

1: Molecular Structure Analysis, German Cancer Research Center, Heidelberg; 2: Systems Biology of Signal

Transduction, German Cancer Research Center, Heidelberg; 3: Tumor and Microenvironment, German Cancer

Research Center, Heidelberg

Einleitung: Analytical proteomics by mass spectrometry currently expands to quantitative analyses, since these

give detailed insights into functional aspects of proteins and their networks. Therefore, numerous quantitative

proteomic methods have been developed over the last years [1]. Phosphorylation degree determination can be

performed at an approximate level without the use of internal standards [2] or by differential stable isotope labeling

in combination with phosphatase treatment [3]. Here a novel method is described for site-specific phosphorylation

degree determination, which is based on the addition of a dual stable isotope labeled standard, so that both the

peptide and the corresponding phosphopeptide cognate are individually standardized. The standard pairs are

characterized by a highly correct molar ratio due to a metamorphic production principle.

Experimenteller Teil: Protein bands were processed following a standard in-gel digestion procedure. Combined

peptide mixtures and standards were analyzed by nanoUPLC (nanoAcquity, Waters) on-line coupled to a LTQ-

Orbitrap mass spectrometer (Thermo) and operated in the data-dependent mode. Survey full scan MS spectra (m/z

350 to 2000) were acquired in the Orbitrap at resolution R = 60 000. The top three ions with charge state ≥ 2 were

fragmented in the LTQ using CID at normalized collision energy of 35. Stable isotope labeled phosphopeptide

standards were prepared using standard Fmoc strategy. Phosphopeptides were dephosphorylated by antarctic

phosphatase, which was then irreversibly heat-inactivated. Standard pairs were mixed from these equimolar stock

solutions at the desired ratio on a volumetric basis.

Ergebnisse und Diskussion: An innovative method for highly accurate, site-specific phosphorylation degree de-

termination by nanoLC-ESI-MS/MS is described. The workflow has some similarity to the preparation of ab-

solutely quantified peptides [Zinn 2010], however, in this study the concept does not require absolute peptide

quantification, but works on a basis of purely relative quantification. The method determines the molar ratio of

peptide/phosphopeptide pairs generated by digestion of phosphoproteins, as obtained in a standard ‘bottom-up’

workflow of analytical proteomics. A stable isotope labeled pair of peptide/phosphopeptide standards with exactly

known molar ratio is used as reference, providing an individual standard for both the target peptide and phos-

phopeptide. The standards are denominated ‘metamorphic’ standard pairs to express that one component - the

labeled peptide - originates from the other - the labeled phosphopeptide. The quantitative metamorphic step per-

formed by enzymatic dephosphorylation assures that the original concentrations of both standard components are

identical, independent of their absolute concentration. By selection of the volumetric mixing ratio, metamorphic

peptide/phosphopeptide standard pairs of any molar ratio can be prepared. It is demonstrated that metamorphic

standard pairs deliver highly accurate phosphorylation degree data both in single point and kinetic measurements

applied to signaling proteins such as ERK1, ERK2, and STAT6. One proof of correctness was obtained by deter-

mination of a site-specific phosphorylation degree in a sample of STAT6 using digestion by AspN or combined

use of AspN+ LysC, respectively, which gave an identical degree of phosphorylation based of two different prote-

olytic peptides. Another proof was obtained by recording an activation kinetic of ERK, which could be following

quantitatively in spite of a maximal degree of phosphorylation of about 10% only.

Neue Aspekte: Production of stable isotope labeled metamorphic peptide/phosphopeptide standards and their use

for accurate phosphorylation degree determination in signaling proteins.

Referenzen: [1] Bantscheff M, Schirle M, Sweetman G, Rick J, Kuster B. Anal Bioanal Chem 2007, 389, 1017-

1031.; [2] Seidler J, Adal M, Kübler D, Bossemeyer D, LehmannWD. Anal Bioanal Chem 2009, 395, 1713-1720.;

[3] Zhang X, Jin QK, Carr SA, Annan RS. Rapid CommunMass Spectrom 2002, 16, 2325-2332.; [4] Zinn N, Hahn

B, Pipkorn R, Schwarzer D, Lehmann WD. J Proteome Res 2009, 8, 4870-4875.


Keywords: phosphorylation degree, stable isotope labeling, dephosphorylation, metamorphic standard pairs,

phosphorylation kinetic


144

P53

Mass spectrometrical analysis of oxidative modifications in ratskeletal muscle proteins

Fedorova, Maria (1); Kuleva, Nadezda (2); Hoffmann, Ralf (1)

1: Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, Faculty of Chemistry andMineralogy, University of Leipzig, Germany; 2: Department of Biochemistry, Faculty of Biology and Soil

Science, St. Petersburg State University, Russia

Einleitung: A number of human diseases, including Alzheimer’s disease, Parkinson‘s disease and diabetes mel-litus, are strongly connected to oxidative stress caused by reactive oxygen species (ROS). ROS can oxidize mostbiomolecules, especially proteins present in high concentrations in the cells. The resulting oxidation of certainamino acid residues can alter the protein structure, which results in a loss of functional activity.Experimenteller Teil: To model oxidative stress in vivo, rats were irradiated by X-ray (5 Gy dose). Proteinswere isolated from skeletal muscle tissues 3, 9 and 24 hours after irradiation. These protein extracts as well as aprotein extract from non-irradiated rats were separated by gel electrophoresis in order to reduce sample complexity.Protein bands of interest were excised, digested with trypsin, and analyzed by nanoUPLC-Orbitrap-MS. Optimizedgas phase fractionation (GPF) was used to improve the detection limits and to sequence more peptides carryingoxidative modifications.Ergebnisse und Diskussion: With GPF technique up to 16 different muscle proteins were identified in eachband of the SDS-PAGE. Additionally, modifications were identified in 14 muscle proteins at tryptophan (kynure-nine, hydroxytryptophan, N-formylkynurenine, hydroxyN-formylkynurenine), in 13 proteins at cysteine (disul-fides, sulfenic, sulfinic and sulfonic acids) and in 15 proteins at methionine residues (sulfoxide and sulfone). Allmodifications were confirmed by tandem mass spectrometry of corresponding peptides. Modified proteins belongto various functional groups and responsible for muscle contraction (actin, myosin light and heavy chains, tro-ponin), energy production (different subunits of ATP synthase complex) and carbohydrate metabolism (glycoliticand TCA cycle enzymes).Neue Aspekte: optimized gas fractionation technique coupled with nanoUPLC-Orbitrap_MS for sensitive identi-fication of muscle proteins oxidation in in vivo animal modelThema: Proteine und PeptideKeywords: Orbitrap, gas phase fractionation, oxidative modificationsKontakt: [email protected]

145

P54

The Role of Increased Resolution and Scan Speed of Ion Traps forTop-Down Proteomics with ETD/PTR

Falter, Ralf; Albers, Christian; Brekenfeld, Andreas; Gebhardt, Christoph; Hartmer, Ralf


Einleitung: Dedicated MS/MS-techniques for top-down proteomics are electron-induced fragmentation processeslike electron capture or electron transfer dissociation. However, the analysis of ETDMS/MS data of highly chargedproteins can be rather complicated, because a plethora of multiply charged and overlaid fragment ions can beexpected. The complexity of the ETD MS/MS-data is significantly reduced when the initial ETD-step is followedby a subsequent proton transfer reaction (PTR) reducing the charge states of the multiple charge fragments. Forcommon ion trap instruments, the charge reduction step is typically optimized leading to singly and doubly chargedfragments. An increase in resolving power of the MS-instrument is strongly demanded if highly charged ETD-fragments (z > 4) have to be analyzedExperimenteller Teil: All measurements were carried out on a modified Bruker ion trap. An improved controlof the non-linear ejection process and the development of the trap environment support faster scan modes aswell as a higher mass resolution. The previously purified proteins were introduced into the ion trap with offlinenanospray. ETD/PTR of the isolated protein is performed with reagent anions dedicated for either ETD or PTR.The formation of the different reagent anion is accomplished from only one neutral compound by altering thevoltage of the negative chemical ionization source.Ergebnisse und Diskussion: We investigate the role of the increased resolution of a modified ion trap massspectrometer on the top-down sequence analysis of larger peptides and proteins with ETD/PTR. The unmatchedresolution of common Bruker ion traps at scan speeds of 8,000 Th/sec is ideally suited for the identificationof fragment ions with charge states equal or less than z = 3. For the sequence analysis of intact proteins withETD/PTR MS/MS, the subsequent charge reduction step with PTR has to be tuned for generating mainly singly,doubly and triply charge fragments. One major drawback of the charge reduction step is the decrease of the totalion signal after the PTR-step. With each proton transfer the m/z of an ETD-fragment will be shifted towards highermasses. Any highly charged ETD-fragment will get out of scope if the final m/z of the ETD-fragment exceeds themaximum accessible m/z of the instrument. The higher accuracy of the resonant ejection process of the modifiedion trap system allows a fivefold faster scan speed compared to the established maximum mode. With the resultingresolving power of the modified ion trap instrument ETD-fragments having five or even six charges are isotopicallyresolved (peak width ∼ 0.1 Th). The scan speed above 4000 Th/sec and the high charge capacity of the modifiedion trap system enables the fast identification of ETD-fragments within a seamless scan (scan range 100-3000). Wewill present the impact of the increased resolution on the sequence analysis of intact proteins (molecular weight upto 16 kDa). With the increased resolution we were able to sequence a purified biomarker protein which has beenpreviously discovered via MALDI imaging of breast cancer tissues.Neue Aspekte: Ionenfalle mit 20,000 Massenauflösung, 52,000 u/s Scan speed und ETD/PTR Fragemtierung für

Proteomikanwendungen

Thema: Instrumentelle Entwicklungen, Proteine und Peptide

Keywords: Ion trap, mass resolution, UPLC, MSn, , ETD, proteomics


146

P55

Fast and Highly Accurate QTOF ETD MS/MS for Top-DownProtein Identification

Wunderlich, Dirk; Störmer, Carsten; Hartmer, Ralf; Lubeck, Markus


Einleitung: The profiling of plasma proteins is a challenge in proteome analyses in terms of the dynamic rangeand the number of analytes present. Once the statistic approach discovers a potential biomarker the major objectiveis the identification and characterization. Most of the biomarkers are too large to obtain substantial sequence infor-mation with common gas-phase protein fragmentation techniques like CID. Higher dedicated MS/MS-techniquesfor top-down proteomic approach are electron-induced fragmentation processes like electron capture or electrontransfer dissociation (ETD). We will present the profiling and top-down identification of plasma proteins utilizinga new type of hybrid quadrupole ETD highly accurate TOF mass spectrometer.Experimenteller Teil: ABruker maXis quadrupole-TOF is equipped with ETDMS/MS capability. The concept ofreagent ion generation and transmission is adapted from the HCT ETD II ion trap. The goal of the present proteinprofiling approach is targeted towards proteins revealing molecular weight below 10 kDa. Plasma proteins withhigher mass were depleted with a 10 kDa cutoff filter. Resulting proteins were subjected to a LC-separation usingmonolithic columns. The eluting and partially separated proteins were split towards the mass spectrometer andfractionated. Afterwards selected fractions were offline infused into the MS. A scheduled list of protein precursorions are then mass selectively introduced into the ETD-reaction cell and fragmented. With the highly accurateTOF-analyzer the resulting ETD-fragments are detected.Ergebnisse und Diskussion: The method is initially evaluated with protein standards spiked into plasma. Dueto the high resolving power of the TOF analyzer the mono isotopic annotation and the determination of chargestates for both precursor and ETD fragment ions is possible. The information of both the accurate precursor andthe ETD fragment masses are combined and submitted to a database search engine (Mascot) optimized for Top-Down data. Sequence tags and Top-Down related sequence analyses were generated in BioTools using a mixtureof automated calculations and interactive sequence assignments. Due to the high informative content of the ETDfragment spectra unambiguous protein identification and high sequence coverage is obtained. Presented will bedata from even low abundant proteins.Neue Aspekte: ETD in einem hochauflösenden, schnellen QTOF zur top-down MS/MS Analyse intakter ProteineThema: Instrumentelle Entwicklungen, Proteine und PeptideKeywords: ETD, Hochauflösung, QTOF, MessgeschwindikeitKontakt: [email protected]

147

P56

SDS-PAGE-MALDI-TDS: Mass Spectrometric Top-DownSequencing of Proteins Extracted from Polyacrylamide Gels

Irrgang, Jessica (1); Dibowski, Harald (1); Evers, Waltraud (2); Resemann, Anja (2); Suckau, Detlev (2);Razunguzwa, Trust R. (3)

1: dichrom - vormals SeQuant GmbH, Germany; 2: Bruker Daltonik GmbH, Germany; 3: Protea Biosciences,Inc., USA

Einleitung: MALDI Top-down sequencing (TDS) using In-Source-Decay (ISD) is a powerful tool to analyzeprotein termini without prior tryptic digestion. Sequence calls up to 80 residues of each terminus can be achieved.In addition, the technique is well suited to detect modifications, truncations, and mutations [1]. The analysisrequires an amount of 10 to 20 pmol of a single, purified protein for each MALDI preparation. When analyzingprotein mixtures or proteins that consist of multiple peptide chains such as antibodies, separation techniques suchas gel electrophoresis have to be applied prior to TDS analysis.Experimenteller Teil: We used a novel chip-based system for improved extraction of proteins from polyacry-lamide gels (GPR-800, Protea Biosciences, Inc.) [2,3]. Standard proteins such as Ubiquitin and RNAseA fromSDS-PAGE spots were extracted and the eluate purified using, e.g., protein precipitation or ultrafiltration. Subse-quently, the samples were analyzed by MALDI-TOF-MS.Ergebnisse und Diskussion: MS spectra of the intact proteins as well as ISD spectra of high quality were obtainedthat provided direct access to detailed sequence information. Finally, a monoclonal antibody was subjected to SDS-PAGE-MALDI-TDS analysis to obtain N- and C-terminal sequence assignments for both, the light (LC) and theheavy (HC) chain. 24µg of the antibody were reduced and the LC and the HC were separated by SDS-PAGE.These were extracted and purified from gel spots and analyzed by ISD. In the resulting MALDI-TDS spectra,approx. 70 N- and 30 C-terminal aminoacid residues were assigned and the N-terminal pyroglutamylation of theHC was confirmed.Neue Aspekte: Theseprotocols can be applied toTDS of proteins after gel-based separation and enable the detailedanalysis of terminal sequences.Referenzen: [1] Bruker Daltonics Technical Note TN-36: Automated Acquisition of MALDI-ISD Spectra forthe N- and C-terminal Sequence Determination of Intact Proteins.; [2] A Novel Chip-based Electroelution Systemfor Rapid and Efficient Recovery of Intact Proteins from Polyacylamide Gels. M.J. Powell., T.T. Razunguzwa,A.D. Biddle, and G. R. Asbury Protea Biosciences, USA, BioTechniques Special Issue, Vol. 46, 5, 2009; [3]Development of a microfluidics-based gel protein recovery system, T.T. Razunguzwa, A. Biddle, H. Andersen, D.Zhan, and M.J. Powell, Protea Biosciences, USA, Electrophoresis, Vol. 30, 24, 2009Thema: Instrumentelle Entwicklungen, Proteine und PeptideKeywords: Gel Protein Recovery (GPR-800), Top-Down Sequencing (TDS), intact protein mass, MALDI-TOF-MS, SDS-PAGEKontakt: [email protected]

148

P57

Top-down protein analysis by ETD, CID and HCD

Scheffler, Kai (1); Damoc, Eugen (2); Möhring, Thomas (2)

1: Thermo Fisher Scientific, Dreieich; 2: Thermo Fisher Scientific, Bremen

Einleitung: Mass spectrometry has drawn more and more attention as an alternative technology to traditionalprotein N-, as well as C-terminal, sequencing. Electron transfer dissociation (ETD) mass spectrometry is partic-ularly advantageous for sequencing applications because ETD is relatively insensible to the size, the amino acidcomposition, and post-translational modifications of proteins, therefore randomly cleaves peptide backbone bonds.ETD of intact proteins is highly efficient, generating very informative, yet extremely complex spectra that containhighly charged product ions that are difficult, or even impossible to resolve at unit resolution. ETD technologywas recently implemented in a hybrid linear ion trap - Orbitrap mass spectrometer whose high resolution and massaccuracy facilitate analysis of intact proteins using ETD.Experimenteller Teil: Proteins were analyzed by direct infusion on an LTQ Orbitrap Velos hybrid mass spectrom-eter equipped with an ETD unit. Experiments were performed using ETD, CID and HCD fragmentation. Criticalparamters such as activation time in case of ETD, energies in the case of HCD and selection of different chargestates were varied and evaluated. All data were acquired with detection at high resolution and mass accuracy inthe orbitrap mass analyzer.Ergebnisse und Diskussion: The data obtained demonstrate the capabilities of the linear ion trap - orbitrap hybridmass spectrometer for protein sequencing. The data discussedemphasize the benefit of high resolution and highmass accuracy fragment ion spectra for unambiguous assignments. Furthermore the spectra show interesting effectswhen acquisition parameters are varied for the different fragmentation types: not all charge states fragment equallywell, with HCD the fragmentation can be strongyl influenced be choosing differnt energies, and with ETD theactivation time duration strongly influences whether fragment ion across the full protein sequence orpredominantlyfrom the sequence termini are obtained.The data obtained also prove the capabilities to replace Edman sequencersfor N-terminal sequencing.Neue Aspekte: Terminal protein sequencing using different dissociation techniquesThema: Proteine und PeptideKeywords: Top-down, protein sequencingKontakt: [email protected]

149

P58

Combination of Micro-High Performance Liquid Chromatographyand High-Resolution Orbitrap Mass Spectrometry: A Powerful

Tool for Intact Protein Analysis

Mohr, Jens (1); Swart, Remco (2); Böhm, Günter (3); Huber, Christian G. (1)

1: Universität Salzburg, Austria; 2: Dionex Benelux, Amsterdam; 3: ThermoFisher Scientific, Switzerland

Einleitung: Highly efficient separations are indispensable for the analysis of complex biological samples. Poly(styrene-

divinylbenzene)- (PS-DVB) based monolithic capillary columns have been shown to be eminently suited for ion-

pair reversed-phase (IP-RP) HPLC of intact proteins [1]. Likewise, the Orbitrap has been recently introduced as a

very effective and sensitive mass analyzer [2]. Although initially it was thought that high-molecular analytes are

not amenable to mass analysis in the Orbitrap, more recent investigations have clearly demonstrated the applica-

bility to MS of proteins having molecular masses in excess of 150 kDa. This presentation reports on our efforts to

combine highly efficient protein separations employing PS-DVB monoliths and high-resolution mass spectrometry

in an Orbitrap mass analyzer as a tool for intact protein analysis.

Experimenteller Teil: IP-RP-HPLC separations were carried out in a fully integrated Accela HPLC system

(ThermoFisher Scientific GmbH, Bremen, Germany) with ProSwift 50 x 1 mm PS-DVB monoliths from Dionex

Benelux (Amsterdam, The Netherlands). Separations were carried out with 10-30-min gradients of acetonitrile

in 0.05% trifluoroacetic acid. Mass spectrometric analysis of intact proteins was carried out on an LTQ Orbitrap

mass analyzer (Thermo Fisher Scientific) using positive mode electrospray for gentle analyte ionization. The mass

spectrometric methods were optimized in a range of 800 to 4000 m/z by infusing different standard proteins at

resolutions of 7,500 to 100,000. The calibration of the mass axis was accomplished with the LTQ FT-Hybrid

positive ion mode calibration solution (Calmix).

Ergebnisse und Diskussion: Characterization of the HPLC-Orbitrap-MS system showed that the chromatographic

performance (peak widths at base between 6 and 24 s) could be fully maintained with Orbitrap-MS detection at fast

scanning rates of 2.5 spectra per s at a resolution of 7.500, while a small increase in peak widths was observed at

maximum resolution for 100.000 and 0.5 Hz scan rate. Isotopic resolution was possible for proteins in the range of

5 to 30 kDa resulting in monoisotopic and average mass determinations having generally less than 10 ppm deviation

from the theoretical mass. Limits of detection in the Orbitrap ranged from 206 to 7 fmol, and in the linear ion trap

from 244 to 49 fmol, depending on the analyzed protein.Examples of application include the analysis of a human

pooled lyophilized IgG digestion fragment (isolated by gel filtration) of approximately 10 kDa. The data revealed

the presence of different proteins in the range of 10-23 kDa. Moreover, a weak (mAB Bet V1 8.3) and a strong

binding intact monoclonal antibody (mAB Bet V1 5.1) against major birch pollen (betula verrucosa) allergen

were analyzed. The results revealed that the strong binding mAB sample contained no impurities or degradation

products resulting in one very sharp peak in the chromatographic run. The molecular mass was determined at

150,399 Da in the Orbitrap and 150,430 Da in the LIT. Limits of detection for the antibody were 1.8 fmol in the

Orbitrap and 1.2 fmol in the linear ion trap. The analysis of the weak binding mAB implied a degradation of the

antibody because several peaks including some characteristic proteins in the 25-kDa range were observed.

Neue Aspekte: We characterized a rapid and highly efficient monolith-based µHPLC-high-resolution-Orbitrap-

MS setup for intact protein analysis including complex real samples.

Referenzen: [1] Premstaller, A.; Oberacher, H.; Walcher, W.; Timperio, A.-M.; Zolla, L.; Chervet, J.-P.; Cavu-

soglu, N.; Van Dorsselaer, A.; Huber, C. G. Anal. Chem. 2001, 73, 2390-2396.; [2] Scigelova, M.; Makarov, A.

Proteomics 2006, 6 Suppl 2, 16-21


Keywords: Orbitrap, HPLC, intact protein analysis


150

P59

Protein stoichiometry of the human SMN complex determined byAQUA

Wiesner, Julia (1); Englbrecht, Clemens (2); Fischer, Utz (2); Sickmann, Albert (1)

1: ISAS - Leibniz-Institut für Analytische Wissenschaften e.V., Germany; 2: Biochemie - Universität Würzburg

Einleitung: The Survival of Motor Neuron (SMN) complex is a macromolecular entity consisting of nine fixedsubunits (SMN, Gemin2-Gemin8, Unrip) and different transient factors. The main function of the housekeepingcomplex comprises the assembly of U snRNPs in the cytoplasm. As a result of mutations in the SMN1 gene thisprocess can be defective and elicits Spinal Muscular Atrophy (SMA). The aim of this study is the characteriza-tion of the human SMN complex composition and stoichiometry by MS. In this context, we compare the nativestoichiometric composition with two recombinant complexes, each incorporating a known mutation involved inSMA (E134K, Y272C). Furthermore, we examine the differences between the cytoplasmic and the nuclear SMNcomplex both derived from immunoprecipitations of HeLa cells.Experimenteller Teil: For the purpose of quantifying the respective subunits we apply absolute quantification

(AQUA). Stable-isotope labeled analogs are spiked to the native sample in defined amounts thus introducing amass difference of 6 (K) to 10 Da (R) during MS analysis. Preliminarily, we chose appropriate peptides thatmeet several conditions which allow for a confident quantification. As MS-analysis is performed by Selected

Reaction Monitoring (SRM) on the QTRAP 4000 (Applied Biosystems), we optimized transitions and parameters(Declustering Potential and Collision Energy) for each respective peptide. Alternatively, we also apply label-freequantitation on the LTQ Orbitrap XL (Thermo Fisher). The high mass accuracy and sensitivity of this hybrid FTMS allows quantifying the respective parent ions directly from the MS1 trace.Ergebnisse und Diskussion: Both strategies, SRM and MS1, provide a linear quantification of peptides downto a concentration of 100 amol over a dynamic range of three to four orders of magnitude. By optimizing thecurrent workflow, we were able to determine the stoichiometry of recombinant SMN core complexes containingseveral known patient mutations within the SMN1 gene as well as the wildtype SMN complex. As expected from1D-PAGE analysis of the three different complexes, namely WT (wildtype), E134K and Y272C, the results showan underrepresentation for Gemin6, Gemin7 and Gemin8 in the Y272C mutant complex. Y272C and E134Kmutants are known to disrupt the self-oligomerization of the SMN protein thus hindering a proper Sm assemblyreaction. In the near future, our analysis will focus the differences between wildtype SMN-complexes originatingeither from cytoplasm or from nucleus. The challenge for this project implies a higher background accompaniedby lower concentrations for the SMN complexes. This is due to the enrichment method by immunoprecipitationout of complete cell lysates.Neue Aspekte: This project may help to reveal the UsnRNP-assembly and provide new targets in diagnosis/therapy for genetic diseases like SMA.Referenzen: [1] Kroiss M, Schultz J, Wiesner J, Chari A, Sickmann A, Fischer U. Evolution of an RNP assemblysystem: a minimal SMN complex facilitates formation of UsnRNPs in Drosophila melanogaster. Proc Natl AcadSci U S A. 2008 Jul 22;105(29):10045-50.Thema: Proteine und PeptideKeywords: AQUA, SRM, stoichiometry, SMNKontakt: [email protected]

151

P60

Untersuchung von Faltungszuständen des Ubiquitins mit Hilfe vonenzymatischer Proteolyse und MALDI-ReToF-MS und ECD vonAngiotensin II und seinem Fluorescein-Kopplungsprodukt mittels

ESI-FTICR-MS

Peters, Jonathan; Grotemeyer, Jürgen

Universität Kiel, Germany

Einleitung: Eine Methode, um Aussagen über den strukturellen Zustand eines Proteins zu treffen, ist die Verwen-

dung von enzymatischem Verdau [1]. Hiermit können nicht nur Strukturdaten über den nativen, sondern auch über

partiell entfaltete Zustände gesammelt werden. Für die Identifizierung eines Proteins oder Peptides ist es weiterhin

wichtig, mit Hilfe experimenteller Verfahren die Sequenz zu ermitteln. Dafür stehen Methoden wie Collisionally

Activated Dissociation (CAD) [2], Infrared Multiphoton Dissociation (IRMPD) [3] und Electron Capture Disso-

ciation (ECD) [4] zur Verfügung. Es wurde das Protein Ubiquitin (8565 Da, 76 Aminosäuren) in seinem nativen

Zustand sowie zwei partiell entfalteten Zuständen mittels Proteolyse untersucht. Zudem war das Octapeptid An-

giotensin II (1045 Da), sowie sein Kopplungsprodukt mit Carboxyfluorescein Gegenstand von ECD-Experimenten.

Experimenteller Teil: Im Rahmen der Untersuchung wurde Ubiquitin durch unterschiedliche Konzentrationen (1

und 2M) an Guanidin-Hydrochlorid (GuHCl) entfaltet und sowohl im nativen als auch in den partiell entfalteten

Zuständen mit der spezifischen Protease Chymotrypsin behandelt. Die auftretenden Verdaufragmente wurden mit-

tels Matrix Assisted Laser Desorption/Ionization an einem ReflexII (Eigenbau, Scout-Ionenquelle von Bruker Dal-

tonics) nachgewiesen. An Angiotensin II und dem Fluorescein-Kopplungsprodukt wurden ECD-Untersuchungen

an einem Fourier Transform Ionen Cyclotron Resonanz-Massenspektrometer (Bruker Daltonics) durchgeführt.

Ergebnisse und Diskussion: Im nativen Zustand des Ubiquitins konnten nur zwei Verdaufragmente nachgewiesen

werden. Dies weist darauf hin, dass die Flexibilität des nativen Proteins und damit die Zugänglichkeit zum aktiven

Zentrum der Protease sehr gering ist. Die gefundenen Proteolysestellen konnten mit zwei Positionen identifiziert

werden, über die Ubiquitin nichtkovalente Wechselwirkungen mit Erkennungsproteinen eingeht [5]. In Gegenwart

von 1 und 2M GuHCl zeigten die partiell entfalteten Zustände eine deutlich höhere Zugänglichkeit. So konnten

Verdaufragmente zu früheren Zeiten und in höherer Anzahl nachgewiesen werden. Zudem ließ die verwendete

Methode eine Unterscheidung dieser beiden Zustände zu. Die ECD-Spektren von reinem Angiotensin II und des

N-terminal mit Carboxyfluorescein gekoppelten Peptids wurden verglichen. Während ersteres deutliche Fragmen-

tierungen zeigte, war im Spektrum des Kopplungsprodukts lediglich eine teilweise Reduktion zu erkennen. Dies

wies auf eine Blockierung der Fragmentierung durch die Addition des Farbstoffs hin.

Neue Aspekte: ECD vom AngiotensinII-Fluorescein-Kopplungsprodukt

Referenzen: [1] H. Yang, X. Li, M. Amft, J. Grotemeyer, Anal. Biochem. 1998, 258, 118.; [2] J. Laskin,

J.H. Futrell, Mass Spectrom. Rev. 2003, 22, 158.; [3] D.P. Little, J.P. Speir, M.W. Senko, P.B. O’Connor, F.W.

McLafferty, Anal.Chem. 1994, 66, 2809.; [4] R.A. Zubarev, N.L. Kelleher, F.W. McLafferty, J. Am. Chem. Soc.

1998, 120, 3265.; [5] Y. He, L. Hicke, I. Radhakrishnan, J. Mol. Biol. 2007, 373, 190.


Keywords: Verdau, Ubiquitin, ECD, AngiotensinII, Fluorescein


152

P61

Interaction Studies of Abeta(1-40) Peptide andAbeta-autotantibodies by Immunoafinity- Mass spectrometry

Cozma, Claudia; Dragusanu, Mihaela; Przybylski, Michael

Universitaet Konstanz, Germany

Einleitung: The presence of autoantibodies against Aß, involved in the inhibition of the fibril formation, wasdeterminate not only in the serum, but also in pooled immunoglobulin fraction (IVIGs preparations). The goal ofthe present study is, after determination of the primary structure of the antibodies separated from human serumimmunoglobulin fraction (IVIG), to characterize their affinity interaction with Aß(1-40) peptide.Experimenteller Teil: Aß-autoantibodies present in IVIG were separated using a column with Aß(12-40) cova-lently immobilized on the matrix. The separated antibodies were quantified using a PIERCE quantification kit.Aß-peptide was synthesized by solid phase peptide synthesis, followed by RP-HPLC purification and MS charac-terization. Aß-peptide was immobilized on as gold surface chip and their interaction with Aß-autoantibodies wasmonitorized by sound acoustic waves (SAW). Using a large domain of Aß-autoantibodies concentrations it waspossible to monitorize the kinetics of the bioaffinity interactions and to assess the kD of Aß-autoantibodies. Theinteraction between these biological partners was characterized also in the reverse system, with Aß-autoantibodiesimmobilized on the gold chip, and the interaction was characterized by K5 Biosensor coupled with ESI- Ion TrapMS.Ergebnisse und Diskussion: Aß-autoantibodies present several working problems: they are in a very low amount,the affinity separation on an epitope containing matrix is damaging for the bioaffinity proprieties of the antibodies,the soluble state of the autoantibodies is not stable at a very wide range of pH (inclusive pH7), the immobilizationin a uniform manner of both Abeta peptides and Abeta autoantibodies is difficult due to the tendency to precipitate,Abeta autoantibodies are polyclonal. To be able to analyze the affinity bonding it was necessary to developa method suitable for this system alone. The antibodies were store d at -28°Cin TFE (trifluoroethanol) in a

stock solution from which the dilutions were made for the affinity and kinetic studies. Synthetic Aß-peptide is

a stable compound under pH oscillating conditions (2.1 to 8) and was immobilized on the chip surface and the

biological partner was added in different concentration in the microfluidic cell. The kD value was determinate

by linear regression using Origin 7.5. To characterize the affinity binding of the biological system an online

coupling was performed of the K5S-sens®biosensor and ESI Ion Trap MS. For this experiment Aß-autoantibodieswere immobilized on the chip and Aß peptides were added. The elution fractions were analyzed online by massspectrometry.Neue Aspekte: Bioaffinity interactions of Abeta(1-40) and Abeta autoantibodies were characterized by SAW andtandem SAW-ESI -MS.Referenzen: [1] M. Przybylski et al., EPA and US Patent applications; Universities of Konstanz and Bonn, 2007;[2] R C Dodel et al., J.Neurol. Neurosurg. Psychiatry 2004;75;1472-1474; [3] I. Perdivara et al , Glycobiology,2009; [4] T.Gronewold et al (2006), Anal. Chem., vol. 78, No.14, 4865Thema: Proteine und PeptideKeywords: Bioaffinity, Aß-autoantibodies, SAW, kD, SAW-ESI-MSKontakt: [email protected]

153

P62

Investigation of Calmodulin/Munc13 Peptide Interaction byChemical Cross-Linking, MALDI-TOF/TOF-MS, and

ESI-LTQ-Orbitrap-MS

Schaks, Sabine (1); Krauth, Fabian (1); Müller, Mathias Q. (1); Ihling, Christian (1); Jahn, Olaf (2); Sinz, Andrea

(1)

1: MLU Halle-Wittenberg, Germany; 2: MPI of Experimental Medicine Göttingen, Germany

Einleitung: Munc13 proteins - as presynaptic regulators - are essential for synaptic vesicle priming and play an

indispensable role in adaptive synaptic mechanisms. Calcium-dependent interaction of Munc13 with calmodulin

(CaM) was recently found to link the changes in residual calcium concentrations to presynaptic vesicle priming

and short-term plasticity. Previous cross-linking and photo-affinity labeling experiments revealed an antiparallel

orientation of Munc13-1 and ubMunc13-2 peptides binding to calmodulin via a 1-5-8-14 binding motif comparable

to other CaM target proteins, such as the neuronal NO synthase or skeletal muscle myosin light chain kinase [1].

Here, we support these results by using an alternative amine/photoreactive cross-linker. The heterobifunctional

reagent SBC is an amine-reactive photo-cross-linker bridging a distance of ca. 10.2 Å [2].Experimenteller Teil: Protein labeling. CaM (8.5 µM ) was employed in 10 mM HEPES buffer (pH 7.2)containing a Ca2+/chelator (EGTA) buffer system, with the free Ca2+ concentration adjusted to 30 nM. CaMwas first reacted with the amine-reactive site of the cross-linker N-succinimidyl-p-benzoyl-dihydro-cinnamate(SBC, 200 or 500µM ) for 30 min. Excess of cross-linker was quenched with 20 mM NH4HCO3 and removed bymicrofiltration. Cross-linking reaction. Munc13-1 and ubMunc13-2 peptide (10 µM ), respectively, were addedto labeled CaM before the reaction mixture was irradiated with UV light (365 nm, irradiation energies 4000 and8000 mJ/cm2). After tryptic in-gel digestion and separation of cross-linked peptides by nano-HPLC (Ultimate3000, Dionex) samples were analyzed by MALDI-TOF/TOF-MS (Ultraflex III, Bruker Daltonik) and nano-ESI-LTQ-Orbitrap-MS (ThermoFisher Scientific).Ergebnisse und Diskussion: In agreement with previous data we found a number of lysine residues in CaM tobe labeled with SBC [2]. In these species, the amine-reactive site of the cross-linker (NHS ester) has reactedwith the amine groups of lysines 21, 30, 77, and 94 in CaM, while the benzophenone moiety of the SBC wasstill intact. Furthermore, intramolecular cross-links within CaM and intermolecular cross-links between CaM andMunc13 peptide were identified, in which the benzophenone had reacted. For Munc13-1, lysines 77 and 94 in CaMwere exclusively found to be labeled with SBC. Cross-links were identified between Lys-75 in CaM and Ala-10in Munc13-1 as well as between Lys-30 in CaM and Ala-3 in Munc13-1. Intramolecular cross-links in CaM werenot detected. For ubMunc13-2, Lys-21 in CaM was found to be both labeled and cross-linked, whereas lysine30 and 94 in CaM were not cross-linked. These findings are in agreement with the proposed structure of Munc13peptide/CaM complexes, in which Lys-94 in CaM is highly solvent exposed. In contrast, Lys-21 in CaM was foundto be involved in both inter- and intramolecular cross-links, underlining that this residue is in the correct distanceto Ala-10 in ubMunc13-2 that can react with the benzophenone of the cross-linker. The same is true for Lys-75 inCaM that was found to be cross-linked with either alanine 5 or 10 in ubMunc13-2. Additionally, intramolecularcross-links were found within CaM. Lys-21 was cross-linked to Met-76, while Thr-110 was cross-linked to eitherPhe-92 or Met-76. The reaction of the amine reactive moiety of the cross-linker with threonine is less likely thanwith lysine, but had been suggested in previous studies [2]. Our experiments provide the basis for creating detailedmodels of CaM/Munc13 complexes, which are crucial for understanding the mechanisms underlying presynapticvesicle priming and short-term plasticity.Neue Aspekte: We confirmed and extended the data of previous structural studies of the CaM/Munc13 peptidecomplex.Referenzen: [1] Dimova K.et al., Biochemistry 2009, 48, 5908-5921; [2] Krauth F. et.al., Rapid Commun MassSpectrom. 2009, 23, 2811-2818Thema: Proteine und PeptideKeywords: Chemical Cross-linking, Calmodulin,Kontakt: [email protected]

154

P63

Mass spectrometric analysis to elucidate the interaction partnersof plant co-chaperones with homology to the human protein HOP

(heat shock organizing protein)

Hedtmann, Christiane (1); Matros, Andrea (1); Kunze, Gotthard (1); de Jaeger, Geert (2); Mock, Hans-Peter (1)

1: IPK-Gatersleben, Germany; 2: VIB/ University of Gent, Belgium

Einleitung: Improved understanding of stress defence systems in plant cells is a prerequisite for generatingimproved crops with increased tolerance towards unfavourable environmental conditions. We are interested in thecharacterization of a stress-induced protein from Nicotiana tabacum (STINT). Initially the protein was found to behighly enriched in tobacco leaf trichomes together with numerous stress-related proteins (1). STINT is homologto the co-chaperone HOP (Hsp90/Hsp70 organizing protein) which acts as linker between Hsp90 and Hsp70 inmammal systems. However, little is known on the functional context of the protein in plants. Specifically, theclient proteins of the co-chaperone/chaperone protein complex are unknown. To elucidate the interaction partnersof plant co-chaperones in larger complexes mass spectrometry analysis is used.Experimenteller Teil: To date, we have obtained a number of data that support the role of STINT within thecontext of stress defence. To obtain a functional proof for this role, we performed an RNAi approach. To study thedynamic distribution of the proteins, localization studies using GFP fusion constructs and subcellular fractionationwere done. In order to isolate the putative protein complexes we used tandem affinity purification method. Eluatefractions from affinity purifications were separated on 1D SDS gels. Resulting protein bands were analyzed by

MALDI-TOF-TOF und LC-ESI-Q-TOF mass spectrometry. Data sets were then used for database searches inorder to identify yet unknown interaction partners.Ergebnisse und Diskussion: Plants containing an RNAi construct of STINT were exposed to various kinds ofstresses, including heat, drought and cold. Whereas under control conditions transgenic plants behave as wildtype, drought and cold stress revealed a stunted growth phenotype. Cold stress had the strongest impact ongrowth, whereas after heat treatment phenotypic effects were similar as in control lines. These data support animportant role of STINT in stress defence. Furthermore, localization studies using GFP fusion constructs andsubcellular fractionation revealed an occurrence of STINT and its homologous proteins of Arabidopsis in nuclei,cytosol and in association to membranes. This strong dynamic distribution of STINT indicates the participation ofthe co-chaperon in various kinds of complexes in different compartments. Initial experiments of tandem affinitypurification method coupled with mass spectrometry analysis of STINT-TAP fusion constructs from Arabidopsiscell culture resulted in the identification of isoforms of Hsp90 protein family. Based on their sequence, cytosoliclocalization of all these identified Hsp90 can be concluded. In parallel we did tandem affinity purification of thehomologous yeast protein STI1 under its endogenous promoter. Mass spectrometric analysis showed interactionto heat shock proteins 90 and 70. For the interaction of STI1 to the Hsp70 protein family the isoform Hsp71 wasclearly identified out of 14 protein family members. Currently it is not possible to distinguish between Hsp90isoforms, due to the 97 % homology of the constitutive isoform HSC82 and the inducible isoform HSP82. Inboth cases the client proteins were not obtained. To define whether STINT interacts with specific isoforms ofHsp90 protein family, proteins have to be studied with additional techniques such as surface plasmon resonancespectroscopy (Biacore).Neue Aspekte: Mass spectrometric analysis revealed members ofHSP90 and 70 protein families as interactionpartners of the HOP homolog protein STINT.Referenzen: [1] 1. Amme S, Rutten T, Melzer M, Sonsmann G, Vissers JPC, Schlesier B, Mock HP. 2005. Aproteome approach defines protective functions of tobacco leaf trichomes. Proteomics 5: 2508-2518Thema: Proteine und PeptideKeywords: TAP, protein-protein interactions, stress induced protein, tobacco, arabidopsisKontakt: [email protected]

155

P64

Alternate Dissociation Pathways Identified in Charge-ReducedProtein Complex Ions

Pagel, Kevin (1,2); Hyung, Suk-Joon (1,3); Ruotolo, Brandon T. (1,3); Robinson, Carol V. (1,2)

1: University of Cambridge, Department of Chemistry, Lensfield Road, Cambridge, CB2 1EW, UK; 2: Universityof Oxford, Department of Chemistry, South Parks Rd, Oxford, OX1 3QZ, UK; 3: University of Michigan,

Department of Chemistry, 930 N. University, Ann Arbor, MI 48109-1055, USA

Einleitung: Tandem mass spectrometry (MS) of large protein complexes has proven to be capable of assessingthe stoichiometry, connectivity, and structural details of multi-protein assemblies.1 While the utility of tandem MSis without question, a deeper understanding of the still controversially discussed mechanism of protein complexdissociation will undoubtedly drive the technology into new areas of enhanced utility and information content.Experimenteller Teil: IM-MS and tandem-MS experiments were performed on a Synapt HDMS (Waters, Manch-ester, UK) quadrupole-ion trap-IM-MS instrument equipped with a nanoflow electrospray (nano-ESI) source. In-strument parameters were adjusted to retain non-covalent interactions in tetrameric TTR. The wide range of TTRcharge states generated in this study were achieved using (i) 10mM NH4OAc solution (15+ to 13+), (ii) 10mMtriethylammonium acetate (TEAA) (12+ to 10+) and (iii) 10 mM TEAA, 250 mM A18C6 (this equates to 100equivalents of CE to TTR) (10+ to 7+).Ergebnisse und Diskussion: We present here the systematic analysis of the charge state dependent decay ofthe non covalently associated complex of human transthyretin (TTR), generated by collision induced dissociation(CID). A novel, crown ether-based charge reduction approach was applied to generate intact transthyretin tetramerswith charge states ranging from 15+ to 7+. These nine charge states were subsequently analyzed by means oftandem MS and ion mobility spectrometry. Three different charge-dependent mechanistic regimes were identified:1) Ions with a charge above 12+ followed the commonly observed CID dissociation pathway2 resulting in highlycharged, unfolded monomers and compact, charge-stripped trimers. 2) Tetramers with charge states between11+ and 9+ still dissociated into monomer and trimers, but the expelled monomers were carrying fewer chargesand retained their compact, native-like conformation. 3) 9+ and 8+ ions primarily yielded C-terminal peptidefragments which were cleaved from intact and fully folded TTR tetramers. Ions with a charge below 8+ werevirtually indestructible in the instrument and neither showed signs of dissociation nor unfolding. Taken together,the results presented highlight the potential of charge state modulation as a method for directing the course of gasphase dissociation2 and unfolding3 of protein complexes. Particularly noteworthy is the fact that under certainconditions the remaining stripped complex, as well as the departing subunit, retain compact, native-like structures.This has implications for various gas phase-spectroscopic and structural biology experiments in which maintainingconformations close to the native state is of paramount importance.Neue Aspekte: Charge state modulation can be used to obtain considerably more information about a proteincomplex from conventional CID activation.Referenzen: [1] Benesch, J. L. P.; Ruotolo, B. T.; Simmons, D. A.; Robinson, C. V., Protein Complexes in the GasPhase: Technology for Structural Genomics and Proteomics. Chem. Rev. 2007, 107, (8), 3544-3567.; [2] Benesch,J. L. P., Collisional Activation of Protein Complexes: Picking Up the Pieces. J. Am. Soc. Mass Spectrom. 2009,20, (3), 341-348.; [3] Ruotolo, B. T.; Hyung, S.-J.; Robinson, P. M.; Giles, K.; Bateman, R. H.; Robinson, C.V., Ion Mobility-Mass Spectrometry Reveals Long-Lived, Unfolded Intermediates in the Dissociation of ProteinComplexes. Angew. Chem. 2007, 119, (42), 8147-8150.Thema: Proteine und PeptideKeywords: protein complexes, collision induced dissociation, charge reduction, ion mobility spectrometryKontakt: [email protected]

156

P65

Identification of lactose binding epitope structure in Galectin-3 byCREDEX-MS and CRD peptide characterization by

SAW-bioaffinity analysis

Eggers, Frederike (1); Moise, Adrian (1); Gabius, Hans-Joachim (2); Przybylski, Michael (1)

1: Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, Department of Chemistry, Universityof Konstanz.; 2: Institut für Physiologische Chemie, Tierärztliche Fakultät, Ludwig-Maximilians-Universität,

München.

Einleitung: Galectins are a family of endogenous lectins with adhesion/growth-regulatory activity binding galacto-sides. They are involved in a large number of processes in cell differentiation, development, fertilization, pathogeninfection, cell-cell recognition, signal transduction, inflammation processes, and cancer cell metastasis. Charac-terization of galectin-carbohydrate interactions and identification of binding sites is of crucial importance for drugdesign. In the present study Galectin-3 peptides containing key amino acids involved in the carbohydrate bindingwere identified by CREDEX-MS of Galectin-3 and lactose. The identified CRD peptides were synthesized andtheir interactions with lactose were characterized by affinity-MS, SAW-bioaffinity measurements.Experimenteller Teil: For CREDEX-MS lactose was immobilised on Sepharose in affinity columns, Galectin-3was added and digested using trypsin. After washing the unbound galectin fragments, the fragments remaining onthe column were eluted. Both fractions were analysed by MALDI-FTICR-MS. The identified binding structuresare in direct agreement with the X-ray structures reported. The identified CRD-peptides were synthesised byFmoc-SPPS and their binding properties characterised by affinity-MS and SAW-bioaffinity-measurements. Theimmobilisation of lactose on bioaffinity-sensor-chips was performed in the form of a glycosylated peptide via anaminooxyacetic linker. SAW measurements were performed with an S-Sens K5 Biosensor. The CRD-peptidesas well as the full-lenght Gal-3 interacted in PBS with the immobilised lactosyl-glycoprobe and were eluted with0.5M Lactose and aqueous solutions of acetonitrile/TFA.Ergebnisse und Diskussion: CREDEX-MS of galectin-3 identified as binding sites for lactose the CRD peptides(152-162) and (177-183) in complete agreement with the X-Ray crystal structure of the Galectin-3 - lactosecomplex. Both peptides were synthesized and their affinity for lactose was demonstrated by affinity-MS. Toget more detailed information about the affinity and interaction kinetics of the CRD peptides with lactose SAW-bioaffinity measurements were performed with two further variant peptides of CRD-(152-162). The peptide (157-163) was synthesized in order to eliminate non-essential amino acids. It represents the shortest peptide that couldhave affinity and contains the residues H158, N160, R162 which are essential for the galactose binding. Thelonger peptide (157-175) was synthesized to include V-172 and N-174, and 12 pmol lactosyl-glycoprobe wereimmobilized on the SAW chip. The synthetic CRD peptides were introduced as 200 µL injections of 10 µMsolutions in 25mM PBS. The phase shifts observed were 5.2 deg for (157-163) and 16.5 deg for (157-175).Injecting a 0.5 µM solution of full-lenght Gal-3 produced a phase shift of 24.7 deg. The signal intensity isconsistent with the size of the ligand. At the identical concentrations the phase difference is higher for the (157-175) peptide relative to the (157-163) peptide. The binding of the full length Gal-3 at 0.5 µM is higher than thatof the peptides at 10 µM . Affinity-MS and SAW-bioaffinity analysis of synthetic CRD peptides (157-163) and(157-175) showed that V172 and N174 are not essential for binding, although they contribute to the increasedaffinity of the (157-175) peptide.Neue Aspekte: Directly analysis of intact peptide/protein carbohydrate interactions. SAW-bioaffinity analysis ofcarbohydrate peptide/protein interactions.Referenzen: [1] Przybylski M., Gabius H.-J., Moise A., Patent application: CREDEX: Mass spectrometric de-termination of carbohydrate recognition structures in proteins. (2007).; [2] Vila-Perelló M., Gutiérrez Gallego R.,Andreu D., ChemBioChem 2005, 6, 1831.; [3] Seetharaman J., Kanigsberg A., Slaaby R., Leffler H., BarondesS.H. and Rini J.M., J. Biol. Chem. 273, 13047-13052 (1998); [4] Marquardt A., Muyldermans S., Przybylski M.Chemistry, 12: 1915-1921 (2006).Thema: Kohlenhydrate, Proteine und PeptideKeywords: CREDEX-MS, CRD, SAW-bioaffinity, FTICR-MS, Affinity-MSKontakt: [email protected]

157

P66

Interaction Studies between Peptides Derived from PhotoreceptorGuanylyl Cyclase and its Activating Protein 2 (GCAP-2) by

Chemical Cross-Linking and MALDI-TOF/TOF MS andESI-LTQ-Orbitrap MS

Pettelkau, Jens (1);Müller, Mathias (1); Ihling, Christian (1); Schröder, Thomas (2); Lange, Christian (2); Sinz,

Andrea (1)

1: Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin Luther University

Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, D-06120 Halle, Germany; 2: Department of Technical

Biochemistry, Institut of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg,

Kurt-Mothes-Str. 3, D-06120 Halle, Germany

Einleitung: We conducted interaction studies between peptides derived from photoreceptor guanylylcyclase (ROS-

GC) and the guanylyl cyclase-activating protein 2 (GCAP-2). ROS-GC is a membrane protein, which increases the

concentration of cyclic guanosine monophosphate (cGMP) and regulates the adaptation of the retina in response to

light. The activity of the enzyme is Ca2+-dependently regulated by GCAP-1, GCAP-2, and adenosine triphosphate

(ATP). GCAP is an N-terminally myristoylated Ca2+-binding protein containing four EF-hand motifs. Disturbed

ROS-GC/GCAP interaction may lead to degenerative retinopathies.

Experimenteller Teil: Cross-linking reactions between ROS-GC peptides and GCAP-2 were performed with

Ca2+ using the homobifunctional amine-reactive, isotope-labeled (d0 and d4) cross-linker bis(sulfosuccinimidyl)

glutarate (BS2G). MALDI-TOF-MS in the linear mode was used to analyze the intact cross-linked complexes.

Cross-linking reaction mixtures were separated by one-dimensional gel electrophoresis (SDS-PAGE). For a de-

tailed structure analysis of the complexes, gel bands of interest were excised and digested with trypsin and en-

doproteinase Glu-C. The peptide mixtures were analyzed by nano-HPLC/MALDI-TOF/TOF-MS (Ultraflex III,

Bruker Daltonik) and nano-HPLC/nano-electrospray ionization (ESI)-linear ion trap (LTQ)-Orbitrap-MS (LTQ-

OrbitrapXL Thermo FisherScientific).

Ergebnisse und Diskussion: Chemical cross-linking and MS presents a novel approach for low-resolution struc-

ture determination of proteins, in which covalent bonds are formed between different molecules (intermolecular)

or between parts of one molecule (intramolecular). The interactions between GCAP-2 and two ROS-GC derived

peptides that represent potential GCAP binding sites (amino acids 503-522 (GC peptide 1) and 965-981 (GC pep-

tide 2) of the full-length protein) were studied by chemical cross-linking in combination with mass spectrometry.

A number of intramolecular cross-links and partially hydrolyzed cross-linkers were identified in GCAP-2. Intrigu-

ingly, one cross-link pointed to an interaction between the N-terminus of GC peptide 2 comprising amino acids

965-981 and lysine at position 129 of GCAP-2. In the excised gel bands, GC peptides 1 and 2, respectively, were

identified proving that an interaction exists between both GC peptides and GCAP-2. Yet, based on the limited

number of cross-links between both binding partners, we have not been able so far to create an exact model of

the GCAP-2/GC peptide complexes. The final aim of the study is to create a structural model of the functional

complex between ROS-GC and GCAP-2 in the lipid membrane.

Neue Aspekte:

Thema: Isotopen-Massenspektrometrie, Proteine und Peptide

Keywords: GCAP-2, Chemical Cross-Linking, MALDI-TOF/TOF MS, ESI-LTQ-Orbitrap MS


158

P67

recent developments in high mass maldi tof ms

Niebel, Jörg

CovalX AG, Switzerland

Einleitung: High mass Maldi ToF mass spectrometry provides valuable information in the field of antibody char-acterization such as Interaction analysis, Sandwich assays and Epitope mapping, further in the area of therapeuticprotein aggregate analysis and in drug discovery. Newer applications and technologies in the high mass Maldi ToFfield are on their way from RandD to practical routine application. Examples will include 1: Studies over a broaderrange of Protein complexes proving usability over a broad KD range 2: New classes of cross linkers with increasedreaction speedExperimenteller Teil: high mass maldi Tof mass spectrometry had been adoped and implemented over the lastview years in the field of Protein complex characterization, Protein Stoechiometry, mutant analysis, integrity elu-cidation, interaction validation Antibody characterization: multibinding, epitope mapping, antigen identification.Therapeutic protein aggregates analysis: Aggregates characterization, stoechiometry Early adopters of high massMaldi ToF are expanding into areas of high mass imaging of proteins up to 110kDa in tissue sections. A newclasses of crosslinkers had been developed with improve yield and significant reduce crosslinking reaction time.Studies had been conducted on model Protein complexes illustrating the usability of the technology for proteincomplex with varous Kds at differnt concentrations.Ergebnisse und Diskussion: tbaNeue Aspekte: - high mass imaging - crosslinker chemistry and reaction kinetics- protein complex Kd studiesThema: Instrumentelle Entwicklungen, Proteine und PeptideKeywords: maldi, crosslinking, interaction, tof,Kontakt: [email protected]

159

P68

A SPE method development platform as a helpful tool for GC-MSanalysis

Uhlschmied, Cindy (1); Krieg, Christof (1); Gjerde, Doug (2); Bonn, Günther (1)

1: Institute of Analytical Chemistry and Radiochemistry, University of Innsbruck, Austria; 2: PhyNexus Inc.,

3670 Charter Park Drive, San Jose, CA 95136, USA

Einleitung: Gas Chromatography coupled to Mass Spectrometry, GC-MS, is a powerful tool in analytical chem-

istry [1]. It combines the high resolution and fast analysis time of Gas Chromatography with the high sensitivity

and the ability of structure elucidation and confident analyte identification of Mass Spectrometry. One disadvan-

tage of Gas Chromatography is the need for time consuming sample preparation and cleanup. To solve these

problems, Solid Phase Extraction, SPE, is often used. Because method development is very time consuming, in

most cases C18 is used as standard material without knowing if it posses the highest efficiency. Therefore detailed

studies with different stationary phases are necessary for optimal recovery rates. Because of the resulting high

number of samples, automation is of crucial importance.

Experimenteller Teil: The extraction of five essential oil components (myrcene, citronellal, p-cymene, menthol

and thymol) out of an ethanolic solution was optimized using automated SPE. An automated robotic system,

the Personal Purification System®

from Phynexus Corp, [2, 3] was adopted for SPE. It allows the simultaneous

automatic extraction of 12 samples with varying SPE materials. 16 different SPE materials, C18Hydra, C18ec, C8,

C4, C2, C6H5, C6H11ec, NO2, CN, NH2C18, Diol, HRP, EASY, PA, Florisil, Davisil, were used; each extraction

was repeated six times. Additionally the extraction procedure was carried out at varied pH-values to investigate

the influence of pH value on recovery rates. Samples were analyzed using GC-MS in Scan Mode with o-cresol as

external standard.

Ergebnisse und Diskussion: This way the most effective SPE materials for the extraction of each of the five

essential oil components were determined. Thymol, menthol and borneol have highest recovery rates in case of

C18ec closely followed by C8, citronellal has highest recovery rates in case of C8followed by C18ec. For p-cymene

and myrcene achieved recovery rates were very low for all materials (<10%). Variation of pH-value did not show

significant changes in recovery rates.

Neue Aspekte: Fast, easy, robust, reproducible automated method development for SPE in combination with GC-

MS

Referenzen: [1] Koek M. et al., Metabolic Profiling of Ultrasmall Sample Volumes with GC/MS: From Microliter

to Nanoliter Samples, Analytical Chemistry, 2010 82 (1), 156-162; [2] Feuerstein, I. et. al., Material-Enhanced

Laser Desorption/Ionization -A New Protein Profiling Tool Utilizing Specific Carrier Materials for Time of Flight

Mass Spectrometric Analysis, Journal of the American Society for Mass Spectrometry, 2006, 17; [3] Feuerstein, I.;

Bonn, G., Gjerde, D., Huck, C., Stecher, G., Automated method and device for preparing an analyte for analysis by

MALDI mass spectrometry using columns in combination with a liquid handling system. PCT Int. Appl. (2008)

Thema: Naturstoffe

Keywords: automated SPE, GC-MS, essential oil, method development


160

P69

Analysis of Whisky by Electrospray FT-ICR Mass Spectrometry:Proof of Origin by Statistical Methods

Witt, Matthias; Paape, Rainer; Fuchser, Jens; Friedrich, Jochen


Einleitung: Whisky is a high-class consumed alcoholic beverage with a several billion dollar market. Due to thehigh value of this liquor counterfeiting and manipulation have been observed. Therefore, the proof of the originof this luxury alcoholic drink is of major interest of distillers and beverage importers. Whisky consists besidewater and alcohol of a variety of volatile and non-volatile chemical components, e.g. organic acids and esters,aldehydes, phenols, polyphenols and lactones. [1-5] Several mass spectrometry techniques have been applied toanalyze volatile compounds in whisky [6,7]. Recently the proof of origin and authenticity of whisky has beenstudied by electrospray mass spectrometry [8].Experimenteller Teil: In our study we used ultra-high resolved mass spectrometry to study whiskies from differentorigins using statistical methods and fingerprinting.The whiskies have been diluted 1:20 in 50% MeOH for directinfusion measurements using electrospay FT-ICR in negative ion mode. The spectra have been acquired with thenew Bruker solariX FTMS platform equipped with a 12T magnet using a resolving power of 300.000 at m/z 400.Repetitive measurements have been performed to proof the concept of the principal component analysis (PCA) forthis kind of samples.Ergebnisse und Diskussion: Several Scottish whiskies from two different origins as well as several whiskiesfrom the japanese distillery Suntory have been analyzed. Using electrospray ionization the most polar componentsof whisky are detected. Beside dominant species like ellagic acid and gluconic acid, the mass spectra of whiskyshow a complex pattern with several peaks at one nominal mass resulting in several thousand peaks in a massspectrum. The molecular formulas of more than thousand compounds have been identified. PCA as well as clusteranalysis have been performed of the full and of a part of the mass spectra with and without using the isotopicfine structure to validate the origin of the studied whiskies and to proof the relevance of the highly resolved massspectra for the characterization of whisky. The importance of the isotopic fine structure for fingerprinting of whiskywill be shown. Additional data interpretation hasbeen applied using van Krevelen analyses to investigate oxygencontaining compounds.Neue Aspekte: ESI-FT-ICR-mass spectrometry has been proven as a powerful tool for the characterization ofcomplex mixtures such as whisky.Referenzen: [1] Fujieda, M., Tanaka, T., Suwa, Y., Koshimizu, S., Kuono, I., J. Agric. Food Chem. 2008, 56,7305.; [2] Kinton, V. R., Adam, L., Abstr. Pap. Am. Chem. Soc. 2002, 224, U90.; [3] Fitzgerald, G., James, K.J., MayNamara, K, Stack, M. A., J. Chromatogr. A 2000, 896, 351.; [4] Møller, J. K. S., Catharino, R. R., Eberlin,M. N., Analyst 2005, 130, 890.Thema: NaturstoffeKeywords: Food analysis, FTMSKontakt: [email protected]

161

P70

Structural Investigation of Furanocoumarins by Positive IonElectrospray Tandem Mass Spectrometry

Heinke, Ramona; Franke, Katrin; Michels, Katharina; Wessjohann, Ludger; Schmidt, Jürgen

Leibniz Institut für Pflanzenbiochemie Halle, Germany

Einleitung: The genus Dorstenia (Moraceae) contains furanocoumarins, an important class of plant phototoxins

[1]. Dorstenia gigas is known as a rich source of prenylated coumarins [2]. A series of linear and angular

prenylated furanocoumarins from leaves and twigs of Dorstenia gigas were investigated by liquid chromatogra-

phy/electrospray tandemmass spectrometry (LC-ESI-MS/MS). The mass spectral behaviour of the furanocoumarins

under positive ion electrospray conditions is discussed using both an ion trap and a triple quadrupole system. The

present paper discusses the possibilities of tandem mass spectrometry as a tool both for a compound-type classifi-

cation and the determination of the substitution pattern.

Experimenteller Teil:

LC-ESI-CID-MS: The positive ion ESI mass spectra and the collision-induced dissociation (CID) mass spectra

were obtained from a TSQ Quantum Ultra AM system (Thermo Electron) equipped with a hot ESI source (HESI)

and coupled with a Surveyor Plus micro-HPLC (RP18-column: 5 µm, 150x1 mm, Hypersil, GOLD, Thermo

Scientific; gradient system: water: acetonitril, each of them containing 0.2% acetic acid, collision gas: argon).

LC-ESI-Ion-Trap-MSn: Positive LC-ESI-IT mass spectra were recorded on a LCQ Deca XP MAX (Finnigan)

coupled with a HPLC system (RP18-column: 5 µm, 150x1 mm, Hypersil GOLD, Thermo Surveyor, gradient

system: water: acetonitril, each of them containing 0.2% acetic acid) and a photodiode array detector (PDA,

Thermo Surveyor).

Ergebnisse und Diskussion: A detailed study of the fragmentation pattern of different prenylated furanocoumarins

from Dorstenia gigas was carried out by employing both an ion trap and a triple quadrupole system.

The main fragmentation of the furanocoumarins investigated is characterized by the appearance of typical neutral

losses allowing a classification of the skeleton (linear, angular) and the type of prenylation at the furanocoumarin

skeleton (C- and O-prenylated) While the MS2 spectra of [M+H]+ ion of the linear C-prenylated furanocoumarins

show a significant loss of C4H8 [3], in the mass spectra of the O-prenylated ones a prominent ion of type [M+H-

C5H8]+ is dominating. Moreover, besides a [M+H-C4H8]

+ ion, reverse C-prenylated compounds additionally

exhibit a fragment ion of type [M+H-C3H6]+ which is characteristic for this kind of prenylation. The fragmenta-

tion of the furanocoumarin scaffold is further characterised by typical successive losses of CO [4,5]. Furthermore,

a differentiation between a C-3 and a C-8-prenylation is possible by tandem mass spectrometry.

The obtained results showed that LC-ESI-MS/MS represents a valuable tool for structural investigations of fura-

nocoumarins.

Neue Aspekte: characterization and classification of prenylated furanocoumarins by liquid chromatography/ elec-

trospray tandem mass spectrometry

Referenzen: [1] Frohne, D., Pfänder, H. J. (1997) Giftpflanzen. Ein Handbuch für Apotheker, Ärzte, Toxikologenund Biologen. Wiss. Verlagsgesellschaft, Stuttgart; [2] Franke, K., Porzel, A., Masaoud, M., Adam, G., Schmidt,J. (2001) Phytochemistry 56, 611-621; [3] Simons, R., Vincken, J.-P., Bakx, E. J., Verbruggen, M. A., Gruppen,H. (2009) Rapid Commun. Mass Spectrom. 23, 3083-3093; [4] Zheng, X., Zhang, X., Sheng, X., Yuan, Z., Yang,W., Wang, Q., Zhang, L. (2010) J. Pharma. Biomed. Anal. 51, 599-605; [5] Kang, J., Zhou, L., Sun, J., Han, J.,Guo, D.-A. (2008) J. Pharma. Biomed. Anal. 47, 778-785Thema: NaturstoffeKeywords: furanocoumarins, Dorstenia gigas, Moraceae, LC-ESI-MS/MSKontakt: [email protected]

162

P71

Mining the myxobacterial secondary metabolome usinghigh-resolution mass spectrometry: natural product discovery as

an analytical challenge

Krug, Daniel (1); Zurek, Gabriela (2); Cortina, Nina (1); Barsch, Aiko (2); Müller, Rolf (1)

1: Universität des Saarlandes, Germany; 2: Bruker Daltonik GmbH, Bremen, Germany

Einleitung: Myxobacteria and other secondary metabolite producers represent an important source of biologi-cally active natural products with considerable promise for human therapy. Several studies have highlighted theenormous and hardly tapped genetic potential of many myxobacterial species for secondary metabolite biosynthe-sis.[1,2] Although more than 100 basic structures from myxobacteria have been characterized to date, the numberof compound classes reported from individual strains clearly falls short of the genetic capabilities. Thus, thediscovery of novel secondary metabolites from genetically proficient bacteria currently constitutes a substantialbottleneck in the discovery process of novel lead structures. Improved analytical methods, based on combined useof LC-coupled high-resolution MS and statistical data evaluation, can significantly enable the process of uncover-ing these "hidden" bacterial secondary metabolomes.[3]Experimenteller Teil: Extracts from cultivations of Myxococcus xanthus were analyzed by a separation of thecomplex samples on a RP-C18 column (1.7µm particle size) under UPLC conditions and coupling to a novelultra-high resolution TOF mass spectrometer. Atmospheric pressure ionization was carried out using positiveand negative ESI. The obtained data were pre-processed using a compound finding algorithm prior to statisticalinterpretation by principal component analysis (PCA) and a t-test model.Ergebnisse und Diskussion: Metabolomics-based experiments employing high-resolution LC-MS measurementsare convenient because they provide the opportunity to apply both targeted queries and unbiased statistical treat-ment to the same dataset. Efficient detection of molecular features in high-resolution LC-MS datasets is a crucialpre-requisite for the susequent mining of complex myxobacterial samples for the presence of novel natural prod-ucts, using statistical tools. Data acquired on the UPLC-ESI UHR-TOF platform are well suited for comprehensiveprofiling applications, enabling the in-depth comparison of myxobacterial secondary metabolomes with previouslyunmatched success. In the present work,M. xanthusDK1622 could be established as a producer of novel secondarymetabolite classes, in addition to the 5 previously known natural products.Neue Aspekte: Genomics meets metabolomics: high-resolution mass spectrometry and statistical interpretationhelpt to uncover previously untraceable natural products in complex myxobacterial metabolomes.Referenzen: [1] Wenzel SC, Müller R (2009). Curr.Opin.DrugDisc.Dev. 12(2), 220-230; [2] Garcia RO, Krug D,

Müller R (2009). Methods in Enzymology 458, 59-91; [3] Krug D, Zurek G, Revermann O, Vos M, Velicer GJ,

Müller R (2008). Appl. Environ.Microbiol.74, 3058-3068


Keywords: high-resolution mass spectrometry, TOF, natural products, myxobacteria, metabolome


163

P72

H/D Exchange Mass Spectrometry in Monitoring Non-covalentInteractions between Polyphenols and biomolecules

Tahrani, Ahmad; Wink, Michael

IPMB, Abt. Biologie, Uni Heidelberg, Deutschland

Einleitung: Dietary polyphenols are often claimed to be responsible for the protective effects against cardiovascu-lar complications, cancer, and many other diseases. A wealth of data exists, that suggests, that most of the relevantmechanisms of disease prevention by polyphenolic are due to the direct interaction with the target biomolecules.Although noncovalent complexes have been studied by many methods, but H/D mass spectrometry has severalpotential advantages over these more conventional methods.Experimenteller Teil: Sample preparation: Studied target was incubated with several candidates in mediumcontaining a plenty of D2O under the following conditions:• pH 7.50 ± 0.1• Incubation time 210 min. in roomtemperature. Sample measurement: • Quenching with 50% aqueous ACN containing 2% FA • Direct injectionusing ESI-MS • Data acquisition in the positive mode. ∆D (Change in deuterium uptake) was calculated incompare with control to confirm weather a non-covalent interaction occurred or not.Ergebnisse und Diskussion: The results highlight, that there is a clear relationship between flavonoids chemicalstructure and the binding affinity. The decrease of the binding affinity is corrolated to: 1. Degree of Glycosylation.2. Absence of C4 keto group, double bond at (C2 and C3) and the phenyl group. 3. Degree of the substitution ofhydroxyl groups.Neue Aspekte: H/DMass Spectrometry methodology offeres a promissing advantages in library and high through-put screening for non-covalent drug discovery.Referenzen: [1] J Am Soc Mass Spectrom 2004, 15, 388-397.; [2] Spectrochimica Acta Part A 73 (2009) 972-975.; [3] International Journal of Mass Spectrometry 222 (2003) 175-187.; [4] Chem. Res. Toxicol. 2009, 22,1689-1698Thema: NaturstoffeKeywords: Polyphenol flavonoids, Ligan-target Non-covalent Interaction, H/D Exchange Mass SpectrometryKontakt: [email protected]

164

P73

Identification of intermediates of Leucin biosynthesis andMethionin chain elongation pathway in knock-out mutants of thelarge subunit of isopropylmalate isomerase in Arabidopsis thaliana

Reichelt, Michael (1); Knill, Tanja (2); Binder, Stefan (2); Gershenzon, Jonathan (1)

1: Max-Planck-Institut für Chemische Ökologie, Germany; 2: Universität Ulm, Institut für Molekulare Botanik

Einleitung: Leu, Val and Ile are essential nutrients in human diets but can be synthesized by plants. In plantsall enzymatic activities required for the formation of branched-chain amino acids have been detected. In themodel species Arabidopsis thaliana, a clear assignment of branched-chain amino acid biosynthetic genes is difficultbecause the same reaction cascade as in Leucin biosynthesis is also found in theMet chain elongation pathway (firstpart of aliphatic glucosinolate biosynthesis). Both pathways involve the condensation of acetyl-CoA and a 2-oxoacid, isomerization of the resulting 2-malate derivative to form a 3-malate derivative, the oxidation-decarboxylationof the 3-malate derivative, and transaminatiom. We have analysed metabolites in Arabidopsis knock-out mutantsof the large subunit of the isopropylmalate isomerase.Experimenteller Teil: The contents of amino acids and glucosinolates were measured in approximately 3 week-old rosette leaves and seeds of wild-type and mutant plants. Additionally, the flow-through fractions of the anionexchange chromatography performed in the course of glucosinolate analysis of leaf extracts of mutant and wild-type plants were compared by LC-MS using a Bruker Esquire 6000 ion trap mass spectrometer coupled to anAgilent 1100 HPLC. Elution was accomplished using a Nucleodur Sphinx RP column (250 x 4.6 mm, 5 um;Macherey-Nagel). Substraction of the mass spectrometer total ion chromatogram of wild-type plants from that ofthe mutants was done using the software package Metabolite Detect 1.1. NMR spectra were recorded on a BrukerAV500 spectrometer equipped with a CryoPlatform.Ergebnisse und Diskussion: The subtraction of the mass spectrometer total ion chromatogram of wild-type plantsfrom that of the mutants identified two additional peaks in mutant ipmi lsu1-3 extracts not found in wild-typeextracts corresponding to molecular masses of 176 (compound1) and 238 (compound 2). The former was unam-biguously identified as 2-isopropylmalate (2-IPM) by identical retention times with an authentic 2-IPM standard(Aldrich). Compound 2 was isolated from aqueous leaf extracts from ipmi lsu1-3 plants by preparative HPLC andanalyzed by mass spectrometry. High resolution electrospray ionization mass spectra and NMR spectra enabledthe identification as 2-(3’-methylsulfinyl)propylmalate. The three ipmi large subunit 1 (LSU1) mutants studiedall exhibit a shift towards shorter chain-length aliphatic glucosinolates. However, there are differences betweenthe individual mutants in other traits. For example, the ipmi lsu1-2 plants showed the smallest chemical differ-ences from wild-type plants. Changes in amino acid and glucosinolate levels were moderate, S-Methylmethionin(SMM) and 2-IPM did not accumulate to detectable levels, and 2-(3’-methylsulfinyl)propylmalate content was thelowest among the three different ipmi lsu1 mutants. A stronger chemical phenotype was observed in the ipmilsu1-1 mutant, with SMM, 2-IPM and 2-(3’-methylsulfinyl) propylmalate accumulating to substantial amounts.The strongest effects on both glucosinolate and amino acid composition as well as on the accumulation of 2-IPMand 2-(3’-methylsulfinyl)propylmalate were observed in ipmi lsu1-3 plants. However, the IPMI LSU1 transcriptlevel in leaves of ipmi lsu1-3 plants was comparable to that in the ipmi lsu1-1 line. Thus the alterations in ipmilsu1-3 cannot be solely explained by reduced transcript levels. The insertion of the T-DNA only 11 bp upstream ofthe ATG might cause changes in spatiotemporal transcription of this gene or alter translation efficiency. Leu levelsare unchanged in all mutants despite the other metabolite alterations observed, indicating that amino acid levelsare extraordinarily well-buffered against fluctuations.Neue Aspekte: Differential LC-MS analysis for characterization of plant mutantsThema: NaturstoffeKeywords: Leucine metabolism, Glucosinolate biosynthesis, Isopropylmalate isomerase, 2-(3’-methylsulfinyl)propylmalateKontakt: [email protected]

165

P74

"TMS-Multisteroid-Analyse": Verbesserter Nachweis adrenalerSteroidhormone in Plasma und Speichel mittels UPLC-MS/MS

Kulle, Alexandra; Holterhus, Paul-Martin; Riepe, Felix

Universitätsklinikum Schleswig-Holstein, Germany

Einleitung: Die Analyse und Quantifizierung von Steroidhormonen in der pädiatrischen Endokrinologie ist für dieDiagnose verschiedener adrenaler und gonadaler Erkrankungen sehr wichtig. Präzision, Geschwindigkeit und Re-produzierbarkeit sind dabei entscheidende Faktoren. Es wurde eine UPLC-MS/MS Methode zur parallelen Erfas-sung der wichtigsten adrenalen Steroidhormone: Progesteron, 11-Desoxycorticosteron, Corticosteron, Aldosteron,17-Hydroxy-progesteron, 21-Desoxycortisol, 11-Desoxycortisol, Cortisol und Cortison entwickelt.Experimenteller Teil: Einer Probenvorbereitung mittels Festphasenextraktion (SPE) folgt eine UPLC-Trennungüber eine C18-Säule mit einem Wasser-Acetonitril-Ameisensäure-Gradienten. Gemessen wird im MRM Modemittels Elektrospray. Zu jedem Steroidhormon werden zwei MRM Übergange gemessen, ein Quantifier und einQualifier.Ergebnisse und Diskussion: Die Methode wurde in einem physiologisch relevanten Bereich von 0.5 bis 200nmol/L kalibriert, Bestimmtheitsmaß der Kalibrierung: r2 > 0.992. Inter- und Intra-Assay Daten für diesen Bere-

ich lagen unter 10% VK. Die Spezifität der Methode wurde überprüft und mittels deuterierter, interner Standards

erhöht. Die Methode erlaubt eine präzise Quantifizierungen der Steroidhormone. Neben einer guten Reproduzier-

barkeit der Methode ist auch das Handling im Labor selbst ein entscheidender Faktor. Aufgrund des geringen

Probenvolumens der pädiatrischen Proben ließen sich die besten Ergebnisse mit einer offline SPE erzielen. Mittels

der vorgestellten Methode wurden Normwerte für Speichel und Plasma generiert.

Neue Aspekte: Quantifier und Qualifier für Steroidhormone

Thema: Naturstoffe

Keywords: Steroidhormone, UPLC


166

P75

Detection of Lipophilic Algal Toxins along the Danish North SeaCoast and Isolation of the Azaspiracid-Producing Dinoflagellate

Azadinium spinosum

Krock, Bernd; Tillmann, Urban; Cembella, Allan D.

Alfred-Wegener-Institut für Polar- und Meeresforschung, Germany

Einleitung: Algal toxins pose a serious threat to human health via the accumulation in shellfish, which are in-

creasingly cultured and harvested worldwide. Among these toxins are domoic acid, a rare amino acid produced

by members of pennate diatoms of the genus Pseudo-Nitzschia, linear polyethers as okadaic acid an deriva-

tives and pectenotoxins, ladder shaped polyethers like yessotoxins, and cyclic imine toxins such as spirolides,

gymnodimines, and azaspiracids. The aim of a research cruise performed in July 2008 was to survey the occur-

rence of these phycotoxins in the North Sea by liquid chromatography coupled to a triple quadrupole tandem mass

spectrometer and the consequent isolation of the producing organisms.

Experimenteller Teil: Plankton samples were collected with a 20 µm-mesh plankton net during the FK Uthörn

cruise in the North Sea from 7th to 18th July 2008. Discrete seawater samples were collected in Niskin entrapment

bottles mounted on an automatically triggered rosette sampler at 3 m and 10 m depth. Toxins were extracted

with methanol and filtered through spin-filters. The filtrates were analyzed on an Agilent 1100/ABI-SCIEX-4000

Q Trap®

instrument. Separation was performed by on a C8 phase (50 × 2 mm, 3 mm Hypersil BDS 120 Å,Phenomenex, Aschaffenburg, Germany). The flow rate was 0.2 mL min−1 and a linear gradient was performedwith water and acetonitrile. Measurements were carried out in positive ion mode by multiple reaction monitoring(MRM) experiments.Ergebnisse und Diskussion: The phycotoxins detected on the cruise through the German Bight and along theDanish west coast, were 13-desmethyl spirolide C, 20-methyl spirolide G, azaspiracid-1 (AZA-1), dinophysistoxin-2 (DTX-2), pectenotoxin-2 (PTX-2) and domoic acid (DA). DTX-2 was detected at all stations and ranged from0.3 to 630 pg/net tow with highest abundances in the > 200 µm size fractions. In contrast pectenotoxin-2 was onlydetected in northern Danish waters in the 55-20 µm fraction to a concentration of 1260 pg/net tow. Both toxinsare known to be produced by the genus Dinophysis, but since there was no correlation between the occurrences ofboth toxins, it can be assumed that they were produced by different Dinophysis species. The most abundant toxindetected on this survey was 20 methyl spirolide G up to concentrations of 11300 pg/net tow. 13-desmethyl spirolideC was also detected, but at much lower levels (max. 88 pg/net tow). Both toxins correlate well geographicallyand are a strong indication for the presence of the marine dinoflagellate Alexandrium ostenfeldii. AZA-1, whichhad been responsible for several human intoxications after shellfish consumption in Europe, was detected in watersamples of the 20-5 µm size fraction only north of latitude 56° N. Concentrations of AZA-1 varied slightly between10 and 100 pg/L sea water among the northern stations except for two stations (56° 35.3362 N, 007° 28.7701 Eand 56° 14.5204 N, 007° 27.5395 E) with very high levels of 927 and 1960 pg/L. From these water samples theazaspiracid-producing dinoflagellate Azadinium spinosum was isolated for the first time from the eastern NorthSea and also a related, non-toxic, hitherto undescribed small dinoflagellate of the genus Azadinium.Neue Aspekte: Tandem mass spectrometry as an efficient tool for detection and isolation of marine phytoplanktonby their secondary metabolitesThema: Organische Massenspektrometrie, NaturstoffeKeywords: Tandem mass spectrometry, phytoplankton, North Sea, phycotoxins, Azadinium spinosumKontakt: [email protected]

167

P76

Metabolomic Profiling of edible Oils by mf MELDI in combinationwith Principal Component Analysis

Krieg, Christof (1); Uhlschmied, Cindy (1); Rainer, Matthias Rainer (1); Abel, Gudrun (2); Popp, Michael (2);Bonn, Günther (1)

1: Leopold Franzens University, Austria; 2: Bionorica AG

Einleitung: MALDI-MS profiling of all kind of samples gained more and more interest during the last years [1-3]. It combines fast data acquisition by MALDI-MS with sophisticated software tools for cluster and PrincipalComponent Analysis, PCA, for the differentiation into classes. Originally developed for proteomic research andbiomarker discovery, this technology found its way into a wide range of other applications. A disadvantage ofMALDI in the low molecular mass range is the high background noise caused by fragments of the used matrixmaterial. To overcome this disadvantage, matrix free Material Enhanced Laser Desorption / Ionisation MassSpectrometry, mf MELDI MS, was introduced [4-5].Experimenteller Teil: The suitability of this material was proven by measuring and analysing different edibleoils, e. g. extra virgin olive oils, soya oils, peanut oils. Data analysis was done by Bruker ClinProTools 2.2. Thissoftware offers Cluster and PCA analysis of MALDI MS data and furthermore cross validation and validation ofthe obtained results. The same measurements were performed with conventional MALDI using DHB as matrixand compared to the mf MELDI MS results.Ergebnisse und Diskussion: mf MELDI shows better differentiation between the different classes of oils and hasreproducibility comparable to MALDI.Neue Aspekte: Simultaneous metaboliomic profiling of free fatty acids, diglycerides and triglycerides in edibleoils.Referenzen: [1] Sabbadin, S., et al, Matrix-assisted laser desorption/ionization mass spectrometry in evaluation ofprotein profiles of infant formulas, Rapid Communications in Mass Spectrometry, 1999, 13(14), 1438-1443.; [2]Nanni P., et al., Serum protein profiling in patients with inflammatory bowel diseases using selective solid-phasebulk extraction, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and chemometric dataanalysis, Rapid Communica; [3] Bonn, G., Feuerstein, I., Huck, C., Stecher, G., Rainer, M., Method for detectingorganic compounds, especially peptides and proteins from blood using MALDI-TOF. PCT Int. Appl. 2007; [4]Hashir M. A. e. al., Identification of carbohydrates by matrix-free material-enhanced laser desorption/ionisationmass spectrometry, 2007, 21(16), 2759-69.; [5] Hashir, M. A. e. al., Identification of amino acids by materialenhanced laser desorption/ionisation mass spectrometry (MELDI-MS) in positive- and negative-ion mode, Inter-national Journal of Mass Spectrometry, 2009, 279(1)Thema: NaturstoffeKeywords: MALDI, edible oils, TAG, PCAKontakt: [email protected]

168

P77

Mass Spectrometry Based Enantiomeric Excess Determination ofNatural Compounds in High Throughput Applications

Fleischer, Heidi (1); Gördes, Dirk (2); Thurow, Kerstin (1)

1: Center for Life Science Automation - celisca, Germany; 2: University of Rostock, Germany

Einleitung: In the past decades, high throughput screenings have been established in various fields of industryand research. The investigation of active chiral compounds is an absorbing research field, especially in drugdevelopment. Fast analysis methods for enantiomeric excess determination are required for efficient chemicaland biological synthesis processes. Classical analysis systems (e.g. HPLC, GC, CE) usually employed in chiralanalytics don’t fulfill the high throughput requirements, such they require relative long analysis times. Massspectrometry affiliated with a suitable previous derivatization using parallel kinetic resolution is an elegant analysismethod without chromatography [1,2,3]. In combination with liquid handlers and additional software this methodwas fully automated [4,5]. In this study a fast method for determination of natural compounds is presented.Experimenteller Teil: The method presented for enantiomeric excess (ee%) determination of natural compoundsuses parallel kinetic resolution. The derivatization of chiral substrates was carried out accordingly to the methodoptimized and adapted to new substrates described in [2]. For the analysis of alcohols and amino alcohols the aux-iliary solution contains (-)-carbobenzyloxy-L-proline and (R)-(+)-1-carbobenzyloxy-2-piperidinecarboxylic acidin the same concentration. (S)-(+)-2-Heptanol and (R)-(-)-2-Octanol were used to analyze carbon acids. For thecalibration stock solutions of the chiral substrates with five ee%-values were prepared. After derivatization foran hour the analysis was carried out with an ESI-TOF/MS. An additional software module was developed forcalibration, enantiomeric excess calculation for unknown samples and visualization the results.Ergebnisse und Diskussion: Up to now natural compounds from various substance classes were successfullytested with the mass spectrometric method presented. Menthol and borneol are representatives for alcohols. Pseu-doephedrine is a representative for amino alcohols, and trolox for carboxylic acids. The derivatization was per-formed in 1-mL vials and in 96-well microtiter plates with a well volume of 500 µL. The substances tested deliv-ered exponential calibration curves with exact variance values. Five stock solutions with defined enantiomeric ex-cess values (100, 50, 0, -50, and -100ee%) were used as representative data points. The standard deviations for theintensity ratios (n=3) measured were about 6%. To verify this measurement method various tests were performed.Intraday measurements were carried out with 25 samples derivatized at the same day. Interday measurementswere carried out at five consecutive days with three replicates. The repeat accuracy of the ESI-TOF/MS used wastested with 10 replicate measurements of the same sample. Finally, the selectivity of the auxiliaries was investi-gated. Combinations of (-)-carbobenzyloxy-L-proline and (R)-(+)-1-carbobenzyloxy-2-piperidinecarboxylic acidand (+)-carbobenzyloxy-D-proline and (S)-(-)-1-carbobenzyloxy-2-piperidinecarboxylic acid as well as (S)-(+)-2-heptanol and (R)-(-)-2-octanol and (R)-(-)-2-heptanol and (S)-(+)-2-octanol delivered calibration curves with anopposite slope. In contrast, auxiliaries with the same chiral constitution lead to horizontal calibration functions andshowed no selectivity. The whole analysis process was automated from the sample pretreatment carried out withliquid handlers over the analysis up to the data processing. For the special requirements of data evaluation in chiralanalytics an additional software module basing on Microsoft Excel VBA was implemented. This enables afterthe worklist run an automated extraction of the processed data files provided by the acquisition software "MassHunter" from Agilent Technologies, creation of the calibration, calculation of the enantiomeric excess, and thefinal data visualization in a 3D-chart.Neue Aspekte: - high throughput able method for enantiomeric excess determination without chromatography -fully automated data processing and visualization - fully automated sample pretreatmentReferenzen: [1] Vedejs, E.: Chen, X. H.: Parallel kinetic resolution. Journal of the American Chemical Society,1997, 119 (10), 2584-2585.; [2] Guo, J. H.; Wu, J. Y.; Siuzdak, G.: Finn, M. G.: Measurement of EnantiomericExcess by Kinetic Resolution and Mass Spectrometry. Angewandte Chemie - International Edition, 1999, 38 (12),1755-1758.; [3] Reetz, M. T.; Becker, M. H.; Klein, H. W.: Stöckigt, D.: A method for high-throughput screeningof enantioselective catalysts. Angewandte Chemie - International Edition, 1999, 38, 1758-1761.; [4] Thurow,K.: Gördes, D.: High-Throughput Screening Application for the Determination of Enantiomeric Excess UsingESI-MS. Journal of the Association for Laboratory Automation, 2006, 11, 128-33.; [5] Fleischer, H.; Gördes,D.: Thurow, K.: High-Throughput Screening Application For Enantiomeric Excess Determination Using ESI-MS.American Laboratory, 2009, 41 (7), 21-24.Thema: Naturstoffe

169

Keywords: enantiomeric excess determination, mass spectrometry, high throughput screening, laboratory automa-tion, parallel kinetic resolutionKontakt: [email protected]

170

P78

Gas-phase Ion Chemistry under ESI(+)/CID Conditions.Unexpected Fragmentation Behaviour of the [M + Na]+ Ions of

Benzylated Natural and Non-natural Polyphenols

Kuck, Dietmar (1); Heitkamp, Sandra (1); Letzel, Matthias C. (1); John, Markus (2); Ahmed, Ishtiaq (3); Krohn,Karsten (3)

1: Bielefeld University, Germany; 2: Macherey-Nagel GmbH and Co KG, Germany; 3: University of Paderborn,Germany

Einleitung: Polyphenols are important ingredients of agricultural products such as wine, tea, fruit, vegetables andherbal medicines (1). Among them, the prodelphinidins form a specific class of proanthocyanidins in which themain building blocks are (-)-gallocatechin and (+)-epigallocatechin. In the course of synthesis work on perbenzy-lated derivatives of the prodelphinidins and their flavonoid precursors, we observed an unexpected fragmentationbehaviour of their [M + Na]+ adduct ions under ESI/CID conditions (2). These multiple benzyl phenyl etherswere found to undergo sequential pairwise losses of C7H7 radicals and specific retro-Diels-Alder reactions. In thiscontribution, we present some of these results in combination with the fragmentation of suitable model cations,e.g., the [M + Na]+ ions of several perbenzylated di- and trihydroxybenzenes.Experimenteller Teil: ESI(+)/CID mass spectra of the pyridinium cations were measured by use of an Esquire3000 ion trap ("ion cage") mass spectrometer equipped with a standard nanoESI source. All samples were dis-solved in CHCl3/MeOH containing NaBF4. The syntheses of the flavonoid "monomeric" and the prodelphinidin"dimeric" polyphenol benzyl ethers have been described in detail (2). The benzylation of the dihydroxy- and tri-hydroxybenzenes giving the 1,2-, 1,3- and 1,4-di(benzyloxy)benzenes and 1,2,3- and 1,3,5-tri(benzyloxy)benzenewas performed by standard procedures. The identity and purity of all compounds were confirmed by mass spec-trometry and NMR spectroscopy.Ergebnisse und Diskussion: The [M + Na]+ ions were generated from the (all-arylic) octabenzyl ether ofcatechin-(4α → 8)-catechin (1), from the two analogous isomeric nonabenzyl ethers of catechin-(4α → 8)-gallocatechin (2) and gallocatechin-(4α→ 8)-catechin (3) and from the corresponding decabenzyl ether of galloca-techin-(4α → 8)-gallocatechin (4). In all cases, consecutive losses of 182 u (apparently corresponding to amolecule of C14H14) were observed as the predominant fragmentation reactions, concomitant with retro-Diels-Alder reactions occurring not only from the [M + Na]+ but also from the [M + Na - "C14H14"]+ ions. The RDA-type channel was found to be open with high specificity within the 3-hydroxyflavan-8-yl moiety but not within the3-hydroxyflavan-4-yl backbone, a finding which, strikingly, enables the distinction between the isomeric prodel-phinidins 2 and 3. The origin of this specificity is tentatively traced to the preferred coordination of the sodiumcation in the inner part of the prodelphinidin framework. The predominance of the formation of ions [M + Na - nC7H7]+ (n = 2, 4, 6 . . . ), giving rise to the losses of 182 u, is attributed to the release of two C7H7 radicals, ratherthan to a conceivable elimination of an intact C14H14 molecule. This is corroborated by model studies on the [M+ Na]+ ions of the three PhCH2O-C6H4-OCH2Ph isomers (o-5, m-5 and p-5) and of 1,2,3-tri(benzyloxy)benzene(6) and 1,3,5-tri(benzyloxy)benzene (7). The consecutive loss of two C7H7 radicals was found to occur exclusivelyfrom ions [o-5 + Na]+, [p-5 + Na]+ and [6 + Na]+, all of which can form stable closed-shell quinoid structuresafter loss of 182 u.Neue Aspekte: Gaseous [M + Na]+ ions of oligobenzyl ethers derived from natural and non-natural polyphenolsundergo unexpected and specific fragmentation reactions.Referenzen: [1] S. Yoneda, H. Kawamoto, F. Nakatsubo, J. Chem. Soc. Perkin Trans. 1 1997, 1025; [2] K. Krohn,I. Ahmed, M. John, M. C. Letzel, D. Kuck, Eur. J. Org. Chem. (submitted).Thema: Organische Massenspektrometrie, NaturstoffeKeywords: Catechin derivatives, benzyl phenyl ether, sodium cation adducts, retro-Diels-Alder reaction, ESI massspectrometryKontakt: [email protected]

171

P79

ANALYSIS OF BLACK TEA THEARUBIGINS FROM SIXDIFFERENT COMMERCIAL TEAS BY ELECTROSPRAY

FT-ICR MASS SPECTROMETRY

Kuhnert, Nikolai (1); Clifford, Michael (2); Fuchser, Jens (3); Witt, Matthias (3)

1: Jacobs University, Bremen, Germany; 2: University of Surrey, Guilford, UK; 3: Bruker Daltonik GmbH,Germany

Einleitung: Thearubigins, the mysterious chemically uncharacterised polyphenolic fraction of black tea compris-ing 60-70 % of its dry weight, isolated from six commercial black teas have for the first time been analyzed byFourier Transform Ion Cyclotron Resonance (FT-ICR) mass spectrometry (MS). Up to 9 000 mass spectral peakshave been detected per sample usingthe ultra-high resolving powerof FT-ICR MS. Due to the high mass accuracybetter than 1 ppmmolecular formulas were assigned to up to 1500 compounds. More than 50 compounds havebeen named by comparison to the available literature. Not allowing for isomers, these thearubigin samples containat least 2500 compounds, thus explaining the inability to resolve individual compounds chromatographically.Experimenteller Teil: Thearubigins were isolated from black tea in a simple method introduced by Roberts[1] in which the major pigmented polyphenols precipitate after complexation with added caffeine leaving mostnon-phenols in solution. For FT-ICR-MS analysis the thearubigins were reconstituted in 1:1 water methanol ata concentration of 2 mg/ml and analyzed with an apex ultra 9.4 T FT-ICR instrument (Bruker Daltonics Inc.,Billerica, USA) by direct infusion measurements in electrospray negative ion mode.Ergebnisse und Diskussion: Data interpretation strategies developed for petrolomics studies (van Krevelen andKendrick analyses) havebeen applied to black tea. A novel software program and a protocol have been developedto refine these procedures for the investigation of polyphenols. Using homologous series analysisoxygenationhas been identifiedarising by nucleophilic addition of water to aromatic CH groups as a key feature. A series ofreaction schemes have been developed linking known precursors with plausible products that match the availableMS data. The accuracy of these predicted structures willbe assessed critically by ion trap MS procedures. Astructural model for thearubigin formation has been developed based on experimental FT-ICR-MS data and noveldata interpretation strategies. ESI FT-ICR-MS provides a powerful tool for the analysis and structure elucidationof extremely complex dietary mixtures such as black tea thearubigins, which are several orders of magnitude toocomplex to be amenable to chromatographic analysis.Neue Aspekte: ESI FT-ICR-MS provides a powerful tool for the analysis and structure elucidation of extremelycomplex dietary mixtures.Referenzen: [1] Roberts, E. A. H., Economic importance of flavanoid substances: tea fermentation, in Chemistryof Flavanoid Compounds ed. Geissmann, T. A., Pergamon, Oxford 1962, 468-512.Thema: NaturstoffeKeywords: Complex Mixture, FTMS, Polyphenols, Food analysisKontakt: [email protected]

172

P80

Identification of alkaloids from fumariaceous plants

Meyer, Achim; Imming, Peter

Instiute of Pharmacy, Martin-Luther-Universität Halle-Wittenberg, Germany

Einleitung: The majority of alkaloids found in plants of fumariaceous species belong to the class of isoquino-

line alkaloids. In the case of fumitory plants, there are mainly protopine, protoberberine, phthalideisoquinoline,

spirobenzylisoquinoline and aporphine alkaloids [1]. Their structural diversity and the similarity of individual

members lead to difficulties in alkaloid analysis and discovery. We established a GC-MS method to separate and

identify alkaloid classes and single members even with minor differences in structure.

Experimenteller Teil: Investigations were made on the alkaloid content of Corydalis cava Schweigg. and Körte

(L.) and Fumaria vaillantii Loisel. Samples were taken from a historic collection compiled by J. Gadamer et al. in

the first half of the 20th century [2,3]. Gas chromatography was performed on a CP-Sil8CB column, and a method

was established by variation of temperature conditions with helium as carrier gas at 7 psi constant flow. The GC

was linked to a quadrupole mass detector in EI mode. The mass spectra were processed and analyzed with HP

ChemStation software supported by NIST MS database.

Ergebnisse und Diskussion: Each isoquinoline alkaloid class showed a specific MS pattern that allowed first

assignments at a glance. The smaller the structural variations were the more fragments had to be taken into

consideration in order to serve as a fingerprint for every single alkaloid. This approach was successful even for

constitutional isomers that showed fragmentation similarity of 95 %. The lacking stereospecificity is the main

limitating factor of our method. The combination of GC and MS was well apt for analyses of complex mixtures

of closely related isoquinoline alkaloids. The relatively high melting point of the alkaloids was not detrimental to

a GC separation and even alkaloids with structural resemblance were fully resolved although overlaps could not

be avoided entirely. We performed a screening over the total substance collection and could identify both rare and

unknown alkaloids [4]. Even oxidation by-products of some alkaloids were detected as impurities which were not

expected under these conditions due to their salt character (and high melting-points).

Neue Aspekte: Identification of new or rare alkaloids. Analysis of a historical alkaloid collection by a modern

method.

Referenzen: [1] Suau, R. et al. (2002) Phytochem Anal 13 (6), 363-7.; [2] Meyer, A. ; Imming, P. in preparation;

[3] Kollmann-Hess, M. (1988), thesis, Dt. Apotheker Verl.; [4] Meyer, A. ; Imming P. (2008) Phytochem Lett 1

(3), 168-70.

Thema: Naturstoffe

Keywords: isoquinoline alkaloids, Corydalis, Fumaria, GC-MS


173

P81

Mass spectrometric determination of ET-1-generating peptidaseactivities in protein fractions

Hildebrand, Diana; Trusch, Maria; Zhao, Xiaoxia; Schlüter, Hartmut

Universitätsklinikum Hamburg Eppendorf, Germany

Einleitung: Peptide hormones like endothelin-1 (ET-1) usually are generated by the proteolytic release frominactive precursor proteins. ET-1 is released by proteolytic cleavage from Big ET-1. For the investigation of the ET-1 generating proteases a reliable protease assay is required. Since mass spectrometry based enzyme assays (MES)are advantageous for the measurement of proteolytic activities, we developed an MES assay for the measurementof ET-1 generating activity.Experimenteller Teil: For the determination of ET-1-generating peptidase activities in complex protein fractionsby the MES method the proteins (for example from tissue extracts or body fluids) where covalently immobilizedonto an affinity chromatography material. Unbound molecules like buffer components were removed by wash-ing thus facilitating the following analysis by mass spectrometry. The immobilized proteins are then incubatedwith Big ET-1, a reaction specific peptide comprising the potential cleavage site of the ET-1-generating protease.Aliquots are removed after defined incubation times and analysed by MALDI-MS or MRM-combined methodslike LC-ESI ion trap-MS or LC-QQQ-MS that enables the relative quantification of ET-1, therefore determiningthe ET-1-generating peptidase activity of the sample.Ergebnisse und Diskussion: The MALDI-MES is well suited to observe additional reaction products beside ET-1deriving from the substrate Big ET-1. In contrast to the MALDI-MES assay the MRM-ESI-MES method providesmore reliable results concerning the quantification of the reaction products and as a result of the ET-1-generatingprotease activity in protein fractions. The combination of MALDI-MES and MRM-based MES-methods is a veryappropriate way to determine of ET-1-generating protease activites in protein fractions. Doing so, we could notonly measure ET-1-generating activities in protein fractions, but observe a couple of other Big ET-1-derived ET-1-peptides. This gives new insights into the generation of ET-1 and the understanding of the complex Endothelinsystem.Neue Aspekte: MADLI and ESI-based enzyme-screening-assays (MES) for the determination of ET-1 generatingpeptidase activities.Referenzen: [1] Yanagisawa M., Kurihara H., Kimura S., Tomobe Y., Kobayishi Y., Mitsui S., Tomobe Y.,Kobayishi Y., Mitsui Y., Yazaki Y., Goto K. and Masaki T., "A novel potent, vasoconstrictor peptide producedby vascular endothelial cell," Nature 332 (1988): 411-415; [2] D‘Orleans-Juste P., Plante M., Honore J.C., CarrierE. and Labonte J., "Synthesis and degradation of endothelin-1," Can. J. Physiol. Pharmacol. 81 (2003): 503-510;[3] Schlüter H., Jankowski J., Rykl J., Thiemann J., Belgardt S., Zidek W., Wittmann B. and Pohl T., "Detectionof protease activities with the massspectrometry-assisted enzyme-screening (MES) system," Analyt. Bioanalyt.Chem. 377 (2003): 1102-1107Thema: Proteine und Peptide, MetabolomicsKeywords: MALDI, ESI, Proteases, Peptidomics, EndothelinKontakt: [email protected]

174

P82

Peptidome and Metabolome Alterations in Pancreatic Cancer

Leichtle, Alexander (1); Ceglarek, Uta (1); Kase, Julia (1); Conrad, Tim (2); Hauss, Johann (3); Witzigmann,Helmut (3); Thiery, Joachim (1); Fiedler, Georg Martin (1)

1: Institute of Laboratory Medicine, University Hospital Leipzig; 2: Department of Mathematics, Free Universityof Berlin; 3: Clinic of Visceral Surgery, University Hospital Leipzig

Einleitung: Pancreatic cancer is one of the leading causes of cancer death. The established serum tumor markersof pancreatic cancer are not well suited for early detection and assessment of tumor stage and prognosis due totheir lack in sensitivity and specificity. Therefore, we investigated peptidome [1] and metabolome [2]alterations tofind new promising diagnostic targets, which might enhance the non-invasive detection and prognostic evaluationof pancreatic cancer.Experimenteller Teil: Serum samples of patients suffering from pancreatic cancer (n=20) and controls (n=20)were standardizedly collected at the University Hospital Leipzig [1]. We performed routine diagnostics, determinedthe concentrations of conventional tumor markers, generated magnetic bead-based MALDI-TOF-MS peptidome(Bruker autoflex) andMS/MS amino acid and acylcarnitine (API 3000) screening profiles. Peptidome profiles wereevaluated using the "proteomics.net" [3] and metabolome profiles using the Analyst 1.4.2 software. General andcomparative statistics were performed by SPSS 17.0 and R 2.9.0. Candidate peptides were quantified by ELISAtechniques.Ergebnisse und Diskussion: Investigating the serum peptidome, we revealed PF4 as a sensitive and specificcandidate in pancreatic cancer. In contrast, metabolome analysis disclosed 18 of 28 amino acids as significantlyaffected, whereas acylcarnitines remained mainly unaltered. The peptidomic approach revealed PF4 as a singlenew discriminating marker peptide for pancreatic cancer. The metabolome investigations resulted in multiplesignificant alterations in amino acid profile.Neue Aspekte: Prospective studies might further elucidate the diagnostic capabilities of high-throughput metabolomescreening and the potential of compound metabolome/peptidome markers.Referenzen: [1] Fiedler GM et al. Serum peptidome profiling revealed platelet factor 4 as a potential discrim-inating Peptide associated with pancreatic cancer. Clin Cancer Res 2009; 15:3812-9.; [2] Sreekumar A et al.Metabolomic profiles delineate potential role for sarcosine in prostate cancer progression. Nature 2009; 457:910-4.; [3] http://msproteomics.net/Thema: Proteine und Peptide, MetabolomicsKeywords: MALDI-TOF, Peptidomics, Bioinformatics, Clinical Study, Pancreatic CarcinomaKontakt: [email protected]

175

P83

Challenges in the investigation of the metabolic changes inNicotiana attenuata during insect herbivory using an improved

HPLC-TOF-MS method

Tellström, Verena (1); Schöttner, Matthias (2); Berger, Beatrice (2); Gaquerel, Emmanuel (2); Rothe, Eva (2);Schneider, Birgit (1); Zurek, Gabriele (1); Baldwin, Ian (2)

1: Bruker Daltonik GmbH, Germany; 2: MPI Chemical Ecology, Jena, Germany

Einleitung: This study investigates the metabolic changes in leaf extracts of Nicotiana attenuata after simu-lated insect herbivory by mechanically wounding and applying oral secretions of Manduca sexta (W+OS) usingHPLC-ESI-TOF-MS. Responses of wild-type (WT) plants are compared with genetically modified plants in whichthe hydroxyproline-rich glycopeptide systemin precursor (ppHypSys) is either over-expressed (OVsys) or down-regulated (IRsys). The challenges inherent to this type of study and strategies for quality control are discussed.Experimenteller Teil: Treated and untreated leaves (control samples) from WT, IRsys, and OVsys plants wereharvested 1, 14, 86, and 120 h after treatment. The flash-frozen plant tissue was extracted with a MeOH/aceticacid buffer 40:60 (v/v). Individual extracts from five biological replicates per time-point, treatment and genotypewere analyzed by HPLC-MS with a 45min binary acetonitrile-water gradient using an ESI-TOF mass spectrometerin electrospray positive mode. Additional MS/MS information for structure elucidation was obtained by analysiswith an ESI-Qq-TOFmass spectrometer. Statistical analysis was performed using ProfileAnalysis software (BrukerDaltonik, Germany) as well as the freely available XCMS and R software. Microsoft Office Excel was used forcalculation of kinetic profiles.Ergebnisse und Diskussion: Preliminary results obtained from a first data set of the same sample origin (7 time-points with three biological replicates per time-point, treatment and genotype analyzed with a 17min gradient)were used to design the experiment presented here. The number of biological replicates was increased from threeto five to access the biological variation. The chromatographic separation was optimized in order to minimizesuppression effects due to coelutions. A pooled sample generated from all individual samples was spiked withinternal standards (reserpine, atropine, D2-jasmonic acid, glycyrhicinic acid) served as quality control sampleto monitor instrumental variation. The samples were analyzed in two different laboratories independently. Thedata sets were analyzed using Principle Component Analysis in order to evaluate the overall variance and filterout changes induced by the treatment and genotype. Quantitative information about relevant ions was extractedindependently and the time dependent changes were summarized. The most time consuming challenge in thisprocess is the identification of the ions of interest. An important key for the identification is the unambiguousassignment of molecular formulae derived from accurate mass and isotopic pattern. The application of accurateand high resolution MS/MS data to support structure elucidation will be demonstratedNeue Aspekte: Statistical analysis of complex metabolomics samples in a high-resolution MSThema: MetabolomicsKeywords: Nicotiana, PCA, QTOF, HochauflösungKontakt: [email protected]

176

P84

GC/APCI-TOF MS: a new valuable tool for analysis of biofluidsin metabolomics studies

Behrens, Marina (1); Carrasco-Pancorbo, Alegria (2); Nevedomskaya, Ekaterina (2); Pacchiarotta, Tiziana (2);Arthen-Engeland, Thomas (1); Zurek, Gabriela (1); Baessmann, Carsten (1); Mayboroda, Oleg (2); Deelder,

Andre (2)

1: Bruker Daltonik GmbH, Germany; 2: Leiden University Medical Center, The Netherlands

Einleitung: Gas chromatography-mass spectrometry (GC-MS) is the major analytical platform in plant metabolomics.More recently, it has also been applied for metabolomics studies in clinical applications like biomarker discovery,disease diagnosis and toxicology. GC-MS is mainly used with electron impact (EI) and chemical ionization (CI)having the major advantage of large commercial and free spectral libraries for compound identification. In EI, frag-mentation during ionization often impairs the identification of the molecular ion. Thus, an important key for com-pound confirmation is missing. An alternative ionization technique is atmospheric pressure chemical ionization(APCI) preserving the molecular ion information. When coupling GC/APCI to a time-of-flight mass spectrometer,high resolution accurate mass and isotopic pattern data can be obtained for molecular formula determinationExperimenteller Teil: Derivatization reaction was based on a two step procedure: methoxyamination and sily-lation. The samples were injected on a HP-5-MS column (30 m, 0.25 mm ID, 0.25 mm film) and analyzed by atemperature gradient of 5ºC/min over 57 min (oven initial T= 70ºC kept over 5 min). MS analysis was carried outusing an ESI-TOF mass spectrometer in positive mode in a scan range from 50-1000m/z and a spectra rate of 1Hz.Human cerebrospinal fluid (CSF) extracts were prepared by cold methanol precipitation (2h) and centrifugation.The supernatant was evaporated under nitrogen and subsequently derivatized.Ergebnisse und Diskussion: The optimization, calculation of the analytical parameters (linearity, detection andquantitation limits) and validation of the method was made with a standard mix of 35 compounds trying to coverthe wider range with regard to polarity and molecular weight of the different analytes. 26 of the 35 compoundswere identified according to the molecular formula of the silylated derivatives with mass accuracies better than3ppm and sigma fit values smaller than 10mSigma being a measure of the isotopic pattern fit. The developedmethod was applied to the analysis of human cerebrospinal fluid (CSF) samples. The analyses of a set of CSFsamples will be considered as a future direction looking for new biomarkers and checking the potential of this newcoupling.Neue Aspekte: GC coupled to an API-TOF for high amss accuracy metabolomic studiesThema: MetabolomicsKeywords: GC/MS, APCI, QTOF, biofluidsKontakt: [email protected]

177

P85

Speziesanalytik von auf Platin basierenden Chemotherapeutika

Brauckmann, Christine; Karst, Uwe

Westfälische Wilhelms-Universität Münster, Germany

Einleitung: Chemotherapiemedikamente auf Platinbasis werden in fast jeder zweiten antineoplastischen Therapieeingesetzt. Cisplatin ist der erste Platinkomplex, dem eine tumorwachstumshemmende Wirkung nachgewiesenwerden konnte. Seit über 30 Jahren wird Cisplatin erfolgreich in Chemotherapien zur Heilung von Keimzell-tumoren, Osteosarkome, Neuroblastome oder Tumoren im Kopf- und Halsbereich verabreicht, obwohl die Ur-sache für schwerwiegende Nebenwirkungen (Nephrotoxizität, Ototoxizität) nicht geklärt ist. Diese Nebenwirkun-gen können nicht durch die Reaktionen von Cisplatin mit der DNA ausgelöst werden. Welche Reaktionen vonCisplatin im menschlichen Körper diese Nebenwirkungen verursachen ist bis heute nicht geklärt. Möglicherweisesind jedoch Intermediate, die aus bioverfügbaren Thiolen und Cisplatin gebildet werden, die Ursache. Aus diesemGrund wurde das Reaktionsverhalten zwischen Cisplatin und biologisch relevanten Thiolen untersucht.Experimenteller Teil: Für die Analyse von Cisplatin und möglichen Reaktionsprodukten wurde eine neue Meth-ode entwickelt. Da stark polare Komplexe vorliegen, wurde für die Trennung eine zwitterionische, hydrophile In-teraktionschromatographiesäule (ZIC

®

-HILIC) verwendet. Für die Detektion wurden komplementär Elektrospray-Ionisations-Massenspektrometrie (ESI-MS) und die induktiv gekoppelte Plasma-Massenspektrometrie (ICP-MS)eingesetzt. Durch den Einsatz beider Detektionstechniken können zum einen die Platinkomplexe identifiziert(LC-ESI-MS) und zum anderen mit sehr niedrigen Nachweisgrenzen und in komplexen Matrices (LC-ICP-MS)nachgewiesen werden. Dies ist vor allem bei Messungen von Realproben ein großer Vorteil.Ergebnisse und Diskussion: Cisplatin ist ein hochreaktiver Komplex. Aus diesem Grund kann das Reaktionsver-halten von Cisplatin während einer Messung von vielen verschiedenen Parametern beeinflusst werden. Zu diesenParametern zählen der pH-Wert und die verwendeten Puffersalze aus der mobilen Phase. Den größten Einflusshat jedoch die Chloridkonzentration im Reaktionsmedium. Eine hohe Chloridkonzentration stabilisiert Cisplatinund hemmt dadurch die Bildung von Hydrolyseprodukten. Auch das Reaktionsverhalten zwischen Cisplatin undbioverfügbaren, thiolhaltigen Verbindungen wird stark beeinflusst. Um Vergleichsmöglichkeiten zu dem Reak-tionsverhalten des Medikamentes im menschlichen Körper zu schaffen, wurden alle Proben unter physiologischenBedingungen angesetzt. Mit Hilfe einer HPLC Trennung in Kombination mit ICP-MS-Detektion konnte die Hem-mung der Reaktionen von Cisplatin durch hohe Chloridkonzentration gut sichtbar gemacht werden. Des Weiterenwar es möglich, eine Adduktbildung zwischen thiolhaltigen Aminosäuren, Peptiden sowie Protein und Cisplatinnachzuweisen und die entstanden Produkte mittels ESI-MS zu identifizieren.Neue Aspekte: Das Reaktions- und Hydrolyseverhalten von auf Platin basierenden Zytostatika wurde unter phys-iologischen Bedingungen untersucht.Thema: Proteine und Peptide, MetabolomicsKeywords: Cisplatin, HILIC, ThioleKontakt: [email protected]

178

P86

Challenges in metabolite identification (Met ID) in-vivo: The useof high resolution TOF MS with sub 1 ppm mass accuracies andextended dynamic range combined with intelligent software tools

Kühne, Stephan (1); Shockor, John (2); Yu, Kate (2); Shion, Henry (2); Marsden-Edwards, Emma (3); Yamada,Yasuhiro (4)

1: Waters GmbH, Eschborn, Germany; 2: Waters Corporation, Milford, USA; 3: Waters MS Technology Center,Manchester, UK; 4: Showa University, Japan

Einleitung: With the current publication from the FDA on the guidance of metabolites in safety testing, there isadded emphasis on the need to have adequate LC/MS platforms to be able to accommodate these new challenges.In order to address these challenges, we present an LC/MS workflow combining efficient data acquisition, TOFMSE functionality with intelligent data processing software. Sufficient knowledge of the metabolic cleavage sitesare crucial for metabolite identifications since the cleavage sites can be the major sites of biotransformation. Inaddition, cleavage fragments can also become sub-parent compounds that are further metabolized. These types ofmetabolites are the most difficult ones to be identified and hence becomes the most time consuming part of the MetID data mining.Experimenteller Teil: Amodel compound Ritonavir (a petidometic inhibitor of the HIV-1 protease) was analyzedin rat plasma, rat urine and rat bile. The animals were administered at 10 mg/kg dose of ritonavir. A WatersACQUITY®UPLC®System was used for LC separations and a Waters Synapt G2 HDMS for MS detection.Ergebnisse und Diskussion: The TOF MSE data acquisition strategy allows both intact and fragment informationto be obtained for the analyte sample. The initial metabolite screening process using the dealkylation/MDF/peakpicking strategy utilizes the low energy acquisition, which contains intact metabolite information. Once identified,the compound’s high energy acquisition is automatically aligned so that the fragment ion information can bereviewed for structural elucidation and confirmation. The structural driven Met ID strategy utilizes the dealkylationtool to predict the possible cleavage sites, then MetaboLynx XS software will automatically build a Mass defectFilter (MDF) protocol to process the raw data file. The MDF filtered file is then used for the peak picking process.By comparing with the control sample, the potential metabolites are then proposed and reported in the MetaboLynxbrowser for interactive review.Neue Aspekte: Obtaining in-vivo metabolite identification results from high resolution UPLC/Synapt G2 MS withintelligent data processing software.Thema: Instrumentelle Entwicklungen, MetabolomicsKeywords: Intelligent Met ID workflow, high resolution UPLC/TOF MS, TOF MSE functionality, Mass defectFilter (MDF), dealkylation toolKontakt: [email protected]

179

P87

Mass Spectrometric Identification of Feeder Signals in PollenEmbryogenesis

Lippmann, Rico; Matros, Andrea; Kumlehn, Jochen; Mock, Hans-Peter

IPK-Gatersleben, Germany

Einleitung: Under certain culture conditions, immature pollen can be switched from gametophytic developmenttowards embryogenesis. The unique potential of pollen embryogenesis (POEM) for plant breeding programs is instrong contrast to the poor understanding of its underlying biological processes and the efficiency of embryogenicdevelopment However, a certain density of pollen is needed for embryogenic development, which indicates aself-generated feeder effect. Interestingly, co-cultivation of pistils can increase or substitute this feeder effect inpollen culture and improve the recovery of regenerable structures. [1,2]. To understand this feeder effect, secretedcompounds are analyzed to identify the responsible molecule(s).Experimenteller Teil: Medium from high density embryogenic pollen cultures (EPC) of barley and mediumconditioned by wheat pistils were analyzed for proteins and metabolites. For the identification of proteins, mediumsamples were filtered through a 10kDa size exclusion tube and an on-filter digestion with trypsin was performed.Peptides were separated with a nanoUPLC system and analyzed with an ESI-qTOFMS to elucidate peptide massand fragmentation data by a data-independent multiplexed LC-MS approach. Extracellular low molecular weightcompounds were analyzed with GC/MS in a first approach, because of the huge range of different chemical groupswhich can be measured within one single run. For details of extraction and derivatisation methods see [3]Ergebnisse und Diskussion: In the medium from both cultivation systems, a large number of metabolites andproteins could be identified. Metabolite analysis confirmed the secretion of different amino acids, organic acidsand sugars. Time course experiments revealed an initially high concentration of proline with a subsequent declinein the following stages of cultivation. Similar patterns were found for most of the secreted metabolites in thekinetic analysis. When media from pistil cultures were analyzed, a couple of metabolites found in EPC mediumwere also detected as well as some metabolites which were unique to this system. Protein identification performedwith different databases revealed a high number of secreted proteins in both systems. Evaluation of sequence datawith SignalP confirmed target motifs for most of the proteins identified in the secretome. All together more than 30proteins and over 20 metabolites could be identified or annotated from the datasets. The elucidation of unknowncompounds is still in progress. Data inspection indicated also an overlap of proteins and metabolites identifiedfrom the both independent systems. These compounds might be potential candidates, responsible for the feedereffect. To investigate these candidates in more detail, a bioassay system was established were only 200 pollen frombarley were cultivated in a millicell, not able to develop alone (negative control) but in the presence of co-cultivatedpistils (positive control).Neue Aspekte: Results raise the thesis that the stimulation of pollen embryogenesis is induced by general stressrelated mechanisms.Referenzen: [1] Broughton, S. (2008) Ovary co-culture improves embryo and green plant production in antherculture of Australian spring wheat (Triticum aestivum L.), Plant Cell Tissue and Organ Culture. 95, 185-195.;[2] Sun, M., Kieft, H., Zhou, C. and van Lammeren, A. (1999) A co-culture system leads to the formation ofmicrocalli derived from microspore protoplasts of Brassica napus L-cv. Topas, Protoplasma. 208, 265-274.; [3]Lippmann, R., Kaspar, S., Rutten, T., Melzer, M., Kumlehn, J., Matros, A. and Mock, H. P. (2009) Protein andMetabolite Analysis Reveals Permanent Induction of Stress Defense and Cell Regeneration Processes in a TobaccoCell Suspension Culture, InternationaThema: Proteine und Peptide, MetabolomicsKeywords: embryogenesis, extracellular, secreted, pollenKontakt: [email protected]

180

P88

Reverse Metabolomics: a new method for direct identification ofbioactive compounds in complex mixtures

Michels, Katharina; Haid, Mark; Gohr, André; Wessjohann, Ludger

Leibniz-Institut für Pflanzenbiochemie, Germany

Einleitung: Natural products are valuable lead compounds for the pharmaceutical industry. However, the tradi-

tional process of natural product based drug discovery is long and tedious. Crude extracts of biological materials

provide complex mixtures of hundreds or thousands of compounds with different chemical properties, which re-

quires excessive dereplication. Accordingly, there is a big demand for new and inventive methods for rapid identi-

fication and simultaneous avoidance of dereplication [1]. How is it possible to identify only the relevant bioactive

compounds?

Here, we present an activity correlation analysis method (AcorA) for the direct identification of unknown bioactive

compounds in crude extracts without the need of prior isolation.

Experimenteller Teil: LC-Ion trap-MS3: The data were obtained using the ion trap system LCQ Deca XP Max

(Finnigan) coupled with a HPLC system (Thermo Surveyor) (Hypersil GOLD, RP18, 150x1 mm, 5 µm, gradient

system: water, acetonitril each containing 0.2% acetic acid). Each sample was measured in triplicates in positive

and negative electrospray ionisation mode.

FTICR-MS: The high resolution ESI mass spectra were obtained from a Bruker Apex III Fourier transform ion

cyclotron resonance (FT-ICR) mass spectrometer (Bruker Daltonics) equipped with an Infinity™ cell, a 7.0 Tesla

superconducting magnet (Bruker), an RF-only hexapole ion guide and an external electrospray ion source (Agilent,

off axis spray).

Data processing: Peakpicking and alignment were carried out by using XCMS [2].

Ergebnisse und Diskussion: This method is based on the correlation of chromatographic and spectroscopic

profiles with biological or other activity data. It is assumed, that those signals caused by the active components

show the most significant correlation to the bioactivity. For the development of a new identification method for

activity relevant metabolites we used data dependent LC-Ion trap-MS3 and FTICR-MS as analytical tools and

crude methanolic extracts of the fungal genus Hygrophorus (Basidiomycetes). The bioactivity was determined

using a quantitative antibacterial growth inhibition assay (Bacillus subtilus), and correlated using known as well

as proprietary software tools.

The ab initio identification enables the directed isolation of only the active natural products towards retention time

and m/z values with avoidance or minimization of dereplication due to additional structural information.

Neue Aspekte: AcorA (activity correlation analysis) - a rapid method for identification of bioactive compounds in

complex mixtures.

Referenzen: [1] M. Heinrich, Phytochemistry Lett. 1, 1-5 (2008).; [2] C.A. Smith et al., Anal. Chem. 78, 779-787

(2006).


Keywords: Reverse metabolomics, metabolite profile, bioactivity, correlation analysis


181

P89

Standardization of Preanalytical Protocols for ClinicalMetabolomic Studies by Tandem Mass Spectrometry

Brauer, Romy; Leichtle, Alexander; Baumann, Sven; Fiedler, Martin; Thiery, Joachim; Ceglarek, Uta

University Hospital Leipzig, Germany

Einleitung: ‘Clinical metabolomics’ aims at evaluating and predicting health and disease risk in an individualby investigating metabolic signatures. However, standardized preanalytical protocols are demanded to minimizein-vitro data variability in large scale clinical studies. Therefore, sample storage and pre-treatment procedureswere optimized to improve accuracy and data reproducibility. Dietary and circadian influences on the metabolomeprofile were investigated. The aim of our investigations was to develop a standardized protocol for reproduciblemetabolite profiling.Experimenteller Teil: An API 3000 tandem mass spectrometer (Applied Biosystems, Germany) using Turbo

Ionspray was used for flow injection analysis. Our established newborn screening method for dried blood samples

was upgraded for the profiling of 27 amino acids and 35 acylcarnitines in 1.5 min. Exogenous variables (e.g.

sampling material, storage conditions and time, batch processing) and endogenous variables (fasting, gender,

age) were investigated with 300 samples from 20 male and 30 female volunteers.

Ergebnisse und Diskussion: The comparison of native and EDTA dried blood results in similar amino acid

and acylcarnitine concentrations. Long chain acylcarnitine pattern significant decreased in serum and plasma

(EDTA, Citrate, Heparin). The storage of EDTA dried whole blood spots up to 4 hours at room temperature

did not significantly influence the metabolite profile. Optimization of the sample-extraction protocol resulted in

between day variability of < 20%. Study samples should be processed in one single batch (sample-pretreatment and

measurement) to minimize data variability. Before blood taking a fasting period of 5 hours has to be considered.

Significant gender specific differences in the metabolism of amino acids and activated fatty acids were observed.

Neue Aspekte: This reliable pretreatment protocol allows standardization of preanalytical modalities and facili-

tates reproducible metabolite profiling in large clinical studies.

Referenzen: [1] Dooley, K. C. (2003). "Tandem mass spectrometry in the clinical chemistry laboratory." ClinBiochem 36(6): 471-81.; [2] Fiedler, G. M., (2004). "Application of mass spectrometry in clinical chemistry andlaboratory medicine " LaboratoriumsMedizin 28: 185-194.; [3] Chace, D. H. (2005). "A biochemical perspectiveon the use of tandem mass spectrometry for newborn screening and clinical testing." Clin Biochem 38: 296-309.;[4] Ceglarek, U. (2008) Challenges and developments in tandem mass spectromtery based clinical metabolomics.Mol Cell Endocrinol. n pressThema: MetabolomicsKeywords: Clinical Metabolomics, Tandem Mass Spectrometry, Preanalytical Protocol, Acylcarnitines, AminoAcidsKontakt: [email protected]

182

P90

Antioxidants in Carob Bean Extracts: Evaluation of ExtractionMethods Using High Resolution Mass Spectrometry

Crone, Catharina (1); Scigelova, Michaela (1); Hornshaw, Martin (1); Duarte, Luis (2); Roseiro, Luísa B. (2);Gírio, Francisco (2); Bernardo-Gil, M. Gabriela Bernardo-Gil (3)

1: Thermo Fisher Scientific, Bremen, Germany; 2: Unidade de Bioenergia, LNEG -Laboratório Nacional de

Energia e Geologia, Lisboa, Portugal; 3: Centro de Engenharia Química e Biológica, IBB, Lisboa, Portugal

Einleitung: Carob beans are a rich source of phenolic compounds with potential use as human dietary supple-

ments. Variations in the extraction procedure employed can influence significantly the kind of extracted phenolic

species and their recovery. A direct coupling of uHPLC to a high mass resolution/accuracy detector with an elec-

trospray source was tested for its suitability to perform both identification and relative quantitation of phenolic

compounds in these very complex natural product extracts

Experimenteller Teil: The samples were extracted using 5 different procedures with the aim to obtain maximum

yield and variety of phenolic compounds in individual extracts. The extracted samples were re-dissolved in water

or water/methanol, sonicated and centrifuged. The compounds were separated on Thermo Hypersil GoldTM PFP

(100x2.1 mm, 1.9 mm particle size). The HPLC was directly coupled to a benchtop Orbitrap mass spectrometer

via an electrospray source operated in negative ionisation mode. The data acquisition consisted of a full scan over

the m/z range 100-2000 with detection at 100,000 resolving power followed by MS/MS using higher collisional

energy fragmentation in a multipole cell (HCD cell). Fragments were detected over the mass range 70-1000 at

25,000 resolving power

Ergebnisse und Diskussion: High mass resolution and high mass accuracy measurements enabled us to detect

many phenolic compounds of interest and compare their relative amounts in various samples. The high mass

resolution enabled reliable accurate mass measurement and precise quantitation by removing potential interfer-

ences. High mass accuracy analysis provides straightforward confirmation of compound identity in many cases

by affording unambiguous elemental composition. Fragmentation spectra further increased confidence of identi-

fication. Finally, multivariate analysis enabled comparison of the extraction procedures with respect to individual

phenolic compound content. The identity of the key differentiators was established using the above mentioned

information. The principal component analysis (PCA) showed relatively tight grouping of four samples, while one

extraction sample was clearly different and resulted in remarkably higher recovery of tricetin-3’,5’-dimethyl ether,

chrysoeriol, ferulic acid and naringenin compared to other methods

Neue Aspekte: High resolution mass spectrometric detection provided an effective tool to evaluate various extrac-

tion procedures yielding phenolic compounds from carob beans

Referenzen: [1] Papagiannopoulos M. et al., J. Agric. Food Chem. 2004, 52, 3784-91.

Thema: Metabolomics

Keywords: Metabolomics, Orbitrap, High Resolution


183

P91

Tandem mass spectrometry-based targeted metabolomics oflow-sinapine Brassica napus seeds

Wolfram, Karina; Frolov, Andrej; Böttcher, Christoph; von Röpenack-Lahaye, Edda; Strack, Dieter

Leibniz Institute of Plant Biochemistry, Germany

Einleitung: Oilseed rape (Brassica napus) is one of the most important oil plants in the world. However, theseeds accumulate high amounts of phenolic compounds, mainly sinapine and other sinapate esters that limit thenutritional value of the protein-rich seed meal (press cakes). UGT84A9 (UDP-Glucose:Sinapate Glucosyltrans-ferase) was identified as a key enzyme of sinapate ester biosynthesis [1]. Accordingly, silencing of UGT84A9gene experession with a dsRNAi vector reduced sinapate ester content in seeds [2]. The aim of this study was tocharacterize qualitative and quantitative changes in phenolic metabolite levels caused by silencing of UGT84A9 inBrassica napus seeds. For this, a targeted RP-HPLC-MS/MS approach was established.Experimenteller Teil: The oilseed rape lines with reduced sinapate ester content were obtained by Agrobac-

terium tumefaciens transformation with a pLH-UGT84A9i construct [1,2]. For evaluation of semi-polar secondarymetabolite patterns, methanolic seed extracts were analyzed by a Waters ACQUITY UPLC system equipped with aHSS T3 C18 (1x10 mm, 1.8 µm) column and coupled online either to a Q-Trap 3200 mass spectrometer (AB Sciex)or a QqTOF mass spectrometer (Bruker Daltonics). For annotation and relative quantification of seed components,a MRM method was established, using the data obtained by the non-targeted QTOF-MS-based metabolomicsapproach. For this, MRM transitions were constructed and optimized on the base of metabolite fragmentationpatterns.Ergebnisse und Diskussion: As reported earlier, silencing of UGT84A9 gene expression resulted in a substantialdecrease of sinapine, the major sinapate ester in seeds of Brassica napus [2]. For T4 seeds, a sinapine reductionof 64% in comparison to wild-type was observed [2]. The quantification of the total sinapate ester content showedfor UGT84A9i seeds a maximal reduction of 76% relative to the wild-type plants [2]. For a more detailed inves-tigation and evaluation of metabolic changes in low-sinapine seeds both targeted and non-targeted metabolomicsapproaches were applied. Using the results of the non-targeted study it was possible to establish a MRM methodfor quantification of 62 annotated phenolic seed constituents of Brassica napus. Almost all of these metaboliteswere quantified by two MRM transitions. Furthermore, 24 unknown analytes that showed a different accumu-lation pattern in low-sinapine seeds in comparison to wild-type, were analyzed by their fragmentation patternand subsequently one MRM transition was choosen for relative quantification. In total, 86 analytes were rela-tively quantified in one 10-min LC-MS/MS run. The analysis of UGT84A9i seeds revealed a strong reductionof many other phenylpropanoid conjugates as well, namely 1-O-sinapoylglucose, sinapoylmalate or 1,2-di-O-sinapoylglucose. Furthermore, a substantial decrease in phenolic choline esters (caffeoylcholine, feruloylcholineand 5-hydroxyferuloylcholine) was found. The strong decrease in sinapate ester content was accompanied by a mi-nor increase of several other phenolic glucosides, namely sinapate 4-O-glucoside, sinapoylcholine 4-O-glucoside,syringate 4-O-glucoside and syringoylcholine 4-O-glucoside. Furthermore, alterations in the content of hydroxy-benzoates and kaempferol derivatives as well as lignin building blocks were observed.Neue Aspekte: Targeted and non-targeted metabolomics revealed far-reaching alterations on phenylpropanoidmetabolism caused by silencing of UGT84A9.Referenzen: [1] Milkowski et al. Plant J. (2004) 38:80-92; [2] Hüsken et al. Mol. Breed. (2005) 16:127-138

Thema: Metabolomics

Keywords: Brassica napus, sinapate ester metabolism, targeted metabolomics, multiple reaction monitoring


184

P92

Identification and quantification of phenolic compounds in cellwalls of Brassica napus seeds

Frolov, Andrej; Wolfram, Karina; Henning, Anja; Böttcher, Christoph; von Röpenack-Lahaye, Edda; Strack,Dieter


Einleitung: Oilseed rape (Brassica napus) is one of the most important oil-yielding crops in the modern agri-

culture. Though the press-cakes, left after the oil production, are rich in valuable proteins, they contain however

antinutritive components, with fiber and sinapine as the most abundant ones [1]. These substances prevent the use

of press-cakes for animal feeding and human nutrition. Hence, their content therein should be reduced. Though

lignin was reported to contribute significantly to the fiber component [2], few information concerning the structure

of cell wall phenolics in Brassica napus seeds is available. The aim of this study was to establish approaches for

the analysis of cell wall phenolics in the seeds of Brassica napus plants with reduced antinutritive contents.

Experimenteller Teil: The seeds were grinded in a ball mill and the cell wall material was prepared. The ester-

bound phenolics were extracted with 1 mol/L NaOH for 24 h at 80ºC. The resulted mixture was separated by

RP-UPLC, coupled online to PDA detector as well as either ESI-Q-Trap or ESI-QqTOF mass spectrometers.

Identification of components was performed by mass spectra and based on comparison with authentic standards,

exact masses and characteristic fragmentation patterns. Relative and absolute quantification was performed in

MS/MS mode by multiple reaction monitoring. For simultaneous identification and quantification of cell-wall

components Information-Dependent Acquisition (IDA) was applied.

Ergebnisse und Diskussion: The chromatographic system for separation of soluble phenolics was optimized with

a standard mixture of 19 standard compounds including hydroxycinnamic and hydroxybenzoic acids, as well as

their aldehydes and alcohols. The best separation of the compounds was achieved by the use of 0.1% aq. formic

acid in methanol as a mobile phase (pH 3.0), while 0.125 mmol/L aq. ammonium acetate in acetonitrile (pH

6.7) was the most favorable for ionization in the mass spectrometer source. The compromise between chromato-

graphic resolution and MS-intensity was found by adjusting the pH of the mobile phase to 4. The HPLC-profiles of

the alkali-extractable phenolic fraction are strongly dominated with 4-hydroxybenzaldehyde, vanillin (4-hydroxy-

3-methoxybenzaldehyde) and ferulic acid (4-hydroxy-3-methoxycinnamic acid). Altogether, 16 phenolics were

identified by their characteristic retention times, UV-VIS spectra, exact masses and characteristic fragmentation

patterns. Further precursors of lignin biosynthesis (e.g. caffeoyl alcohol and 5-hydroxyferuloyl alcohol) were

tentatively identified by prediction of their fragmentation pathways. Also 47 unknowns were annotated and ten-

tatively identified as lignin cleavage products by their fragmentation. These results were additionally confirmed

in the course of explorative Information-Depentent Acquisition experiments, utilizing an MRM experiment as a

survey scan and Q-Trap-MS/MS as dependent ones. The developed targeted metabolomics workflow was applied

to characterization of plants with reduced antinutritive (sinapine and fiber) contents. The corresponding metabolite

patterns of cell-wall phenolics in seeds demonstrated clear difference from those observed in wild type plants.

Besides, correlation between cell-wall composition and seed color was observed.

Neue Aspekte: A set of methods for chromatography-mass spectrometry analysis of cell-wall bound phenolics

was established for the seeds of oilseed rape.

Referenzen: [1] 1. Font R, Wittkop B., Badani A.G., del Rio-Celestino M., Friedt W., LühsW. and de Haro-Bailon

A. The measurement of acid detergent fibre in rapeseed by visible and near-infrared spectroscopy (2005). Plant

Breeding 124: 410-412.; [2] 2. Bell J.M. Nutrients and toxicants in rapeseed meal: a review (1984). J. Anim Sci.

58: 996-1010.


Keywords: cell wall phenolics, tandem mass spectrometry, Q-Trap-MS, targeted metabolomics


185

P93

Massenspektrometrische Analyse vonPhenylpropan-Polyaminkonjugaten in Pollen von Arabidopsis

thaliana

Handrick, Vinzenz; Vogt, Thomas; Frolov, Andrej


Einleitung: Pollenwände sind extrem widerstandsfähig. Sie schützen die Keimzellen vor äußeren Einflüssen. For-mal setzt sich die Pollenwand aus dem äußeren Exine und dem inneren Intine zusammen. Neben dem für dieextreme Widerstandsfähigkeit beteiligtem Sporopollenin sind zahlreiche Metabolite, Polysaccharide, Kohlenwas-serstoffe, Lipide, Lignin-ähnliche Substanzen und Proteine in Pollenwänden vorhanden. Obwohl verschiedenebiochemische und analytische Untersuchungen zu Pollen vorliegen [1], ist die Zusammensetzung des Pollens vonArabidopsis thaliana auf Metabolitenebene bislang kaum untersucht. Besonderes Interesse weckt die Gruppe vonPhenylpropan-Polyaminkonjugaten. Diese Substanzen sind in Blütenknospen bereits nachgewiesen. Ihre biologi-sche Funktion ist bislang unbekannt [2]. Das Ziel dieser Arbeit ist eine möglichst vollständige Charakterisierung

dieser Substanzklasse in Pollen unter Verwendung von RP-UPLC-MS und Tandemmassenspektrometrie (MS/MS).

Experimenteller Teil: Für die Analysen wurden jeweils 5-10 mg reiner A. thaliana Wildtyp-Columbia-0 Pol-

len verwendet. Dieser wurde mit 80%igem Methanol extrahiert. Der Extrakt wurde hinsichtlich phenolischer Se-

kundärmetabolite untersucht. Mit UPLC-QTOF-MS erfolgte eine ungerichtete Analyse von Sekundärstoffen. Die

im Mittelpunkt des Interesses stehenden Phenylpropan-Polyaminkonjugate wurden mittels UPLC-QTrap-MS/MS

gerichtet untersucht. Dafür wurden die Massen von möglichen Polyaminkonjugaten theoretisch berechnet und

entsprechende MS/MS-Scans aufgenommen. Aufgrund der Fragmentierungsmuster wurde eine MRM (Multiple

Reaction Monitoring)-Methode etabliert.

Ergebnisse und Diskussion: Sowohl die gerichtete als auch die ungerichtete Metabolomuntersuchung des me-

thanolischen Pollenextraktes lieferte reiche Strukturinformationen über die Zusammensetzung der Phenylpropan-

Polyaminkonjugate-Fraktion. Auf Basis der UPLC-QTof-MS Läufe wurden mehrere mögliche Polyaminkonjugate

aufgrund ihrer Retentionszeiten und genauenMassen (mit Massengenauigkeit < 5 ppm) annotiert. Mittels der Frag-

mentierungsmuster der Tandem-Massenspektrometrie ließen sich konjugierte Polyamine eindeutig nachweisen.

Weiterführend soll die Pollenwand schrittweise in mehreren Extraktionsschritten gelöst werden und neben ande-

ren Substanzklassen insbesondere die Phenylpropan-Polyaminkonjugat-Zusammensetzung geklärt werden. Diese

Untersuchungen sollen auch die noch ungelösten Fragen zur Akkumulation dieser Substanzen auf der Pollenober-

fläche beantworten.

Neue Aspekte: Die vorgestellte Methode zur Charakterisierung des Pollens ermöglicht mit geringen Ausgangs-

mengen eine effiziente und zuverlässige Identifizierung der komplexen Pollenbestandteile.

Referenzen: [1]Arráez-Roman, D., Zurek, G.,Bäßmann, C., Almaraz-Abarca, N., Quirantes, R., Segura-Carretero,A., Fernandez-Gutiérrez, A., 2007. Identification of phenolic compounds from pollen extracs using capillaryelectropheresis-electrospray time-of-flight mass spe; [2] Fellenberg, C., Böttcher, C., Vogt, T., 2009. Phenylpropa-noid polyamine conjugate biosynthesis in Arabidopsis thaliana flower buds, Phytochemistry 70, 1392-1400.Thema: Naturstoffe, MetabolomicsKeywords: Arabidopsis, Pollen, QTrap-MS, Tandemmassenspektrometrie, PolyaminkonjugateKontakt: [email protected]

186

P94

Toward glycosphingolipid-based cancer diagnosis: first insightsinto nano-HPLC/MS data of human serum samples

Kirsch, Stephan; Souady, Jamal; Peter-Katalinic, Jasna; Bindila, Laura

Westfälische Wilhelms-Universität Münster, Institut für Medizinische Physik und Biophysik, Münster, Germany

Einleitung: Glycosphingolipids (GSLs) are biomolecules present in the outer membrane leaflet of almost allmammalian cells. Given their participation in processes like cell-development, cell-cell-interaction and immuneresponse they are of high interest in biological and clinical research [1]. Moreover, their expression has beenreported to change with malignant transformation [2] making them promising candidates for cancer diagnosis. Inthis context, different levels of abberant glycosylation have been observed so far, e.g. overexpression of gangliosidespecies, elevated or downregulated levels of GSLs supporting metastasis or reduced apoptosis of tumor cells [3].Due to shedding processes occurring at cell surfaces, GSLs are removed from the cell membrane promoting theiraccumulation in the blood stream [4].Experimenteller Teil: With respect to their molecular structure GSLs are amphipathic molecules comprisingan unpolar ceramide moiety attached to a polar glycan chain. Because of this antagonism purification and/orchromatographic analysis is a challenging task. Additionally, their very low abundance in biological mixturescompared to that of phospholipids mandate for even more efforts toward glyco-biomarker discovery. We developeda set of techniques for the small-scale purification of GSLs involving liquid extraction and phospholipase digestionfollowed by solid phase extraction. Fractions obtained were subsequently analyzed by nano-high performanceliquid chromatography (nano-HPLC) interfaced with MS as reported previously [5].Ergebnisse und Diskussion: Here we report the application of this methodology to the analysis of human serumGSLs. In order to investigate how GSL patterns change with cancer progression, samples obtained from patientssuffering from pancreatic and stomach cancer were examined and compared with those of control volunteers.Neue Aspekte: nano-HPLC screening for glycosphingolipid-based biomarker discovery in cancerReferenzen: [1] Hakomori S (2003), Curr Opin Hematol 10, 16; [2] Distler U, Souady J, Hülsewig M, Drmic-Hofman I, Haier J, Denz A, Grützmann R, Pilarsky C, Senninger N, Dreisewerd K, Berkenkamp S, Schmidt MA,Peter-Katalinic J, Müthing J (2008), Mol. Cancer Ther. 7, 2464; [3] Hakomori S (2002), PNAS 99, 10231; [4]Lauc G, Heffer-Lauc M (2006), Biochim Biophys Acta 1760, 584; [5] Kirsch S, Müthing J, Peter-Katalinic J,Bindila L (2009), Biol Chem 390, 657Thema: Kohlenhydrate, LipideKeywords: nano-HPLC, glycosphingolipid, human serum, pancreatic cancer, stomach cancerKontakt: [email protected]

187

P95

Existiert eine spezifische Bindung von (Phospho)-Lipiden anKieselgel-Oberflächen? Eine TLC, MALDI-TOF MS und

NMR-Untersuchung

Teuber, Kristin; Riemer, Thomas; Süß, Rosmarie; Schiller, Jürgen

Universität Leipzig, Germany

Einleitung:DieMALDI-TOFMSwird zunehmend für die Analytik von Lipiden verwendet, da mit dieser Technik-auch an Gemischen-schnelle und zuverlässige Informationen über die Zusammensetzung erhältlich sind. Dennochsind verschiedene Lipidklassen mit unterschiedlicher Sensitivität nachweisbar [1]: In der Regel werden z.B. diePositiv-Ionen-Massenspektren von den Phosphatidylcholinen (PC) dominiert. Obwohl dies nicht zwangsläufig einNachteil ist (der zudem durch die überlegte Wahl der Matrix überwunden werden kann [2]), ist für eine detail-liertere Analyse vorherige Trennung erforderlich, wozu sich insbesondere die Dünnschichtchromatografie (TLC)anbietet. Für die anschließende (quantitative) Analyse ist jedoch das Wissen erforderlich, ob alle Lipidklassen ingleichem Maße eluiert werden können, oder ob bestimmt Lipidklassen eine bevorzugte Absorption an die statio-näre Phase zeigen.Experimenteller Teil: Hühnereigelb wurde mit einem CHCl3/CH3OH Gemisch extrahiert. Das erhaltene Lipid-gemisch wurde mit einem internen Standard versetzt und anschließend mittels TLC auf kommerziell erhältlichenHPTLC 60 Platten (Merck) getrennt. Die einzelnen Spots wurden dann (mit unterschiedlichen Mengen an Lö-sungsmitteln) von der Platte eluiert und die einzelnen Spots mittels MALDI-TOFMS (Bruker Äutoflex", Reflektor-Modus) hinsichtlich der Fettsäurezusammensetzung charakterisiert. DHB oder 9-Aminoacridin (9-AA) wurden alsMatrices verwendet. Zusätzlich erfolgte die Charakterisierung der einzelnen Phospholipid-Klassen direkt auf derTLC-Platte gemäß [3]. Die Bestimmung der absoluten Phospholipidmengen erfolgte zusätzlich mittels 31P-NMR-(nuclear magnetic resonance)-Spektroskopie sowie mittels SStewart-Assay", bei dem die Bindung von Lipiden anEisenthiocyanat für die quantitative, fotometrische Bestimmung verwendet wird [4].Ergebnisse und Diskussion: Im Zusammenhang mit der TLC ist bekannt, daß eine Probe um so stärker in diestationäre Phase eindringt, je weiter sie migriert. Es wäre somit logisch anzunehmen, daß sich Lipide, die stark un-terschiedliche Rf-("Retardation Factors")-Werte aufweisen, auch in ihrem Extraktionsverhalten unterscheiden. Um

dies zu untersuchen wurden die einzelnen Banden (ohne Differenzierung der einzelnen Spots) mit unterschiedli-

chenMengen an Lösungsmitteln eluiert. Als Kontrolle diente der Gesamtextrakt ohne Trennung. Erwartungsgemäß

zeigte sich eine starke Abhängigkeit der Lipid-Ausbeute vom eingesetzten Volumen an Lösungsmittel. Es war je-

doch unter keinen Bedingungen möglich das gesamte Lipid zu eluieren, sondern ein beträchtlicher Teil blieb am

Kieselgel gebunden. Bemerkenswerterweise war jedoch die relative Zusammensetzung der einzelnen Lipidklassen

unverändert, so daß von keiner bevorzugten Absorption einer bestimmten Lipidklasse an das Kieselgel ausgegan-

gen werden kann. In der Literatur finden sich außerdem Hinweise auf einen sogen. Ëffect of Chromatography", d.h.die einzelnen Lipidklassen unterscheiden sich vor und nach der TLC in ihrer Fettsäurezusammensetzung [5], wo-bei besonders Lipide mit gesättigten Fettsäureresten eine verstärkte Bindung an die Kieselgeloberfläche eingehensollen. Ein solcher Effekt konnte jedoch nicht gefunden werden und die Zusammensetzung war vor und nach TLC-Trennung identisch. Zusammengefaßt ergeben sich damit keine signifikanten Änderungen bei der TLC-Trennung:Alle Lipidklassen werden mit identischer Fettsäurezusammensetzung und im gleichen relativen Verhältnis zuein-ander gefunden. Allerdings tritt ein sehr beträchtlicher Verlust der absoluten Menge an Lipid ein. Dies wirft einrealistisches Bild auf unsere Versuche die gekoppelte TLC/MALDI für quantitative Aussagen einzusetzen, da derpotentielle Absolutverlust von Lipid in Gegenwart eines internen Standards leicht korrigiert werden kann. Einespezifische Bindung von Lipiden an Kieselgeloberflächen kann ausgeschlossen werden.Neue Aspekte: Diese Arbeit untersuchte erstmalig detailliert die Veränderungen an Lipidgemischen (Fettsäure-zusammensetzung, absolute Ausbeuten einzelne Klassen) bei der TLC.Referenzen: [1] Petkovic, M., et al.: Anal. Biochem. 2001, 289:202-216.; [2] Sun, G., et al.: Anal. Chem. 2008,80:7576-7585.; [3] Fuchs, B., et al.: Anal. Bioanal. Chem. 2007, 389:827-834.; [4] Stewart, J.: Anal. Biochem.1980, 104:10-14.; [5] DeLong, C.J., et al.: J. Lipid Res. 2001, 42:1959-1968.Thema: LipideKeywords: TLC, MALDI-TOF MS, Phospholipide, Effect of ChromatographyKontakt: [email protected]

188

P96

(TLC)-MALDI-TOF MS OF LIPIDS: IS THERE A "PERFECT"MATRIX?

Eibisch, Mandy (1); Teuber, Kristin (1); Fuchs, Beate (1); Süß, Rosmarie (1); Schürenberg, Martin (2); Suckau,Detlev (2); Schiller, Jürgen (1)

1: Universität Leipzig, Germany; 2: Bruker Daltonics GmbH, Germany

Einleitung: The interest in lipid analysis by matrix-assisted laser desorption ionization time-of-flight mass spec-trometry (MALDI-TOF) MS is continuously increasing. Although 2,5-dihydroxybenzoic acid (DHB) is the sofar most established matrix, DHB detects different lipids with strongly different sensitivities. To overcome thisproblem, several new matrices such as 9-aminoacridine (9-AA), 2-mercaptobenzothiazole (MBT) and some otherswere recently introduced. Here we will discuss some characteristics of these matrices as well as their potentialapplicability to directly combined thin-layer chromatography TLC-MALDI-TOF MS.Experimenteller Teil: Crude lipid mixtures were obtained by extraction (CHCl3/CH3OH) from different cellcultures and hen egg yolk. All matrices were used as previously described. Samples were mixed 1:1 (v/v) withthe matrix prior to deposition onto a commercially available MALDI target. Commercial HPTLC60 plates withaluminum backs (Merck) were used for lipid separation. A novel spraying technique that allows covering theentire TLC plate with matrix was used. TLC plates were mounted onto a TLC-adapter target and automaticallyscanned using TLC-MALDI software (both Bruker). All measurements were performed on a Bruker "Autoflex"MALDI-TOF device.Ergebnisse und Diskussion: Many different lipid classes, for instance, phosphatidylcholine (PC), phosphatidyl-ethanolamine (PE), phosphatidylinositol (PI), sphingomyelin (SM) and different lysolipids (lacking one fatty acylresidue) are present in crude organic tissue or cell extracts. In the presence of standard DHBmatrix, it is impossibleto detect all phospholipid classes neither as positive nor as negative ions. This problem can be overcome by (a)separation of the individual PL classes prior to MALDI MS or (b) by using less acidic matrices than DHB. 9-AAoffers among all tested matrices the highest sensitivity and is, thus, the matrix of choice for detailed phospholipidanalysis. Additionally, 9-AA provided high sensitivity in some initial TLC-MALDI MS analyses. The negative iondetection of the individual PL classes turned out as a suitable alternative to positive ion detection because negativeion spectra are much simpler to interpret and there is no superposition between different adducts and differencesin the fatty acyl compositions of the phospholipids.Neue Aspekte: With the choise of the right matrix you can optimize your results and be more sensitive.Thema: LipideKeywords: MALDI-TOF MS, Phospholipide, MatricesKontakt: [email protected]

189

P97

High throughput sequencing of polysialylated gangliosides bychip-nanoelectrospray multistage collision-induced dissociation

Schiopu, Catalin (1); Serb, Alina (2); Flangea, Corina (1); Sisu, Eugen (2); Zamfir, Alina (1,3)

1: National Institute for Research and Development in Electrochemistry, Timisoara, Romania; 2: "Victor Babes"University of Medicine and Pharmacy, Timisoara, Romania; 3: "Aurel Vlaicu" University of Arad, Romania

Einleitung: Gangliosides encompass a large family of sialylated glycosphingolipids that occur in the outer layerof the plasma membrane in all eukaryotic cells [1,2]. Ganglioside oligosaccharide core differs in glycosidic link-age position, sugar configuration and the content of neutral residues and sialic acid. Oligosaccharide chain isattached to a hydrophobic ceramide, which anchors the whole molecule to the plasma membrane. Recent develop-ment [3,4] of chip-based nanoelectrospray (nanoESI) mass spectrometry (MS) proficient not only in accurate massmeasurement but also in generating specific fragmentation of selected precursor ions opened new perspectives inganglioside analysis field. However, several limitations of single stage fragmentation among which the lack of suf-ficient diagnostic ions for unequivocal determination of sialylation site(s) and ceramide structure characterizationwere reported.Experimenteller Teil: Ganglioside sample used in this study was a GT1 fraction from bovine brain purchased fromSigma (Taufkirchen, Germany). An aliquot was dissolved in pure methanol to the final working concentration of0.5 pmol/µL calculated for an average relative molecular mass (Mr) of 2000. Mass spectrometry was conducted innegative ion mode on a High Capacity Ion Trap Ultra (HCT Ultra, PTM discovery) mass spectrometer from BrukerDaltonics (Bremen, Germany) coupled via a custom-made interface to a NanoMate robot incorporating ESI 400Chip technology from Advion BioSciences (Ithaca, USA). Multistage MS (MSn) was carried out by collision-induced dissociation (CID) using He as the collision gas. Fragmentation spectra were obtained by accumulatingscans at variable RF signal amplitudes within 0.6-1.0 V.Ergebnisse und Diskussion: We report here on the development of a straightforward approach for high-throughputtop-down glycolipidomics based on NanoMate-HCT MS and CID MSn. The method was optimized and tested ona polysialylated ganglioside fraction (GT1) which was profiled by MS1 and sequenced in tandemMS up to MS6 inone and the same experiment. Screening of the fraction in the MS1 mode indicated the occurrence of six [M-2H]2−

ions which, according to calculation, support ten GT1 variants differing in their relative molecular mass due to dis-similar ceramide (Cer) constitutions. To test the feasibility of a top-down experiment for polysialylated gangliosidespecies, the doubly deprotonated molecule at m/z 1077.20 corresponding according to calculation to a ubiquitousGT1 form known to contain mostly ceramide of (d18:1/20:0) or (d18:0/20:1) compositions, was isolated withinan isolation window of 2u and submitted to stepwise fragmentation by low energy multistage collision-induceddissociation. By stepwise CID MS2-MS5 the complete characterization of its oligosaccharide core including theidentification of sialylation sites was achieved. Structure of the lipid moiety was further elucidated by CID MS6

analysis carried out using as a precursor Y0 fragment ion detected in MS5. MS6 fragmentation resulted in a patternsupporting aCer form having the less common (d20:1/18:0) configuration. MS1-MS6 were performed in one andthe same experiment, in a high-throughput mode, within a total acquisition time of only 2.66 minutes. In view ofthe flow rate provided by NanoMate chip technology, the employed solvent and working sample concentration, weestimate that only 0.133 pmols of material were used. Consequently, we succeeded in designing the first top-downapproach applicable to glycolipidomics surveys at sub-picomolar range of sensitivity that is functional in highthroughput mode without the need of additional separation techniques on-line coupled to MS.Neue Aspekte: We developed here the first approach for high-throughput top-down glycolipidomics based onfully automated chip-nanoelectrospray CID MS1-MS6.Referenzen: [1] Yu RK, Nakatani Y, Yanagisawa M., J. Lipid Res. 50, S440, 2009.; [2] Todeschini AR, HakomoriSI., Biochim. Biophys. Acta 1780, 421, 2008.; [3] Serb A, Schiopu C, Flangea C, Vukelic Ž, Sisu E, Zagrean L,Zamfir AD., Eur. J. Mass Spectrom.15, 541, 2009.; [4] Serb A, Schiopu C, Flangea C, Sisu E, Zamfir AD., J. MassSpectrom. 44, 1432, 2009.Thema: Kohlenhydrate, LipideKeywords: top-down glycolipidomics; NanoMate; multistage collision-induced dissociation; gangliosides; high-throughputKontakt: [email protected]

190

P98

HPTLC-MALDI MS and MS/MS Analysis of Lipid Mixtures

Anders, Ulrike (1); Fuchs, Beate (2); Bischoff, Andrea (2); Süß, Rosemarie (2); Suckau, Detlev (1); Morlock,Gerda (3); Schiller, Jürgen (2)

1: Bruker Daltonik GmbH, Germany; 2: University of Leipzig, Institute of Medical Physics and Biophysics,Leipzig, Germany; 3: University of Hohenheim, Institute of Food Chemistry, Stuttgart, Germany

Einleitung: Thin layer chromatography (TLC) is a well established chromatographic analysis method that permitsa robust, parallel analysis with analyte specific detection. Analytes such as lipids, that are difficult to be analyzed bycolumn chromatography, can very well be analysed by TLC. However, molecular identification of TLC-separatedbands is currently an awkward "scratch-elution" process. We coupled TLC offline with MALDI to permit a highresolution molecular readout that perpetuates the initial high chromatographic resolution for high performanceTLC-MALDI analysis of lipids.Experimenteller Teil: Model lipids were applied to 7.5*5 cm HPTLC60 plates with aluminum backing (Merck)using pneumatic spray assisted sample preparation. Developed TLC plates were uniformly covered by MALDImatrix. TLC plates were mounted onto a TLC- adapter target and automatically scanned using dedicated TLC-MALDI software on a MALDI-TOF/TOF with 100 µm spot raster along the chromatographic trace. LC-traceswere generated in the software permitting direct access to the TLC-MALDIMS data and to directly trigger MS/MSspectra acquisition from bands of interest.Ergebnisse und Diskussion: Various lipid classes (constant head groups, variable chain length and degree ofsaturation) were well separated and class specific MALDI spectra were observed. In addition, individual molec-ular species were selected in the software for acquisition of MS/MS spectra right from the surface of the TLCplates. These MS/MS spectra provide further specificity in assigning lipids with regard to their respective sn1/sn2substituents and their head groups. In combination, automated samples application and high resolution MALDIrastering of HPTLC plates offers a new powerful chromatographic system that may provide a solution for sam-ples that currently provide significant challenges in lipid analysis, lipidomics or in industrial applications such asherbals, food or cosmetics.Neue Aspekte: High resolution TLC-MALDI MS and MS/MS platform for lipid and industrial applications.Thema: Lipide, ImagingKeywords: TLC, MALDI, MS/MS, Lipide, TOFKontakt: [email protected]

191

P99

Benchmarking of shotgun lipidomics software tools

Herzog, Ronny (1); Schwudke, Dominik (2); Shevchenko, Andrej (1)

1: Max Planck Institute for Cell Biology and Genetics, Germany; 2: NCBS, Tata Institute of FundamentalResearch, Indea

Einleitung: Lipidomics aims at quantifying the full lipid complement in cells, tissues or organisms. Shotgunlipidomics relies on the direct analysis of total lipid extracts, in which the molecular species of lipids are recognizedby either accurately determined m/z in survey MS spectra or by structure-specific "signature ions" in MS/MSspectra of prospective precursors. Although the shotgun approach has been widely applied in biological research,the currently available software falls short of users expectations, since the programs only support particular typesof shotgun experiments. Here we report on a systematic comparison of the available software with our softwareLipidX that supports the currently broadest range of top-down and bottom-up shotgun experiments.Experimenteller Teil: Total lipid extracts of E.coli and bovine heart were analyzed by automated direct infusionexperiments on a QSTAR Pulsar I quadrupole time-of-flight (MDS Sciex) and LTQ Orbitrap XL mass spectrome-ters (Thermo Fisher Scientific) equipped with a robotic nanoflow ion source NanoMate HD (Advion BioSciences).The raw data files were converted into the *.mzXML format and imported by LipidX. Spectra interpretation rou-tines employed by LipidX are programmed in the molecular fragmentation query language (MFQL) enabling thesoftware to adapt to specific data types, instruments features and known fragmentation pathways without the needto modify the workflow.Ergebnisse und Diskussion: Conventional approaches to the interpretation of MS and MS/MS spectra rely upona database of reference spectra that are compared with the acquired spectra. Since lipids have just a small numberof signature fragments, direct spectrum to spectrum comparison lacks the identification specificity. Therefore,most lipidomics software compare peak lists with pre-computed intact lipid masses stored in a database or text file.However, such databases are rarely complete and miss structural variations within already known classes, as well asnovel lipids. Contrary, MFQL allows designing and applying straightforward algorithms to distinguish structuralsignatures of particular lipid classes in MS and MS/MS spectra. Therefore, many known and putative lipid classescan be profiled in parallel in a single comprehensive shotgun dataset. Importantly, LipidX aligns spectra acquiredwithin the series of experiments, hence allowing flexible sorting of signals according to their abundance, or foldchanges etc., while sorting criteria can be bundled by Boolean operations. To benchmark LipidX performanceagainst the dedicated lipidomics application software, we first developed an approach to estimate the false positiverate of lipid species identifications and demonstrate how it is affected by the resolution and mass accuracy ofmost common types of instruments. Using complex standard lipid extracts analyzed on QSTAR and LTQ Orbitrapmass spectrometers in different operation modes, we benchmarked LipidX performance against popular shotgunsoftware LipidProfiler[1], LipidQA[2] and LipidMaps[3].Neue Aspekte: Generic approach to benchmark lipidomics software.Referenzen: [1] [1]C. Ejsing et. al, Anal. Chem. 2006, 78, 6202-6214; [2] [2]H. Song et al, Am. Soc. MassSpectrom. 2007, 18, 1848; [3] [3]K. Schmelzer at al, Methods Enzymol. 2007, 432, 171-83Thema: LipideKeywords: Shotgun lipidomics, bioinformaticsKontakt: [email protected]

192

P100

Analysis of arachidonic acid and related fatty acid metabolites byhybrid QTrap mass spectrometry

Kortz, Linda; Bruegel, Mathias; Leichtle, Alexander; Fiedler, Georg Martin; Thiery, Joachim; Ceglarek, Uta

Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig,Liebigstraße 27, 04103 Leipzig, Germany

Einleitung: Eicosanoids are products of arachidonic acid (AA) bound in cell membranes to phospholipids. Theyplay important functional roles in inflammation, cellular proliferation,and intracellular signalling. Much lessisknown about the non-enzymatic metabolites, e.g. isoprostanes. Our aim was to establish a fast analytical methodfor the simultaneous quantification and structural verification of known eicosanoids as well as structural elucidationof unknown AA metabolites by combining liquid chromatography (LC) and hybrid triple quadrupole/linear iontrap mass spectrometry (QTrap MS). This will allow us to study the metabolic fate of eicosanoids in patients withcardiovascular disease and to investigate the genetic diversity of eicosanoid metabolism in large scale populationstudies.Experimenteller Teil: 200 µL of human EDTA-plasma was used after protein precipitation and off-line solidphase extraction. The LC-QTrap MS method was developed using an API 4000 QTrap tandem mass spectrometerwith electrospray ionization in negative ion mode. Our method currently contains about 50 multiple reactionmonitoring (MRM) transitions of eicosanoids for quantitation. Product ion spectra are generated by changing toion trap mode during a run, thus allowing verification of each lipid through the characteristic fragment pattern.Ergebnisse und Diskussion: Amass spectrometric library containing characteristic fragment pattern of prostaglan-dins, thromboxanes and hydroxy-eicosatetraenoic acids (HETEs) was created from our QTrap experiments. LC-QTrap MS showed comparable sensitivity as conventional LC-MS/MS. Storage and freeze-thaw cycles have beeninvestigated with regard to stability of the analyzed lipids. Significant differences in the stability of the eicosanoidsrequest strict preanalytical standardization. Our analytical method using LC-QTrap MS technology [1] allows nowthe simultaneous acquisition of both quantitative data and structural information of a multitude of AA metabolitesin human biological fluids. The simultaneous analysis of eicosanoids will help to unravel molecularmechanismsunderlying the complex AA metabolic system and the functional relevance of eicosanoid gene polymorphisms.Neue Aspekte: LC-QTrap MS method for analysis of eicosanoids, preanalytical data for measurements fromplasma samplesReferenzen: [1] Kortz et al., J Lab Med 2009;33(6):341-348Thema: Lipide, MetabolomicsKeywords: arachidonic acid metabolites, eicosanoids, hybrid triple quadrupole/linear ion trap mass spectrometryKontakt: [email protected]

193

P101

Elucidation of the Fragmentation Pathways of DifferentPhosphatidylinositol Phosphate Species (PIPx) using IRMPD and

CID Implemented on a FT-ICR MS

Zehethofer, Nicole; Lindner, Buko

Research Center Borstel, Germany

Einleitung: PIPx species play an important role in various cellular processes. [1] However, only low amountsare present in biological samples making their quantification quite difficult. PIPx profiling can be done by meansof tandem mass spectrometry (MS/MS) [2] or by liquid chromatography in combination with tandem mass spec-trometry (LC-MS/MS) to resolve regioisomers.[3] We noted, however, that a detailed comparative study of thefragmentation of PIPx species using infrared multiphoton dissociation (IRMPD) and collision induced dissocia-tion (CID) has not been conducted and that there is no LC-MS/MS method published which uses data dependantacquisition.Experimenteller Teil: In this work, we studied the fragmentation of 17:0-20:4 PI, 17:0-20:4 PI(1)P, 17:0-20:4PI(2)P, and 17:0-20:4 PI(3)P using MS/MS and MS³ experiments on a hybrid apex-Qe Fourier Transform-IonCyclotron Resonance Mass Spectrometer (FT-ICR MS) using IRMPD and CID for fragmentation. The separationof PIPx on a reversed-phase (RP) C8 column was recently published [4] and the method was modified to fit ourexperimental requirements. Based on these results, an LC-MS/MS method for the quantification and identificationof PIPx species was designed.Ergebnisse und Diskussion: The fragmentation behaviour of the singly charged PIPx species was investigatedin detail using CID and IRMPD. The high mass accuracy of the FT-ICR MS allowed assignment of molecularformulae to product ions of given m/z values. IRMPD was more efficient than CID for fragmentation of thesingly charged species and was chosen as fragmentation technique. Fragmentation of PIPx with IRMPD yieldedabundant product ions and neutral losses specific for the acyl chains and the number of phosphates. The IRMPDlaser energy needed to obtain high quality spectra was different for the investigated PIPx species. Therefore,CID was used for the development of a data dependant LC-MS/MS method as the collision energy could beadjusted during the LC run depending on the mass of the precursor ion of interest. Fragmentation conditions wereoptimized for the fragmentation of the doubly charged PIPx species and used for the development of a LC-MS/MSmethod allowing the identification of PIPx species eluting from the column. Limits of detection (LOD, definedas S/N=3) were below 1 pmol on column. The LC-MS/MS method was also used for the separation of somephospholipids (phosphatidylserine, phosphatidyl glycerol, phosphatidylinositol, ceramide) which could be foundas contaminations in biological isolates. These phospholipids were successfully separated under the conditionsdescribed for PIPx separation. AcknowledgementThis work was financially supported by the DFG SFB-TR 22 Project Z01Neue Aspekte: Detailed study of the IRMPD fragmentation of PIPx species for the development of a data depen-dant LC-MS/MS method.Referenzen: [1] Wymann et al. Nature Rev. Mol. Cell Biol. 2009, 9: 162-172; [2] Milne et al. J. Lipid Res.2005, 46: 1796-1802; [3] Pettitt et al. J. Lipid Res. 2006, 47: 1588-1596; [4] Ogiso et al. Anal. Chem. 2008, 80:9226-9232Thema: LipideKeywords: PIPx, IRMPD, LC-MS/MS, data dependant acquisitionKontakt: [email protected]

194

P102

Kombinierter Einsatz von zwei komplementärenmassenspektrometrischen Techniken und multivariater

Datenanalyse zur Charakterisierung pflanzlicher Öle und Fette

Krist, Sabine (1); Stübiger, Gerald (2); Belgacem, Omar (3); Werther, Wolfgang (4)

1: Department für Klinische Pharmazie und Diagnostik, Universität Wien; 2: Zentrum für BiomolekulareMedizin und Pharmakologie, Medizinische Universität Wien; 3: Shimadzu Biotech, Manchester, UK; 4: Labor

Massenspektrometrie Prof. Schmid, Wien

Einleitung: Die Qualität von Ölen von hohem Wert für die Ernährung (z.B. Olivenöl) oder für pharmazeutis-che Anwendungen (z.B. Mandelöl) kann durch Verfälschung ernstlich gemindert werden. Routinemethoden zurQualitätskontrolle von Pflanzenölen (z.B. Bestimmung der Fettsäurezusammensetzung) liefern oft keine zufrieden-stellenden Aussagen, insbesondere wenn zur Verfälschung Öle ähnlicher Fettsäurezusammensetzung verwendetwerden. Die Triacylglycerin-Muster (TAGs) der Öle besitzen einen höheren Informationsgehalt als die Fettsäure-muster allein, da die Fettsäurepositionen bei der Biosynthese der TAGs in Pflanzen spezifisch besetzt werden.Während die TAGs die Hauptbestandteile der Pflanzenöle mit artspezifischen Mustern bilden, kann Vorhandenseinund Menge der flüchtigen Verbindungen durch die Verarbeitungsbedingungen (z.B. Presstemperatur, vorherigesRösten) stark beeinflusst werden. Daher bietet die kombinierte Analyse beider Parameter mehr Information zurCharakterisierung von hochwertigen Pflanzenölen.Experimenteller Teil: MALDI-MS(/MS):. Die in Chloroform gelösten Öle wurden in einer binären Natriumfer-rocyanid (Na4[Fe(CN)6]) - Glycerin Matrix auf einem AXIMA-CFR (Shimadzu Biotech) Reflektor-TOF Massen-spektrometer vermessen. Die Identifikation der Triglyceride wurde durch eine selbst entwickelte Software (Li-pidIdent) unterstützt. Headspace-SPME-GC-MS: Die flüchtigen Verbindungen im Dampfraum über den Ölenwurden mittels einer SPME - DVB/Carboxen/PDMS Faser bei Raumtemperatur extrahiert und mittels GC-MS aufeiner 60m RTX-5 Kapillarsäule vermessen. Die Identifikation der Verbindungen erfolgte über MS-Datenbanken,Retentionsindices und Vergleichsmessungen von Referenzsubstanzen. Multivariate Datenanalyse (MVDA): Lin-eare Mapping-Methoden wie etwa die Hauptkompontenanalyse und der PLS-Diskriminanzplot wurden auf Daten-sätze von GC- und MALDI-Peakintensitäten angewandt. Die resultierenden Mappings wurden visuell auf Clusteruntersucht und die verantwortlichen Analyten durch Interpretation der Faktorloadings detektiert.Ergebnisse und Diskussion: Über 300 verschiedene flüchtige Verbindungen wurden imDampfraum der Pflanzenölegefunden, vor allem Carbonylverbindungen, Alkane, Alkene, Alkohole, Carbonsäuren und Terpene. Pyrazine undFuranverbindungen treten dann auf, wenn während der Produktion eine Hitzebehandlung erfolgt ist. Individu-elle TAGs werden in den MALDI Massenspektren anhand ihrer zugehörigen Molekülmassen erfasst. MS/MS-Analyse (PSD or CID) ermöglicht eine Unterscheidung isobarer TAGs mit gleicher Molekülmasse, aber unter-schiedlicher Fettsäurezusammensetzung. Regioisomere mit identer Fettsäurezusammensetzung aber veschiedenerPosition (sn-1,2,3) am Glycerin können nicht unterschieden werden. Mappings der TAG-Muster zeigen Clusterder einzelnen Ölspecies. Hingegen kann die Gruppierung der Proben bei Mappings der Headspace-SPME-Datenverschiedene Gründe haben: So ist etwa ein Cluster von Nussölen durch die gemeinsame Vorbehandlung (Rösten)und die dabei entstehenden Pyrazine bedingt. Es zeigt sich, daß manche Verfälschungen eher bei durch Unter-suchung der TAG-Profile erkannt werden können (z.B. ein verfälschtes Senföl) während andere Zusätze sich beiden flüchtigen Verbindungen zeigen (z.B. Verfälschung von Mohnöl durch Sonnenblumenöl). Darüber hinaus kon-nten durch die gemeinsame multivariate Datenanalyse der MALDI- und GC-MS-Daten Korrelationen zwischen54:9/54:8/54:7-TAGs (enthalten Linolensäure) und den Carbonylverbindungen 3,5-Octadienone, 2,4-Heptdienalund 2-Butenal gefunden werden. Bei letzteren dürfte es sich um Autooxidationsprodukte der Linolensäure han-deln. Die gemeinsame Analyse von Ölen mittels HS-SPME-GC-MS und MALDI-MS kann Informationen überAroma und Fettzusammensetzung, Anbau, Verfälschung und Alterung, Verarbeitung (z.B. Rösten), geographis-chen Ursprung und umweltbedingten Kontaminationen liefern. Die Multivariate Datenanalyse unterstützt dieUnterscheidung zwischen Ölspecies, macht Veränderungen (z.B. Verfälschungen) über die Detektion einzelnerMarker-Verbindungen hinaus sichtbar und liefert Information über den Ursprung von flüchtigen Verbingungen(etwa von Autooxidationsprodukten). Eine Datenbank von TAG-Profilen und Headspace-Profilen für Pflanzenöleist im Aufbau.Neue Aspekte: Die Kombination von HS-SPME-GC-MS und MALDI-MS mit multivariater Datenanalyse ist eineinnovative Strategie zur raschen Charakterisierung von Ölen und Fetten.

195

Referenzen: [1] S. Bail, G. Stuebiger, H. Unterweger, G. Buchbauer, S. Krist. Characterization of volatilecompounds and triacylglycerol profiles of nut oils using SPME-GC-MS and MALDI-TOF-MS. Eur. J. LipidSci. Technol. (2009), 111, 170-182.; [2] K. Varmuza und P. Filzmoser. Varmuza. Introduction to MultivariateStatistical Analysis in Chemometrics, CRC Press, Boca Raton, FL (2009).Thema: LipideKeywords: MALDI-MS, SPME-GC-MS, Pflanzenöle, multivariate DatenanalyseKontakt: [email protected]

196

P103

Identifikation ausgewählter Glykosaminoglykan-Oligosaccharide mittels

kombinierter TLC/UV-MALDI-TOF MS

Nimptsch, Kathrin (1); Süß, Rosmarie; Riemer, Thomas; Nimptsch, Ariane; Schiller, Jürgen

Universität Leipzig - Medizinische Fakultät, Institut für Medizinische Physik und Biophysik, Deutschland

Einleitung: Lange wurde angenommen, dass Polysaccharide in lebenden Organismen lediglich für die Energiespe-icherung, bei der Ernährung und als Stützmaterialien von Bedeutung sind. Neben einfachen Kohlenhydraten, wiez.B. Dextranen und Alginaten, finden sich jedoch auch weitaus komplexere Polysaccharide. Glykosaminoglykane(GAG), wie beispielsweise Hyaluronan (HA) und Chondroitinsulfat (CS), sind vor allem wichtige Bestandteile derextrazellulären Matrix (EZM) des Bindegewebes. Diese Zucker werden zunehmend auch als Moleküle mit Mes-sengerfunktion diskutiert und es verdichten sich Hinweise auf die Relevanz des sogenannten "Sulfatierungscodes"[1]. Leider ist die Analytik dieser Molekülenur nach enzymatischen bzw. chemischen Abbau und Trennungder entstandenen Produkte möglich [2]. Wir beschreiben hier eine einfache Methode auf der Grundlage derTLC/MALDI [3] für die Analytik unterschiedlicher Kohlenhydrate sowie zur Generierung definierter Oligosac-charide.Experimenteller Teil: Die eingesetzten, natürlich vorkommenden Kohlenhydrate wurden in der Regel kom-merziell erworben, während die artifiziell übersulfatierten Zucker von Dr. Schnabelrauch (Innovent Jena) zurVerfügung gestellt wurden. Der Abbau der eingesetzten Polysacchariden erfolgte enzymatisch (Chondroitinase,Hyaluronidase) oder durch saure Hydrolyse der wäßrigen Lösungen mit halbkonzentrierter HCl. Die Charak-terisierung der entstandenen Oligosaccharide erfolgte an einem Bruker AutoflexTM MALDI-TOF MS sowiemit Methoden der hochauflösenden NMR. Im Positivionenmodus wurde 2,5 Dihydroxybenzoesäure (DHB), fürdie Detektion von Negativionen dagegen 9-Aminoacridin (9-AA) als Matrix verwendet. Für die Trennung derOligosaccharide wurde die Normalphasen Dünnschichtchromatographie (TLC) und ein unlängst beschriebenesLösungsmittelsystem genutzt [4]. Die identifizierten Spots wurden anschließend mit Matrixlösung versetzt unddirekt mittels MALDI-TOF MS charakterisiert. Die Aufnahme von PSD-Spektren erfolgte mit Standard BrukerSoftware.Ergebnisse und Diskussion: Die Enzyme Chondroitinase ABC und testikuläre Hyaluronidase spalten die na-tiven Glykosaminoglykane Chondroitinsulfat und Hyaluronan mit unterschiedlichen Selektivitäten. Die Charak-terisierung der entstehenden Produkte mittels MALDI-TOF MS ist problemlos möglich [5], wobei die Neg-ativionendetektion das Verfahren der Wahl ist, da hier insbesondere bei Anwendung der basischen Matrix 9-Aminoacridin (9-AA) eine höhere Sensitivität als im Positivionenmodus erzielt werden kann. Erwartungsgemäßwerden kleinere Oligosaccharide im Gemisch sensitiver als größere detektiert. Es stellte sich heraus, dass eineTrennung der Oligosaccharidgemische mittels TLC möglich ist. Auch wenn nur eine eingeschränkte Trennqualitätbei zunehmend größeren Oligosacchariden erreicht wird, so ist die Detektion aller erwarteten Produkte mittelskombinierter TLC/MALDI ohne weiteres möglich. Leider versagt der enzymatische Verdau bei "übersulfatierten"

GAG, da Sulfatreste als Inhibitoren für die Enzyme wirken. Um dennoch einen Abbau zu erreichen, setzten wir die

saure Hydrolyse mit HCl ein. Unter den verwendeten Bedingungen wird nur ein geringer Grad der N-Acetylreste

bzw. der Sulfatreste hydrolysiert, während die glykosidische Bindung das bevorzugte Ziel für HCl ist. Dennoch

sind die beiden glykosidischen Bindungen (1-3 bzw. 1-4 Verknüpfung) in unterschiedlichem Maße anfällig gegen

den Abbau, da lediglich Oligosaccharide mit einer geraden Anzahl von Monosaccharid-Bausteinen entstehen.

Würde der Abbau statistisch erfolgen, so sollten sich auch Verbindungen mit ungeradzahligen Oligosacchariden

bilden. Durch gezielte Variation der Reaktionsbedingungen können darüber hinaus die Massen der Oligosaccha-

ride definiert beeinflusst werden. Somit ist die salzsäureinduzierte Hydrolyse ein bequemes Verfahren für den

gezielten Abbau unbekannter Kohlenhydrate. Der Verlust der Sulfatreste ist das bislang größte Problem bei der

Charakterisierung sulfatierter Kohlenhydrate. Auch wenn es nach den uns bislang vorliegenden Ergebnissen na-

hezu unmöglich ist, den Verlust der Sulfatgruppen vollständig zu unterdrücken, so ist doch durch geschickte Wahl

der experimentellen Bedingungen eine Detektion der entsprechendenMolekülionen in allen Fällen möglich. Durch

weitergehende PSD-(Post Source Decay)- Untersuchungen ist zusätzlich die Bestimmung der Positionen der Sul-

fatgruppen möglich.

Neue Aspekte: Die durch saure Hydrolyse von Glykosaminoglykanen erhaltenen Oligosaccharide können mittels

TLC/ MALDI-TOF MS einfach und schnell charakterisiert werden.

197

Referenzen: [1] J. Kovensky, Curr. Med. Chem. 16 (2009) 2338.; [2] J. Schiller, B. Fuchs, K. Arnold, Curr. Org.Chem. 10 (2006) 1771.; [3] B. Fuchs, R. Süß, A. Nimptsch, J. Schiller, Chromatographia 69 (2009) 95.; [4] Z.

Zhang, J. Xie, F. Zhang, R.J. Linhardt, Anal. Biochem. 371 (2007) 118.; [5] J. Schiller, J. Arnhold, S. Benard, S.

Reichl, K. Arnold, Carbohydr. Res. 318 (1999) 116.

Thema: Kohlenhydrate

Keywords: Oligosaccharide, Chondroitinsulfat, Hyaluronsäure, TLC, MALDI-TOF


198

P104

Graphitized Carbon HPLC-Chip mass spectrometry of N-linkedglycans

Bank, Stephanie; Kapková, Petra

Universität Würzburg, Germany

Einleitung: Nowadays, about 50% of all proteins are supposed to be glycosylated. The glycans possess highvariety and complexity and are involved in a number of cell processes. Depending on the structure, oligosaccharidechains do not only participate in physiological activities. Moreover, they are associated with pathological processesas well [1]. Therefore, there is constant interest in improvement of analytical methods and development of theinstrumentation, which would be able to produce reliable and specific data about the protein glycosylation and itschanges. During this study, glycans from different glycoproteins have been analysed by means of a nano-LC/Chip-mass spectrometry; among them ovalbumin as a model protein for the study of glycosylation.Experimenteller Teil: After PNGase F-deglycosylation, glycans were extracted and purified by means of theGlycoClean

®

Cartridges. Further, an aliquot of the sample was labelled with 2-aminobenzamide. Analysis wasperformed using an Agilent 1200 nano-LC-system coupled to a chipcube and electrospray-ion trap mass spectrom-eter. Separation of N-glycans was acquired on a graphitized carbon chip. Chromatography was performed usingan ACN/water solvent system containing 0.1% formic acid.Ergebnisse und Diskussion: Ovalbumin is known to possess one glycosylation site with about twenty differentoligosaccharide structures [2]. The glycans showed up in the ESI-spectrum as single-charged ions and as doubly-charged ions as well. Especially high-mass glycans produced doubly-charged ions. This showed up for both,the labelled and the unmodified samples. The on-chip enrichment and separation brought the required sensitiv-ity and allowed analysis of samples with concentrations in the low picomolar range. Furthermore the MS/MS-spectrometry permitted useful insights into the structure of the present glycans.Neue Aspekte: The presented approach allowed for rapid and sensitive analysis of glycans and can be furtherelaborated for in-depth glycomic studies.Referenzen: [1] Gabius HJ, The Sugar Code, Willey -VCH 2009, p. 365; [2] Harvey DJ. J. Am. Soc. MassSpectrom. 2000; 11:564-571Thema: KohlenhydrateKeywords: -glycosylation, glycoprotein, HPLC/Chip - mass spectrometryKontakt: [email protected]

199

P105

Analysis of Sialylated Glycans with the MALDI LTQ OrbitrapMass Spectrometer using DHB/N,N-Dimethylaniline Matrix

Saba, Julian (1); Snovida, Sergei (2); Charych, Deb (3); Strupat, Kerstin (1); Viner, Rosa (1)

1: Thermo Fisher Scientific, San Jose, CA and Bremen, Germany; 2: University of Manitoba, Canada; 3: FivePrime Therapeutics, CA, USA

Einleitung: Studies have shown that the alterations in glycan structures are associated with various developmentaland pathological states of glycoproteins and have great biological significance.[1,2] Particular attention is beingpaid to sialylated glycans due to their involvement in many important biological phenomena, including cell-celladhesion and cell-pathogen interaction.[3] Ionic signal suppression is often a major issue in the analysis of sialy-lated glycans by MS, particularly due to the presence of a carboxyl group at the anomeric carbon, which is usuallyionized at physiological pH, thus resulting in a negative charge above pH value of ∼ 2.6. Here, we examined theutility of 2,5-dihydroxybenzoic acid/N,N-dimethylaniline (DHB/DMA) matrix [4]to improve analysis of N-linkedsialylated glycans from human α1-acid glycoprotein (AGP) by MALDI.Experimenteller Teil: PNGase F released glycans from human AGP were labeled with 2-aminobenzoic acid.DHB/DMA matrix solution was prepared by initially preparing solution of the DHB matrix in 1:1, ACN/water.This was followed by adding 4 µL of DMA to the DHB solution, such that the molar ratio of DHB to DMA was3:1. Samples were deposited onto a stainless steel MALDI sample plate by mixing the analyte and matrix solutions(0.5 µL each) on plate and allowing it to dry at room temperature. All experiments were performed on a ThermoScientific MALDI LTQ Orbitrap XL mass spectrometer in either negative or positive modes. MS/MS analysis wasconducted in the linear ion trap with Orbitrap mass analyzer or ion trap detection.Ergebnisse und Diskussion: Human AGP was chosen as the model glycoprotein because its glycan contenthas been extensively studied over the years and there is wide availability of data in the literature pertaining toits structural characterization.[5] To evaluate the DHB/DMA matrix, the N-linked glycans released from humanAGP were divided into three equimolar proportions and spotted onto the MALDI target using three differentmatrices, DHB, DHB in 70% ethanol, and DHB/DMA. Mass spectra were acquired in the positive mode toevaluate ionization efficiency of all three matrices. In contrast to these two matrices, DHB/DMA provided muchmore complete glycan characterization in terms of the number of sialyl and asialyl glycans detected. DHB/DMAprovided much more complete glycan characterization in terms of the number of sialyl and asialyl glycans detected.In order to detect sialylated glycans that were observed using DHB/DMA in the positive ion mode, negativeion mode MALDI was required using DHB. Molecular masses of the glycans were measured within 2 ppm oftheir calculated monoisotopic mass values allowing for structural assignment without acquiring MS/MS spectra.However, we did perform MS/MS analysis for structural confirmation. Others have explored quantification ofsialylated glycans in the past, but majority of these approaches have relied on exploring sialylation in the absenceof asialylated glycans due to the inability to see both while using the same MS ionization mode. Typically thesequantification results have been reported on the basis of comparing sialylations relative to each other. Thanksto our ability to profile both sialylated and asialylated in the same spectrum, we were able to quantify sialylatedglycans relative to the total amount of glycans present in a sample. Overall, the majority of the glycans detected inthe study were sialylated, which is in agreement with what is observed in literature.Neue Aspekte: The use of DHB/N,N-Dimethylaniline matrix for enhanced sensitivity in the analysis of sialylatedglycans by MALDI LTQ OrbitrapReferenzen: [1] Gagneux, P.; Varki, A., Evolutionary considerations in relating oligosaccharide diversity tobiological function. Glycobiology 1999, 9, (8), 747-55.; [2] Freeze, H. H.; Aebi, M., Altered glycan structures: themolecular basis of congenital disorders of glycosylation. Curr Opin Struct Biol 2005, 15, (5), 490-8.; [3] Lamari,F. N.; Karamanos, N. K., Separation methods for sialic acids and critical evaluation of their biologic relevance.J Chromatogr B Analy Technol Biomed Life Sci 2002, 781, (1-2), 3-19.; [4] Snovida, S. I.; Perreault, H., A2,5-dihydroxybenzoic acid/N,Ndimethylaniline matrix for the analysis of oligosaccharides by matrix assisted laserdesorption/ionization mass spectrometry. Rapid Commun Mass Spectrom 2007, 21, (22), 3711-5.; [5] Treuheit,M. J.; Costello, C. E.; Halsall, H. B., Analysis of the five glycosylation sites of human alpha 1-acid glycoprotein.Biochem J 1992, 283 ( Pt 1), 105-12.Thema: KohlenhydrateKeywords: MALDI, sialylierte Zucker, Glykane, OrbitrapKontakt: [email protected]

200

P106

Structural analysis of chondroitin/dermatan sulfate oligosaccharides from

biglycan by fully automated chip-nanoelectrospray ion trap mass spectrometry

Flangea, Corina (1,2); Sisu, Eugen (1); Seidler, Daniela (3); Zamfir, Alina D. (2)

1: "Victor Babes" University of Medicine and Pharmacy, Timisoara, Romania; 2: Department of Chemical andBiological Sciences, "Aurel Vlaicu" University of Arad, Romania; 3: Institute for Physiological Chemistry and

Pathobiochemistry, University of Münster, Germany

Einleitung: Biglycan (BGN) is a small leucine-rich repeat proteoglycan (SLRP) which is found in a variety ofextracellular matrix tissues, including bones, cartilages and tendons. BGN consists of a protein core containingleucine-rich repeat regions and two glycosaminoglycan (GAG) chains consisting of either chondroitin sulfate (CS)or dermatan sulfate (DS), with DS more abundant in most connective tissues [1]. For structure investigation ofCS/DS glycoforms, development of high performance specific methods was lately required, among which, chip-based nanoelectrospray ionization (ESI) mass spectrometry (MS) contributed an essential progress to the field[2-4]. Although MS demonstrated its ability to offer distinctive insights into the domain structure of GAGs, so farno structural analysis of biglycan GAGs was pursued by employing mass spectrometry-based methodologies.Experimenteller Teil: 293 BGN cell line medium was colected and applied on DEAE-Tris-Acryl M anion ex-change column. Released CS/DS were eluted and digested with 50 mU/assay chondroitin AC I lyase. Sizefractionation of oligosaccharides was performed on a Superdex Peptide HR10/30 column. Tetrasaccharide anddisaccharide fractions were pooled and desalted on a prepacked D-Salt column. MS experiments were conductedin the negative ion mode on a High Capacity Ion Trap Ultra mass spectrometer (HCT MS, Bruker Daltonics).Multistage MS was carried out by collision-induced dissociation (CID) using He as the collision gas. Sampleswere infused using a fully automated chip-based nanoelectrospray system (NanoMate robot, Advion BioSciences)incorporating ESI 400 Chip technology coupled to the HCT MS via an in-laboratory made mounting system.Ergebnisse und Diskussion: We have released by β-elimination and isolated a hybrid BGN CS/DS domain,which was subsequently purified and partially depolymerized using chondroitin AC I lyase. Based on enzymespecificity, we generated a mixture of oligosaccharides with known hexuronate (HexA) epimerization. Further,oligosaccharide mixture was fractionated and disaccharide and hybrid tetrasaccharide fractions were collected andsubmitted to chip-based nanoESI MS and CID MS2-MS3 analysis for the determination and characterization ofregularly and irregularly sulfated species. Chip-nanoESI MS1 screening of the BGN CS disaccharide fractionindicated the presence of an unsaturated mono-sulfated species and of an atypical bisulfated sequence detected as[M-H+]− and [M-2H++Na+]− ions, respectively. Fragmentation analysis carried out by employing CID MS2-MS3 demonstrated that in the bisulfated BGN disaccharide, GlcA is sulfated in position 2 and GalNAc in position 6,which is consistent with a ∆-4,5-GlcA(2S)-GalNAc(6S) structural motif. By chip-nanoESI MS1screening underidentical experiment conditions, BGN CS/DS tetrasaccharide fraction was found to contain species of variablesulfation degree. CID MS2 experiment on the [M-H+]− ions related, according to mass calculation, to a bisulfatedCS/DS tetramer, resulted in a fragmentation spectrum indicating that one sulfate group is situated at GlcA residueand the other one at the first GalNAc moiety from the non-reducing end. The site of sulfation within each monomerring was determined by further sequencing in CID MS3 mode of the fragment ions corresponding to sulfated-GlcA and sulfated-GalNAc. Obtained fragmentation pattern corroborated a ∆-4,5-GlcA(2S)-GalNAc(6S)-IdoA-GalNAc structure. These findings demonstrate the occurrence in BGN CS/DS chains of an unusual ∆-4,5-GlcA(2S)-GalNAc(6S) motif, which was identified in both disaccharide and tetrasaccharide fractions that were so farinvestigated.Neue Aspekte: Chondroitin/dermatan sulfate oligosaccharides from biglycan are here for the first time investigatedby fully automated chip-nanoelectrospray CID MSn.Referenzen: [1] Scott PG, Dodd CM, Bergmann EM, Sheehan JK, Bishop PN. Crystal structure of the biglycandimer and evidence that dimerization is essential for folding and stability of class I small leucine-rich repeatproteoglycans. J. Biol. Chem. 281, 13324, 2006; [2] Flangea C, Serb AF, Schiopu C, Tudor S, Sisu E, Seidler DG,Zamfir AD. Discrimination of GalNAc (4S/6S) sulfation sites in chondroitin sulfate disaccharides by multistagemass spectrometry. Cent. Eur. J. Chem. 7, 752, 2009; [3] Zamfir AD, Flangea C, Serb AF, Sisu E, Dinca N,Bruckner P, Seidler DG. Analysis of novel over- and under-sulfated glycosaminoglycan sequences by enzymecleavage and multiple stage mass spectrometry. Proteomics 9, 3435, 2009; [4] Flangea C, Schiopu C, Sisu E,Serb A, Przybylski M, Seidler DG, Zamfir AD. Determination of sulfation pattern in brain glycosaminoglycans bychip-based electrospray ionization ion trap mass spectrometry. Anal. Bioanal. Chem. 395, 2489, 2009

201

Thema: KohlenhydrateKeywords: Chondroitin/dermatan sulfate; Biglycan; Fully automated chip-based nanoelectrospray; Ion trap massspectrometry; collision-induced dissociationKontakt: [email protected]

202

P107

Electron Capture Dissociation und Infrared MultiphotonDissociation von Monomeren, Dimeren und Trimeren von

Oligosacchariden unter Zugabe von Additiven.

Hahn, Andrea; Grotemeyer, Jürgen

CAU Kiel, Germany

Einleitung: Bei Oligosacchariden ist die Spaltung der glykosidischen Bindung der vorherrschende Fragmen-tierungsmechanismus. Die entstandenen Fragmente geben Aufschluss über die Reihenfolge der verbundenenMonosaccharide, wohingegen Ringspaltungen Informationen über die Verknüpfungspositionen liefern. Die An-wesenheit von Alkali- und Erdalkaliionen beeinflusst die Ionisierung und Fragmentierung der Oligosaccharide [1].Der Einfluss dieser Ionen auf die Bildung und Fragmentierung vonMonomeren, Dimeren und Trimeren von Lacto-n-fucopentaose I und II (LNFP-I, LNFP-II) wurde mit Hilfe von IRMPD-FT-ICR und ECD-FT-ICR untersucht.Experimenteller Teil: Die Experimente wurden mit einem APEX Qe FT-ICR-Massenspektrometer (Bruker Dal-tonics, Bremen) mit einer Apollo II Combi Quelle, ESI / MALDI-Version durchgeführt. Im ESI-Modus wurdendie Proben mit einer Flussrate von 2 µL/min injiziert. Die Fragmentierungen wurden in einer ICR Infinity Celldurchgeführt. Für stoßinduzierte Fragmentierungen wurde Argon als Stoßgas verwendet, IRMPD Experimente

wurden mit einem CO2-Laser (λ = 10,6 µm) durchgeführt. ECD wurde mit einer Hohlkathode durchgeführt.

Ergebnisse und Diskussion: Bei Zugabe von Alkali- und Erdalkaliionen zu LNFP-I und II bilden sich neben den

Monomeraddukten auch Dimer- und Trimercluster z.B. [2M+Mg]2+, [2M+K+H]2+, [2M+Ca]2+, [2M+2Li]2+,

[3M+K+H]2+. MS/MS Spektren der zweifach geladenen Cluster wurden nach Fragmentierung mit ECD aufgenom-

men. Die Isomere LNFP-I und II zeigten bei der Fregmentierung unterschiedliches Verhalten. Die Spektren gaben

Hinweise auf den Anlagerungsort der Adduktionen im Molekül. Bei IRMPD und SORI traten bei den MS/MS

Spektren der Di- und Trimere weniger Fragmente auf als bei den Spektren der Monomere. Eine Ausnahme stellen

die Cluster [2M+K+H]2+und [3M+K+H]2+ dar. Es traten in den Spektren Fragmente auf, die bei [M+K]+ und

[M+H]+ nicht zu sehen waren. Anhand der MS/MS Spektren von [2M+K+H]2+ konnten die Isomere LNFP-I und

II unterschieden werden. MS/MS Spektren von [2M+K+H]2+und [3M+K+H]2+ zeigten mit hoher Intensität die

Fragmente [2M+K+H-Fuc]2+ und [2M+K+H-2Fuc]2+, bei denen der zweifach geladene Cluster erhalten blieb.Neue Aspekte: IRMPD und ECD von Monomeren, Dimeren und Trimeren von Oligosacchariden unter Zugabevon Additiven.Referenzen: [1] [1] D.J. Harvey, Ionization and collision-induced fragmentation of N-linked and related carbohy-drates using divalent cations, J. Am. Soc. Mass Spectrom., 2001, 12(8):926-937.; [2] [2] J.T. Adamson, ElectronCapture Dissociation of Oligosaccharides Ionized with Alkali, Alkaline Earth, and TransitionMetals, Anal. Chem.,2007, 79(7):2901-2910.Thema: KohlenhydrateKeywords: Oligosaccharide, FT-ICR, IRMPD, ECD,Kontakt: [email protected]

203

P108

Detection methods and theoretical studies for FT-ICR massspectrometry

Heck, Michael (1); Blaum, Klaus (1); Cakirli, R.Burcu (1,2); Kretzschmar, Martin (3); Rodriguez, Daniel (4);Schweikhard, Lutz (5); Stahl, Stefan (1); Ubieto-Diaz, Marta (1)

1: Max-Planck-Institute for Nuclear Physics, Saupfercheckweg 1, 69117 Heidelberg, Germany; 2: Department ofPhysics, University of Istanbul, Istanbul, Turkey; 3: Institut für Physik, Johannes-Gutenberg-Universität, 55099Mainz, Germany; 4: Universidad de Granada, 18071 Granada, Spain; 5: Institut of Physics, Ernst-Moritz-Arndt

University Greifswald, 17487 Greifswald, Germany

Einleitung: Fourier-Transform Ion-Cyclotron-Resonance (FT-ICR) is nowadays a standard technique in Chem-istry used for mass identification with very high resolution after molecular and stable ions are confined in a Penningtrap. It is a non-destructive detection technique, opposite to other methods applied on rare isotopes for identifica-tion and precision mass measurements. Therefore, the use of this technique arouses interest since those decayingisotopes can be re-used for other experiments after mass determination. Moreover, the technique has not been usedyet for high-precision mass measurements on radionuclides at a level required for fundamental physic studies,where any aspect of ion motion and detection signal must be taken into account.Experimenteller Teil: The fundamental studies of coherent ion motion in Penning traps and non-destructive iondetection provide structured examination of the observed FT-ICR signals. In the experiment the typically welldetection methods for FT-ICR studies are usedErgebnisse und Diskussion: The basic distinctions of the FT-ICR spectra between the dipole-detection, in whichthe pickup signals of the electrodes are subtracted, and the quadrupole-detection, in which the pickup signals areadded, will be explained. In this contribution, recent results obtained at the Max-Planck-Institute for NuclearPhysics in Heidelberg using 7Li+ ions and a broad-band FT-ICR Penning trap system will be presented, andcompared with the simulation results and the theoretical predictions.Neue Aspekte: Knowledge of coherent ion motion in Penning traps and FT-ICR detection methods provideexamination of the observed FT-ICR signals.Thema: Instrumentelle Entwicklungen, Element-MassenspektrometrieKeywords: Penning trap, FT-ICR ion detection, coherent ion motion, ion storage,frequency shiftKontakt: [email protected]

204

P109

Zweiphotonen- und Einphotonen-Circulardichroismus in einemLaser-Massenspektrometer

Logé, Christoph; Boesl, Ulrich

Technische Universitaet Muenchen, Germany

Einleitung: Nichtlineare Photonen-Prozesse sind aufgrund ihrer besonderen und zu linearen Prozessen unter-

schiedlichen Charakteristika interessant. Neben Ramanstreuung und SHG (second harmonic generation) zählt dazuvor allem die Zweiphotonenabsorption. Die simultane Absorption von zwei Photonen statt eines einzelnen Photonseröffnet eine Vielzahl neuer Möglichkeiten. So werden angeregte Zustände, die wegen der Auswahlregeln der Ein-photonenabsorption nicht besetzt werden können, mittels Zweiphotonenabsorption erreichbar. Die Verknüpfungvon Zweiphotonenabsorption mit Circulardichroismus lässt eine Reihe neuer Effekte erwarten. Für den Circulardi-chroismus in der Einphotonenabsorption spielen elektrische und magnetische Übergangsdipolmomente eine Rolle.Für den Zweiphotonen-Circulardichroismus sind zusätzlich die permanenten Dipolmomente des Moleküls von Be-deutung. Ein weiterer ganz besondere Vorteil ist es, dass spektrale Bereiche im VUV mit sichtbarem und nahemUV-Licht erreichbar sind.Experimenteller Teil: Die vorliegenden Untersuchungen wurden mittels enantiosensitiver Lasermassenspektro-metrie, der Kombination aus resonanter Multiphotonenionisation mit zirkularpolarisiertem gepulstem Laserlichtund Flugzeitmassenspektrometrie, durchgeführt. Diese Methode verknüpft den Circulardichroismus (unterschied-liche Absorption von links und rechts zirkular polarisiertem Licht in einem chiralen Molekül) mit der Selektivitäteines Massenspektrometers [1]. So kann Chiralität auch in Stoffgemischen ohne vorherige Auftrennung bestimmtwerden. Der Einsatz von frequenzverdoppelten und frequenz-verdreifachten Farbstofflasern eröffnet die Möglich-keit, Licht geeigneter Wellenlänge sowohl für Ein- als auch Zweiphotonenabsorbtion zu erhalten. Die untersuchteSubstanz ist das Molekül 3-Methylcycopentanon. Es ist sowohl experimentell als auch in quantenmechanischenBerechnungen charakterisiert worden. Der Einphotonen-Circulardichroismus wird über eine (1+2)- oder (1+1)-Multiphotonenionisation durchgeführt. Für den Zweiphotonen-Circulardichroismus erfolgt die Anregung über eine(2+1)-Multiphonenabsorption.Ergebnisse und Diskussion: Erste Hinweise auf die Besonderheiten des Zweiphotonen-Circulardichroismus wer-den von theoretischen Betrachtungen vorhergesagt. Aktuelle quantenmechanische Berechnungen dieser Effek-te zeigen, dass bei Zweiphotonen-Circulardichroismus teilweise wesentlich höhere Effekte als bei Einphotonen-Circulardichroismus auftreten können [2-4]. Bisher wurden jedoch kaum experimentelle Untersuchungen diesesPhänomens angestellt [5]. Daher wurde gezielt versucht, mit ein und derselben Apparatur den Circulardichrois-mus sowohl in der Einphotonenabsorption als auch in der Zweiphotonenabsorption zu bestimmen. Die Einpho-tonenabsorption kann für die ersten beiden angeregten Zustände von 3-Methylcycopentanon durchgeführt wer-den. Die Ergebnisse für den Einphotonen-Circulardichroismus sind im Einklang mit Literaturwerten. Die Zwei-photonenabsorption konnte für die ersten drei angeregten Zustände durchgeführt werden. Bei der Bestimmungdes Zweiphotonen-Circulardichroismus tritt auf Grund der hohen notwendigen Laserintensitäten eine wesent-lich stärkere Fragmentierung der Molekülionen auf, sodass deren Circulardichroismus schwieriger zu bestim-men ist. Im Einphotonen-Circulardichroismus können dagegen hohe Signalintensitäten erreicht werden, sodasshier eine präzise Messung auch kleinerer Effekte möglich ist. Es konnte gezeigt werden, dass der Zweiphotonen-Circulardichroismus teilweise um Größenordnungen über dem Einphotonen-Circulardichroismus liegt. Der Effektist besonders interessant für die Untersuchung elektronisch erlaubter Übergänge, deren linearer Circulardichro-ismus normalerweise gering ist. Weitere Untersuchungen sind vorgesehen, den Circulardichroismus weiterer Ab-sorptionsprozesse wie zum Beispiel bei der Absorption und Fragmentierung von Molekülionen zu bestimmen. Au-ßerdem sollen die Untersuchungen auf andere, biologisch interessante Moleküle wie die Aminosäuren ausgedehntwerden. Zu diesen existieren ebenfalls bereits quantenmechanische Berechnungen, die auf besondere Vorteile desZweiphotonen-Circulardichroismus hinweisen.Neue Aspekte: Zweiphotonen-Circulardichroismus ist eine neue kaum untersuchte Spektroskopieart chiraler Mo-leküle mit Bedeutung für physikalische Grundlagenforschung wie für analytische AnwendungenReferenzen: [1] Ch.Logé, A. Bornschlegl, U. Boesl, Anal. Bioanal. Chem., 2009, 395, 1631; [2] R. Li, R. Sullivan,

W. Al-Basheer, R.M. Pagni, R.N. Compton, J. Chem.Phys., 2006, 125, 144304; [3] B. Jansik, A. Rizzo, H. Agren,

J. Phys. Chem. B, 2007, 111, 446; [4] A. Rizzo , N. Lin, K. Ruud, J. Chem. Phys., 2008, 128, 164312; [5] N. Lin,

F. Sntoro, A. Rizzo, Y. Luo, X. Zhao, V. Barone, J. Phys. Chem. A, 2009, 113, 4198

Thema: Instrumentelle Entwicklungen, Ionen-Molekül-Reaktionen

205

Keywords: Chirale Moleküle, Multiphotonenionisation, Circulardichroismus, Zweiphotonenabsorption, Laser-

massenspektrometrie


206

P110

Parametric Excitation of Ions in an ICR-Trap by Axialization usingtwo Electrodes

Martinez, Franklin (1); Herlert, Alexander (2); Marx, Gerrit (1); Schweikhard, Lutz (1); Walsh, Noelle (1)

1: University of Greifswald, Germany; 2: CERN, Geneva, Switzerland

Einleitung: In ICR mass spectrometry radial quadrupolar excitation is combined with buffer gas cooling, in orderto achieve axialization of the trapped ions [1]. Ideally, the excitation signal is applied to two pairs of oppositelocated ring segments, using inverted phases. If the excitation signal is applied to only one pair of electrodes,i.e. with one phase, parametric resonances at the frequencies 2ωz and ωp = ω+ - ω- can cause a correspondingexcitation of the ion motion.Experimenteller Teil: The parametric resonances have been investigated theoretically and experimentally. Mul-tipole components of different excitation schemes can be deduced from a simple vector representation, and thusparametric components can be quickly identified [2]. The effect of one-phase quadrupolar excitation has beendemonstrated by experiments with gold and aluminum cluster ions stored in a Penning trap.Ergebnisse und Diskussion: At axialization of cluster size Al_70ˆ- the loss of Al_29ˆ- and Al_63ˆ- has beenobserved and could be explained by the vector representation approach. In the case of Au_51ˆ- clusters at aparticular trapping voltage the one-phase quadrupolar excitation caused ion loss rather than axialization.Neue Aspekte: Application of one-phase quadrupolar excitation is investigated experimentally and by a newvector-representation approach.Referenzen: [1] G. Savard et al., Phys. Lett. A 158 (1991) 247; [2] F. Martinez et al., Eur. J. Mass Spectrom. 15(2009) 283Thema: Instrumentelle EntwicklungenKeywords: axialization, ICR MS, ion storage, unintended ion ejectionKontakt: [email protected]

207

P111

Pushing toward to next generation Ion-Trap-MS: consequentadoption of newest technology in ion formation, transfer and

trapping

Sauer, Jörg; Albers, Christian; Brekenfeld, Andreas; Gebhardt, Christoph; Hartmer, Ralf


Einleitung: Radio-frequency Ion-Trap mass spectrometers have achieved a leading position in life science appli-cations. This is mainly due to their flexibility, robustness, the extraordinary multiple MS/MS capabilities and tothe unmatched MS/MS sensitivity at high duty cycle. The commercial availability of ETD strongly increased theiruse in detailed structure elucidation of proteins and peptides. Especially for these applications there is a strong de-mand to resolve the isotopic structure of highly charged ions over a large seamless m/z-range at analytical feasiblescan-speed and sensitivity.Preparing Ion-Traps for the expanding ultra-fast chromatography, for the requirementsof top-down ETD/PTR workflows and to sharpen their position with respect to alternative techniques it is crucialto push the limits of their key performance factors.Experimenteller Teil: The presented development is based on the Bruker amazon ion trap system. The introducedtwo-stage funnel ion guide results in a substantially increased ion flux and MS/MS sensitivity. The improvedcontrol of the non-linear ejection process and the trap environment supports faster scan modes as well as a highermass resolution. The increased spectral rate can even be maintained during positive / negative ion mode switchingdue to a novel zero-dead time ion inlet system. The performance factors (sensitivity, resolution, scan rate andpolarity switching) have been investigated systematically in several applications: peptide identification in complexmatrices, clinical screening applications, and top down ETD/PTR workflows.Ergebnisse und Diskussion: Based on the predicted improvement in ion transfer yield due to the incorporated dualstage ion funnel the modified Ion-Trap MS achieves one decade improvement in MS/MS sensitivity, measured at alevel of 250 fg Reserpine injected. Taking advantage of the 10-times increased ion flux and increased scan speed,high protein identification yields can be achieved at much lower sample amounts for example in the analysis ofcomplex samples like complete organism extracts. The increased duty cycle allows selecting more of the generallyincompletely covered precursor ensemble. The further enhanced trap-control allows doubling the highest scanspeed while still meeting the resolution specifications of standard HCTultra ETD II for the Ultra scan mode witha peak width of less than 0.6 Th. The system is furthermore able to clearly resolve the isotopic pattern of six-fold charged ions at a scan speed 5-times higher than the former Maximum Resolution scan mode, making thisresolving power available for a routine, wide m/z-range, full spectrum acquisition, as required for the ETD-baseddetailed structure elucidation of peptides or proteins larger the 10 kDa. In alternating polarity acquisition mode alevel of 15 spectra/s is surpassed, especially suitable for small-molecule applications like rapid drug and metaboliteidentification and quantification in body liquids in conjunction with ultra fast chromatography.Neue Aspekte: Consequent combination of ion formation, ion transfer and control of trap environment: pushingapplications beyond their current limits.Thema: Instrumentelle EntwicklungenKeywords: Ion trap, MSn, mass resolution, ion funnelKontakt: [email protected]

208

P112

A Different Way of Measuring Ion Mobility for Absolute CrossSections at Ultra-High Resolution TOF MS

Schneider, Andrea; Baykut, Gökhan; von Halem, Oliver; Räther, Oliver


Einleitung: In the classical drift tube-IMS an electric field accelerates ions while collisions with a stationarybuffer gas lead to a frictional force: Ions drift with constant velocities and get separated depending on mobilities.In our dynamic method using a tapered stacked ring ion guide in combination with a UHR-QqTOF design, ionsare carried by the gas flow into the mass spectrometer and move against an electrical barrier potential introducedto decelerate them. The barrier repels the ions and permits only those below a certain mobility threshold to enterthe mass spectrometer. With increasing barrier voltage, this threshold moves from high to low mobilities. Whenthe threshold passes an ion’s mobility its signal decreases. Derivatives of decreasing signal intensities form peaks.Experimenteller Teil: In a derivative plot the position of each peak on the barrier voltage axis (the blockingvoltage) is related to the mobility of this ion. For mobility measurements, ions were generated in an electrosprayion source from solutions prepared in methanol/water (1:1 in vol.) with 1% formic acid. Solutions were introducedto the sprayer by direct infusion with a syringe pump. The barrier voltage was increased stepwise and the massspectra were recorded. From derivative plots blocking voltages were determined. To calibrate the barrier voltagescale, ions generated from reference peptides bradykinin, angiotensin I, neurotensin, and fibrinopeptide A wereused, of which absolute cross sections in nitrogen were known.Ergebnisse und Diskussion: From absolute cross sections of the reference ions the K0 values in nitrogen werecalculated and the barrier voltage scale was calibrated. The calibration curve of the barrier voltage vs. K0 waslinear (with a negative slope) in the range of mobilities measured. Ion mobilities, as well as cross sections of variouspeptide ions were determined with IMS resolving power values of 20-30. As preliminary examples, mobilities andcross sections of ions generated from bombesin, substance P, melittin, insulin B chain, and ACTH were determinedusing this dynamic method.Neue Aspekte: Neuer Ansatz für IMS in einem QTOF zur Berechnung von StossquerschnittenThema: Instrumentelle EntwicklungenKeywords: IMS, QTOF, Ionenmobilität, HochauflösungKontakt: [email protected]

209

P113

SO2 als oxidationsbeständiger Tracer für die Messung desÖlverbrauchs von Verbrennungsmotoren

Sellmeier, Stefan; Boesl, Ulrich

Technische Universität München, Germany

Einleitung: Möglichkeiten den Ölverbrauch von Verbrennungsmotoren schnell und exakt messen zu können, sindheute in der Motorenentwicklung von größtem Interesse. Wie bereits vor einiger Zeit gezeigt werden konnte, sindhierfür insbesondere Tracer basierte Methoden prädestiniert[1] Hierbei wird ein Tracer aus dem Öl im Abgas desMotors quantifiziert und daraus der aktuelle Ölverbrauch errechnet. Ein hierbei verwendeter Tracer, welcher sichinsbesondere durch seine Stabilität auszeichnet, ist SO2. Dieses wird aus Schwefelkohlenwasserstoffen im Abgasmittels nachgeschalteter Oxidation erzeugt und anschließend selektiv und hochsensitiv detektiert. Hierfür eignensich beispielsweise die selektive laserinduzierte Fluoreszenz oder die Laser-Massenspektrometrie.Experimenteller Teil: Um SO2 überhaupt als Tracer verwenden zu können muss zuvor die quantitative Oxidationaller im Abgas vorkommender Schwefelkohlenwasserstoffe zu SO2 sicher gewährleistet sein. Dies geschiehtherkömmlicherweise durch Erhitzen des Abgases in einem Pyrolyseofen auf ca. 1000°C. Hierbei wird nebeneiner großen Menge Energie auch eine starke Wärmeisolation im Gerät benötigt. Eine elegantere Alternativeder Oxidation stellt der Einsatz einer Glimmentladungszelle dar. Hierbei kann bei vergleichsweise niedrigenTemperaturen gearbeitet werden ohne Einbußen in der Effizienz der Oxidation hinnehmen zu müssen. Zusätzlichbietet die kleine Entladezelle die Möglichkeit kompakte und mobile Messapparaturen zu realisieren, wie sie in derIndustrie benötigt werdenErgebnisse und Diskussion: In mehreren Experimenten wurde die Glimmentladung auf ihre mögliche Anwen-dung zur quantitativen Oxidation von Schwefelkohlenwasserstoffen zu SO2 untersucht. Zunächst wurde die Ent-ladungszelle in einer Reihe von Experimenten charakterisiert. So wurden der Einfluss des Druckes, der Spannungund des Stromflusses auf die Stabilität der Entladung untersucht. Um die Oxidationswirkung zu bestimmen wurdenverschiedene schwefelhaltige Testsubstanzen mit der Zelle oxidiert und die Produktzusammensetzung mit einemQuadrupol-Massenspektrometer untersucht. Die so erhaltenen Ergebnisse wurden den Oxidationsergebnissen beiAnwendung des Hochtemperaturofens verglichen. Hierbei konnte gezeigt werden, dass die Glimmentladung beiallen untersuchten Stoffen mindestens dieselbe Oxidationswirkung aufweist, wie der Ofen. Somit stehen denVorteilen hinsichtlich Größe und Energieeffizienz keine Nachteile bei der Funktion gegenüber. Einer Verwendungin Apparaten zur schnellen Ölverbrauchsmessung an Verbrennungsmotoren steht somit nichts im Wege.Neue Aspekte: Die Glimmentladungszelle bietet eine optimale Möglichkeit verschiedenste Schwefelkohlen-wasserstoffe quantitativ zu SO2 zu oxidieren.Referenzen: [1] Püffel, P. K.; Thiel, W.; Frey, R.; Boesl, U.; SAE SP, 1998, 1389, p 27-33.Thema: Instrumentelle EntwicklungenKeywords: Ölverbrauch, Glimmentladung, Schwefeldioxid, TracerKontakt: [email protected]

210

P114

A Monolithic Trypsin Reactor for Protein Digestion by off-lineand on-line nano-LC/MS

Sproß, Jens; Sinz, Andrea

Martin-Luther-University Halle-Wittenberg, Germany

Einleitung: In the field of proteome research there is a great interest in new techniques for a high throughputidentification of minute protein amounts. Currently, enzymatic cleavage followed by MS-based identification ofproteins is state-of-the-art. Enzymatic cleavage of proteins is usually performed in-gel or in-solution by the serineprotease trypsin. In order to prevent autodigestion of trypsin, which complicates the interpretation of mass spectra,most protocols employ a low concentration of protease in combination with long incubation times. Immobilizationof the digestion enzyme via a covalent bond to the stationary phase is a useful tool to overcome these limitations.[1]

In case monolithic materials are used proteins are digested within minutes because of the excellent mass transferof these materials.Experimenteller Teil: Trypsin was immobilized via a glutardialdehyde spacer on a poly(glycidyl methacrylate-co-acrylamide-co-ethylene glycol dimethycrylate) monolith, which was prepared in an 100 µm ID fused silicacapillary by heat-induced polymerization.[2] To prevent autodigestion during the immobilization the competitivetrypsin inhibitor benzamidine was added. The monolithic trypsin reactors were characterized by capillary elec-trophoresis (CE) using the prototype substrate Na-benzoyl-L-arginine ethyl ester (BAEE). The digestion efficiencywas compared to tryptic digests of BAEE in solution. Proteins were digested with the monolithic trypsin reactor(MTR) and digests were separated by nano-HPLC and analyzed off-line by MALDI-TOF/TOF-MS (Ultraflex III,Bruker Daltonik) and on-line by nano-ESI-MS/MS (LTQ-Orbitrap XL, ThermoFisher). Protein database searcheswere performed with BioTools 3.1 (Bruker Daltonik) and BioWorks Browser 3.3.1 (ThermoFisher).Ergebnisse und Diskussion: The effects of denaturing agents, such as urea, and digestion temperature wereevaluated using myoglobin and bovine serum albumin. Digestion efficiencies were determined based on theobtained sequence coverages. In on-line LC/MS experiments cytochrome c was digested with the MTR and loadedon a C18 pre-column. Afterwards, the peptide mixture was separated on a C18-particle column with a lineargradient (5 to 52.5% acetonitrile containing 0.1% formic acid in 50 min) and analyzed by nano-ESI-MS/MS. Theresults were compared with in-solution digested cytochrome c.[3] After optimization of the digestion parameters, across-linked protein/peptide complex was digested in the MTR and the resulting peptides were analyzed in off-lineESI-MS experiments. Also, a mixture of five model proteins (BSA in 1000-fold excess, cytochrome c, hPPARa[4], lysozyme, and b-lactoglubulin) was digested with the MTR and analyzed by off-line nano-HPLC/MALDI-TOF/TOF-MS.[5] Enzymatic reactors with trypsin immobilized on a monolithic stationary phase were shown tobe of great use for a rapid and efficient protein digestion. The low backpressure of the monolith (∼ 50 bar at 300nL/min) makes immobilized monolithic enzyme reactors most suitable for off-line and on-line coupling with MSmethods. Moreover, the high surface area of the MTR allows rapid protein identification with digestion times inthe range of minutes.Neue Aspekte: Also, at a 1000-fold molar excess of one protein, low abundant proteins (femtomol level) werereadily identified by MS/MS experiments.Referenzen: [1] F. Svec, Electrophoresis, 2006, 27, 947-961; [2] J. Duan, et al., Proteomics, 2006, 6, 412-419; [3]Sproß, J.; Sinz, A. Anal. Bioanal. Chem. 2009, 395, 1583-1588; [4] Müller, M. Q.; Roth, C.; Sträter, N.; Sinz, A.Prot. Expr. Purif. 2008, 62, 185-189; [5] Sproß, J.; Sinz, A. Anal. Chem. in pressThema: Instrumentelle Entwicklungen, Proteine und PeptideKeywords: monolith, enzyme immobilization, nano-HPLC, MALDI-MS, ESI-MSKontakt: [email protected]

211

P115

The implementation and characterization of electron transferdissociation (ETD) on an ion mobility enabled Q-TOF mass

spectrometer

Desor, Michael (1); Campuzano, Iain (2); Brown, Jeff (2); Langridge, James (2); Williams, Jon (3)

1: Waters GmbH, Eschborn, Germany; 2: Waters Corporation, Manchester, UK; 3: University of Warwick,Warwick, UK

Einleitung: ETD is a MS/MS technique in which precursor cations are reacted with radical reagent anions toinduce fragmentation. Electron transfer from anion to cation, promotes fast and randomised dissociation comparedto collision induced dissociation (CID) and hence ETD product ion spectra contain predominantly "c" and "z" typeions and post translational modifications often remain intact providing valuable sequence information. Here weshow how ETD can be implemented and performed on a hybrid Q-IMS-TOF (Waters Synapt).Experimenteller Teil: A supply of reagent from a sealed ampule is delivered to the intermediate pressure regionof the nano ESI source and a high voltage discharge pin was added to the ion source to generate the reagent anions.Analyte cations were generated using nanospray source via infusion or nanoACQUITY UPLC. For ETD, the ionsource polarity and the quadrupole set mass were sequentially switched to deliver anions and cations into the TRAPtravelling wave (TWAVE) ion guide where they reacted to form ETD product ions. Product ions were optionallyseparated by ion mobility in the IMS TWAVE ion guide or were accelerated into the TRANSFER TWAVE ionguide to cause 2nd generation CID ions prior to mass analysis in the TOF.Ergebnisse und Diskussion: Tryptic peptides from a variety of biological sources have been separated by nanoAc-quity UPLC and a selection of triply charged precursor masses were subsequently made to generate LC-ETD spec-tra. Data were acquired at 1 spectra/second and the fragment ions were database searched. High quality data wasobserved for single tryptic digests at the 20-50fm injection level, with ETD data acquired at 1 spectra/second. Ingeneral, cleavage was observed at almost every amide bond in the peptide backbone, yielding easy-to-interpretsequence ladders of c and z-ions. This coupled with the inherent mass measurement accuracy and resolution of theoa-TOF mass analyser makes the data easily amenable to de novo sequencing. The signal intensity of the fragmentions seemed to diminish with decreasing m/z, as previously reported. In addition to the ETD experiments the massspectrometer can acquire alternate scans in CID, and as such data will be compared and contrasted between ETDand CID on a variety of peptides produced and separated by nanoscale chromatography.Neue Aspekte: Implementation of ETD on a hybrid Q-IMS-TOF shown. This technology will allow for directcombination of both fragmentation techniques.Thema: Instrumentelle Entwicklungen, Proteine und PeptideKeywords: Ionenmobilität, Q-TOF mass spectrometer, ETD, Tryptic peptides, CID


212

P116

Photodetachment-Spektroskopie von Anionen in derIonenmobilitäts-Spektrometrie

Riebe, Daniel; Zenichowski, Karl; Beitz, Toralf; Löhmannsröben, Hans-Gerd

Universität Potsdam, Germany

Einleitung: Die Ionenmobilitäts (IM)-Spektrometrie wird als Sensortechnik zur Detektion flüchtiger organischerVerbindungen wie chemischer Kampfstoffe, Drogen, toxischer Industriechemikalien und Sprengstoffen angewandt.Der Einsatz der IM-Spektrometrie in Durchgangsportalen von Flughäfen zum Nachweis von Sprengstoffen führtzu falsch-positiven Alarmen, die durch interferierende Substanzen hervorgerufen werden. Eine Erhöhung der Se-lektivität kann über die spektroskopische Charakterisierung der Anionen in der Driftröhre erreicht werden. Diegeringe Dichte der Anionen erschwert allerdings die Anwendung von absorptionsspektrometrischen Methoden.Als spektroskopische Methode, die auf der Detektion von Ladungen beruht, wird die Integration der Photodetach-ment (PD)-Spektroskopie in die IM-Spektrometrie mittels der Charakterisierung ausgewählter Anionen wie z.B.SF6, 1,4-Dinitrotoluol (DNT) und 2,4,6-Trinitrotoluol (TNT) vorgestellt.Experimenteller Teil: Die Molekülanionen (M−) wurden durch Laserionisation (1+1 REMPI) von Toluol undnachfolgendem Elektronentransfer auf SF6, DNT und TNT gebildet. Alternativ wurde das [DNT-H]− untersucht,das durch Protonenabstraktion von DNT durch Chloridionen bzw. O2

− erzeugt wurde. Die Darstellung der Chlori-dionen erfolgte durch dissoziative Elektronenanlagerung an Methylenchlorid. Die in der Ionisationsregion gebilde-ten Ionen wandern unter dem Einfluß eines elektrischen Feldes durch die Driftröhre zur Faraday-Elektrode. Nachca. 2/3 der Driftstrecke sind Laserfenster in die Driftröhre integriert, die die Einkopplung des PD-Lasers ermögli-chen. Die durch das Photodetachment gebildeten Elektronen, Fragmentionen und überlebenden Anionen werdenim letzten Drittel der Driftstrecke aufgetrennt und an der Faraday-Elektrode nachgewiesen. Der Nachweis der ge-bildeten Anionen erfolgte parallel mittels einer IM-Spektrometrie-Massenspektrometrie-Kopplung (LTQ XL, Fa.Thermo).Ergebnisse und Diskussion: Die PD-IM-Spektren einer Reihe von Verbindungen wie SF6, DNT, TNT, 2,4-Dinitrophenol, Pentachlorphenol und 4-Nitrotoluol zeigen bei den beiden PD-Wellenlängen von 355/532 nm signi-fikante Unterschiede in der PD-Effizienz der Anionen. Diese Unterschiede können zur Identifikation von Anionen,die ähnliche Ionenmobilität besitzen und im IM-Spektrum nicht aufgetrennt werden können, genutzt werden. DiePD-Effizienz wird quantitativ durch den PD-Querschnitt (PDQS) beschrieben. Zur Bestimmung des PDQS wurdedieWechselwirkung von Laserstrahl und Anionenwolke systematisch charakterisiert. Dazu gehören die Abhängig-keit des PD-Signals von der Laserenergie, dem Strahldurchmesser, der Strahlform und die räumliche Abbildungder Anionenwolke. Aus diesen Experimenten wurde eine Methode zur Berechnung des absoluten PDQS abgeleitet.Die absoluten PDQS werden für einzelne Anionen berichtet. Die PD-Experimente bei den beiden Wellenlängenλ = 355/532 nmwurden um die Aufnahme von PD-Spektren (PDQS(λ)) erweitert. Der Vergleich der experimentel-len PD-Spektren von SF6− und DNT− mit bereits publizierten PD-Spektren [1,2], die durch eine andere Methodebestimmt wurden, zeigt eine sehr gute Übereinstimmung. Die PD-Spektren von [DNT-H]− und TNT− werdenerstmals vorgestellt. Die Interpretation der erhaltenen PD-Spektren erfolgt auf der Basis quantenchemischer Rech-nungen.Neue Aspekte: Kombination von PD-Spektroskopie und IM-Spektrometrie Bestimmung absoluter Photodetach-mentquerschnitte Photodetachmentspektren von [DNT-H]− und TNT−

Referenzen: [1] R. S. Mock, E. P. Grimsrud J. Am. Chem. Soc. 1989, 111, 2861-2870.; [2] R. S. Mock, E. P.Grimsrud Chem Phys. Lett. 1991, 184, 99-101.Thema: Instrumentelle Entwicklungen, Ionen-Molekül-ReaktionenKeywords: Photodetachment, Anionen, Ionenmobilitätsspektrometrie, TNT, SF6Kontakt: [email protected]

213

P117

Authentic Open Formats in Mass Spectrometry

Hester, Alfons; Spengler, Bernhard

JLU Giessen, Germany

Einleitung: When data is stored electronically a set of methods are needed to guarantee integrity and authenticityof the data. A standardized data format should implement such methods directly within its specification. Further-more the implementation of authenticity needs to be flexible enough to be well-prepared for future needs. Severalinvestigative methods, for example compliance monitoring, define principles to ensure consistency and reliabilityof results, covering also the electronic storage of acquired data [3]. Using the standardized open formats imzML[1] and mzML [2] as a test frame, an authenticity framework was implemented. This framework can easily beextended to other XML (eXtensible Markup Language)[4] based formats. An authentic work flow from data originto the expert is feasible by implementing it.Experimenteller Teil: A digital signature method following rules and principles defined in RFC4880 was adaptedto ms data and ms related data stored in imzML or mzML. RFCs (Request For Comments) are memos published byIETF (Internet Engineering Task Force). They deal with subjects like research, innovations and methods applicableto the Internet and Internet-connected systems. RFC4880 describes the OpenPGP format and procedure. OpenPGPstands for open pretty good privacy, which covers methods for digital signature and encryption.Ergebnisse und Diskussion: Data derived from our mass spectrometers is stored in authentic imzML and thenprocessed and visualized by imaging software Mirion V2.Neue Aspekte: Authentic data format and authentic work flow in imaging of mass spectra.Referenzen: [1] http://www.computis.org; [2] http://www.psidev.info;[3] http://www.oecd.org/document/63/0,3343,en_2649_34381_2346175_1_1_1_1,00.html;[4] http://www.w3.org/XML/Thema: Instrumentelle EntwicklungenKeywords: Authenticity, GLP, ImzML, Open FormatKontakt: [email protected]

214

P118

Direct skin analysis by desorption electrospray ionization

Dénes, Júlia (1); Katona, Mária (2); Skoumal, Réka (3); Tóth, Miklós (3); Takáts, Zoltán (1)

1: JLU Gießen, Institut für Anorganische und Analytische Chemie, Germany; 2: The Institute of Enzymology of

Hungarian Academy of Sciences; 3: Semmelweis University, Hungary

Einleitung: The applicability of the desorption electrospray ionization method was already demonstrated for

arbitrary type of surfaces including the biological tissues. The use of the technique for intact skin analysis was

introduced in the very first report of the DESI [1]. The potential application areas of this intact skin analysis could

be toxicology, pharmacology, forensic analysis and airport security as well. Some of these were presented below.

Experimenteller Teil: The analysis were performed with home-built DESI sources for the Thermo LCQ and

LTQ instruments. The human skin tests were executed with volunteers and since the model experiments for

toxicology were successfull we made the following tests with laboratory animals. We tested the excretion of

the pharmaceuticals of the animals and we observed that the rats have the sweat glands on the sole.

Ergebnisse und Diskussion: This method is based on DESI technique and it is a non-invasive testing technique

of human skin with 1-5 s analysis time. This is absolutely painless for the patient, involves no electric shocking.

We can get real-time information of the composition of any material excreted by sweat glands, and long-term

information of any material excreted by oil glands. An aqueous-alcohol DESI spray was directed onto the finger

of a person who had taken some medicine or had smoke, or onto a rat skin which was dosed with a pharmaceutical

and the molecules became detectable directly on the skin. We developed a model system for toxicology and this

is smoking. We made nicotine measurements from human skin in different times, before and after smoking and

we could determinate the kinetic of metabolism of the nicotine. We developed pharmacokinetic analysis on rat

skin as well. The animal was anesthetized by ketamine-xylazine combination and these species were measurables

from the skin of the rat sole. The control kinetic measurements were performed from blood by HPLC-MS. We

have developed in vivo metabolism studies as well. The model was the oxidation of dimethylthiourea (DMTU)

in oxidative stress. Wistar rats were dosed with DMTU, which can tranform to S-oxide form. The effect of

Angiotensin II was tested which induces the formation of reactive oxidative species, DMTU captures OH radicals

and is oxidized into its S-oxide. Both DMTU and DMTU S-oxide were detected from the sole of rats.

Neue Aspekte: We developed a direct DESI analysis for rapid determination of pharmaceuticals from human skin

or skin of treated animals.

Referenzen: [1] Takats, Z.; Wiseman, J.M.; Gologan, B.; Cooks, R.G., Science, 306(5695), 471-473. 2004.

Thema: Instrumentelle Entwicklungen

Keywords: DESI, direct analysis, pharmacokinetics, therapeutic drug monitoring


215

P119

Ambient pressure mass spectrometric analysis of live Drosophilamelanogaster (fruit flies)

Pirkl, Alexander (1,2); Dreisewerd, Klaus (2); Yew, Joanne Y. (2); König, Simone (1)

1: Integrierte Funktionelle Genomik, Interdisziplinäres Zentrum für Klinische Forschung, WestfälischeWilhelms-Universität Münster, Germany; 2: Institut für Medizinische Physik und Biophysik, Westfälische

Wilhelms-Universität Münster, Germany

Einleitung: Direct analysis of whole animals (e.g., insects) by mass spectrometry is lethal for the animal in mostcases. This drawback prevents the measurement of the same organism before and after experimental manipulation.Recently we showed that with minimal sample preparation, mass spectra can be generated from the cuticular sur-face of live fruit flies using field-based ion generation (FBIG) performed at atmospheric pressure (AP) conditions[1]. This technique is based on the application of a high voltage between the fly and the inlet capillary of themass spectrometer. In order to better understand the underlying mechanisms of this new method, we comparedionization products generated under FBIG conditions with those produced by direct laser desorption/Ionizationmass spectrometry (UV-LDI-MS) [2].Experimenteller Teil: Analyses were performed with an Esquire3000 ion trap mass spectrometer (Bruker) and aPremier QTOF-MS (Waters). Individual Drosophila melanogaster were taped to their backs on a sample holderusing adhesive. For FBIG, flies were positioned about 1 mm in front of the inlet capillary of the mass spectrometer.A voltage of 2-3 kV was applied to the ion extraction capillary. Several artificial FBIG-emitters and syntheticcompounds were studied for comparison. For AP-UV-LDI measurements, flies were irradiated by the focusedbeam of an N2-laser (λ = 337 nm, τ = 3 ns). Extraction voltages were lowered to avoid FBIG conditions. Controlexperiments were furthermore carried out with an o-TOF mass spectrometer, operated with background pressureof ∼ 2 mbar of Ar [2].Ergebnisse und Diskussion: Ions could be detected from living and dead flies over extended periods of time bysimply exposing them to an electric field typically used for nano-ESI or AP-MALDI. The spectra were highlycomplex and in some cases exhibited ions up to ∼ m/z 1800. Live flies could be re-interrogated after experimentalbreaks of several hours. The findings suggest that endogenous compounds can be profiled with this method fromlive animals behaving in real time.Scanning electron microscopy revealed hairs on fly legs which are spaced at∼ 10-25 µm distance and are∼ 25-30µm in length. Radii at the tip were ∼ 80 nm. This suggests that high electrical field strengths produced at the tipof the hairs may play a role in the ion generation. Artificial emitters which reflect this geometry were examinedin order to investigate whether such features are sufficient for field-based ion generation (FBIG). Various synthetichydrocarbons (HCs) and other small molecules were detected using sharp single-point emitters (e.g., needle tips) orcommercial FD emitters. Tentative assignment suggests the presence of a series of oxygen-containing hydrocarbonspecies in the FBIG mass spectra acquired from the flies. Cuticular HCs are expressed by the flies to preventdesiccation and to act as communication cues. Comparing the FBIG data with results obtained by direct UV-LDI mass spectrometry of single flies [2], similarities but also significant differences are found, in particular withrespect to the detection of oxygen-containing HCs. AP-UV-LDI MS experiments were performed with the ion trapinstrument, bridging the AP-FBIG and the UV-LDI-o-TOF configurations.Neue Aspekte: MS analysis of living fruit flies under atmospheric pressure field-based ion generation conditions;first results of AP-UV-LDI mass spectrometric analysisReferenzen: [1] A. Pirkl, K. Dreisewerd, J.Y. Yew, S. König, Field-based ion generation from microscale emitterson natural and artificial objects for atmospheric pressure mass spectrometry, Anal. Bioanal. Chem., Publishedonline: 17 October 2009; [2] J. Yew, K. Dreisewerd, H. Luftmann, J. Müthing, G. Pohlentz, E. Kravitz, A NewMale Sex Pheromone and Novel Cuticular Cues for Chemical Communication in Drosophila, Curr. Biol., 2009,19, 1245-1254Thema: Instrumentelle Entwicklungen, LipideKeywords: Drosophila melanogaster, atmospheric pressure ionization, fruit fly, microscale emitter, field-supportedionizationKontakt: [email protected]

216

P120

Improvements of mass accuracy using novel calibration conceptson Orbitrap instruments

Zeller, Martin; Damoc, Eugen; Lange, Oliver; Kuipers, Hartmut; Möhring, Thomas

Thermo Fisher Scientific, Germany

Einleitung: Mass accuracy is one of the key parameters for the characterization of mass spectrometers. Usuallythis parameter is evaluated under nearly ideal conditions with direct infusion of a sample of low complexity.However, the level of mass accuracy can change for complex samples with a high dynamic range and it may alsodependent on changes in environmental conditions of the mass spectrometer such as temperature or drifts in theelectronics over the time etc. In this work we evaluate the effect of a novel calibration routine and concept ofinternal recalibration during data acquisition.Experimenteller Teil: All data was acquired using a LTQ Orbitrap XL ETD or LTQ Orbitrap Velos ETD (ThermoFisher Scientific, Bremen, Germany). All samples were separated for each experiment via Surveyor MS PumpPlus LC equipped with MicroAS Autosampler (all Thermo Fisher Scientific) using a peptide trap (C18 Biobasic,100 µm inner diameter, 2 cm length) and a C18 analytical column (C18 Biobasic, 75 µm inner diameter, 10 cmlength, both NanoSeparations, NL), at a flow rate of 250 nl/min using standard data-dependent acquisition methodswith lockmass functionality enabled and disabled. Data analysis was done using Proteome Discoverer 1.1.Ergebnisse und Diskussion: The new "non-linear" calibration routine for external mass calibration makes useof the observation that the precision of the mass measurements is even higher than the mass accuracy. The newcalibration routine does not discriminate the masses over the m/z scale and therefore improves the overall statisticalmass accuracy. For the LTQ Orbitrap Velos the concept of internal mass recalibration has changed: If an internalcalibrant is not found in the high resolution spectrum, the calibration parameters of the last found recalibrationare used. This is possible as the mass accuracy drifts slowly over time. The internal calibrant is not injectedinto the C-trap via SIM injection and therefore the overall cycle time is improved. The database search for eachsample resulted in several thousand identified peptides (1% FDR). The statistical analysis of the mass deviationof the identified peptides for each sample revealed that the new calibration routine improves the overall statisticalmass accuracy as well as the maximum mass deviation. The mass deviation is almost independent from the signalintensity and m/z.Neue Aspekte: Improved mass accuracy for real life samples using a novel external and internal calibrationconcept.Thema: Instrumentelle Entwicklungen, Proteine und PeptideKeywords: mass accuracy, Orbitrap, protein and peptide identificationKontakt: [email protected]

217

P121

Einsatz der LC-MS/MS im medizinischen Routinelabor

Vogeser, Michael; -, AG "LC-MS/MS in der Medizin" der DGKL

Klinikum der Universität München, Germany

Einleitung: Die GC-MS-Technologie hat sich in einigen Bereichen derMedizin lange als Schlüsseltechnik etabliert,insbesondere in der Toxikologie. In typische Hochdurchsatz-Labors der allgemeinen Krankenversorgung konntediese Technologie vor allem auf Grund der komplexen Anwendungscharakteristika keinen Einzug halten. Seitnunmehr etwa 10 Jahren findet dagegen die LC-MS/MS in einer ständig wachsenden Zahl klinischer Labors An-wendung.Experimenteller Teil: Die LC-MS/MS bereichert das methodische Spektrum der Labormedizin komplementär zuden bisherigen Standardtechniken der Photometrie und des Immuno-Assays wesentlich. Die Technik erlaubt eineflexible Methodenentwicklung unabhängig von der Diagnostika-Industrie; dies ist insbesondere von Relevanz fürdas therapeutische Drug-Monitoring. Sie bietet im Vergleich zur Immunoassay-Technik eine überlegene Spezifitätbei vielen kritischen Analyten wie Steroidhormonen und eröffnet die Möglichkeit multi-parametrischer Analysenwie etwa beim Neugeborenen-Screening auf angeborene Stoffwechselstörungen und für künftige metabolomische

Analysen. Praktikabilität und Robustheit der LC-MS/MS-Technologie sind wesentlich besser mit den Anforderun-

gen und Arbeitsabläufen klinischer Labors (inklusive 24 Stunden-Betrieb) vereinbar, als dies für die GC-MS gilt.

Des Weiteren ist die Probenvorbereitung ungleich einfacher und der mögliche Probendurchsatz wesentlich höher.Ergebnisse und Diskussion: Dennoch ist der Standard der Bedienerfreundlichkeit und des Probendurchsatzesvon LC-MS/MS-Analysen noch sehr weit von den Verhältnissen bei den gegenwärtigen Standardtechniken Pho-

tometrie und Immunoassay entfernt. Insbesondere die Neu-Implementierung von LC-MS/MS-Methoden erfordert

Expertenwissen, das nur in wenigen Kliniklabors verfügbar ist. Die breite Anwendung der LC-MS/MS in der

Labordiagnostik bietet zweifellos die Perspektive, die Leistungsfähigkeit der diagnostischenMedizin wesentlich zu

steigern; Voraussetzung ist jedoch die Entwicklung voll-automatisierter Analysensysteme auf Grundlage der LC-

MS/MS, die ebenso bedienerfreundlich sind, wie derzeit eingesetzte konventionelle Analyzer-Systeme, die durch

angelerntes Personal bedient werden können. Dabei stehen die Automation von Probenhandling und -vorbereitungsowie derErgebnisauswertung im Mittelpunkt der notwendigen medizintechnischen Entwicklungsarbeiten.Neue Aspekte: Die LC-MS/MS-Technologie könnte in den kommenden Jahren zu einer weiteren Grundtechnolo-gie der Labormedizin entwickelt werden.Referenzen: [1] Vogeser M, Seger C. A decade of HPLC-MS/MS in the routine clinical laboratory–goals forfurther developments. Clin Biochem 2008;41:649-62.Thema: Instrumentelle EntwicklungenKeywords: Klinische Chemie, Labormedizin, medizinische Diagnostik, in-vitro-Diagnostik, LC-MS/MSKontakt: [email protected]

218

P122

Direct coupling of normal- and reversed-phase HPTLC withUV-spectroscopy and IR-MALDI-o-TOF mass spectrometry for

the detection of tetracycline antibiotics

Meisen, Iris (1); Wisholzer, Sebastian (1); Soltwitsch, Jens (2); Dreisewerd, Klaus (2); Mormann, Michael (2);Müthing, Johannes (1); Karch, Helge (1); Friedrich, Alexander (1)

1: Institute for Hygiene, University of Münster, Robert-Koch-Str. 41, D-48149 Münster, Germany; 2: Institute of

Medical Physics and Biophysics, University of Münster, Robert-Koch-Str. 31, D-48149 Münster, Germany

Einleitung: Tetracyclines (TCs) are a group of bacteriostatic antibiotics with activity against a wide variety of

gram-positive and gram-negative bacteria. They have been widely employed as therapeutic agent in human and

veterinary medicine but also as growth promotor in animal husbandry. Hence, it is not surprising that the emergence

of bacterial resistances to these antibiotics is increasing. For monitoring the exposure of humans, animals and

environment with residual TCs sensitive analytical methods are required. Here, we introduce an approach based

on the direct coupling ofHPTLC, UV-spectroscopy andIR-MALDI-o-TOF-MS. The liquid matrix glycerol was

used for the mass spectrometric detection of tetracycline (TC), oxytetracycline (OTC), chlorotetracycline (CTC)

and doxycycline (DC) on normal- and reversed-phase HPTLC plates.

Experimenteller Teil: Antibiotic mixtures were separated on silica gel 60 and RP-18W HPTLC plates. After

development TCs were either stained with Fast Blue B salt or detected and quantified by UV-spectroscopy at a

wavelength of λ = 360 nm. Mass spectrometric analysis was performed from unstained TCs bands with a modified

o-TOF mass spectrometer equipped with an Er:YAG infrared laser emitting pulses of ∼ 100 ns duration at a

wavelength of 2.94 µm and a repetition rate of 2 Hz. All spectra were recorded in the positive ion mode. Glycerol

was used as liquid matrix.

Ergebnisse und Diskussion: The analysis of the tetracycline antibiotics involves the application of three comple-

mentary methods (i) HPTLC separation of analytes, (ii) their detection and quantification by UV-spectroscopy at

λ = 360 nm, and (iii) direct identification of separated analytes on the HPTLC plate by IR-MALDI-o-TOF-MS.

Since TCs are known to form chelate complexes with metal ions a special pretreatment of the HPTLC plates is

required prior to application of the analytes. For silica gel plates a predevelopment step with EDTA solution is per-

formed, additionally, solvent systems containing EDTA are employed and for RP plates solvent systems containing

oxalic acid are used to control the undesired metal ion complexation of TCs. Subsequently, antibiotics analyzed

by HPTLC and UV-spectroscopy are detected as well-separated bands and peaks, respectively. The analysis of

various TCs mixtures with different concentrations (1 µg - 10 ng) revealed a linear relationship of amount of an-

alyte applied for HPTLC and UV-absorption atλ = 360 nm. The coefficients of determination obtained by linear

regression ranged from R2 = 0.97 - 0.99. Desorption/ionization of antibiotics directly from the silica gel HPTLC

plate resulted in quite complex mass spectra. Due to the EDTA predevelopment step the spectra showed increased

background signals, thus hampering the detection of small amounts of sample. The analytes were detected as

sodiated and doubly sodiated species. Additionally, analyte-glycerol adducts of high abundance were observed. In

contrast, the mass spectra acquired from TCs separated on RP-HPTLC plates exhibited high abundant protonated

species. Cationized species were not detected and analyte-matrix adducts were only present to a minor extent. The

limits of detection were 20 ng for UV-spectroscopy and 5 ng for mass spectrometric analysis of TCs from RP-18W

HPTLC plates.

Neue Aspekte: This combinatorial HPTLC-MS technique offers high potential for the sensitive detection of

residual antibiotics in human, animal and environmental samples.

Thema: Organische Massenspektrometrie, Umweltanalytik und Aerosole

Keywords: IR-MALDI MS, HPTLC, antibiotics, tetracycline


219

P123

Distinguishing isomers and entering the ppqv detection limitregion - latest developments in PTR-MS instruments

Jordan, Alfons (1); Sulzer, Philipp (1); Jaksch, Stefan (1); Hanel, Gernot (1); Hartungen, Eugen (1);Seehauser, Hans (1); Märk, Lukas (1); Haidacher, Stefan (1); Schottkowsky, Ralf (1); Märk, Tilmann D. (1,2)

1: Ionicon Analytik GmbH, Technikerstr. 21a, 6020 Innsbruck, Austria; 2: Institut für Ionenphysik und

Angewandte Physik, Universität Innsbruck, Technikerstr. 25, 6020 Innsbruck, Austria

Einleitung: Here we report on very recent instrumental developments in proton-transfer-reaction mass spectrom-

etry (PTR-MS), namely i) the improvement of the detection limit that now allows for measuring trace gas com-

pounds in a concentration range from several ppmv down to the ppqv (parts-per-quadrillion) region with a typical

response time well below 100ms and, in case a TOF mass analyzer is used, a mass resolution up to 8.000 m/∆m

and ii) the so called "switchable reagent ions (SRI)" feature, i.e. possibility to switch between H3O+, NO+ andO2

+ as reagent ions [1].Experimenteller Teil: The PTR-MS technology was invented and developed by scientists of the "Institut für

Ionenphysik" at the Leopold-Franzens University in Innsbruck and made commercially available by the spin-off

company IONICON Analytik GmbH (for details see [2] and very recently [3]). However, in its original version

only H3O+ reagent ions can be used, which is the preferable choice for many applications but also has some

disadvantages (e.g. only substances with a proton affinity higher than water can be ionized). Therefore we

developed an extension to these instruments that allows for switching between H3O+, NO+ and O2

+ as reagent

ions in less than 10 s. In the framework of this development we also improved significantly the overall sensitivity

of the PTR-MS instrument.

Ergebnisse und Diskussion: We present data that show that the sensitivities obtained with NO+ and O2+ are

comparable or even better to the outstanding sensitivity of the established PTR-MS instruments using H3O+

reagent ions. For several compounds like e.g. chlorobenzene we obtain a sensitivity close to 1000 cps/ppbv which

leads to a detection limit of several hundred ppqv and is therefore much better than those from e.g. SIFT-MS

instruments. To demonstrate the advantages of the SRI setup we e.g. measured acetone and propanal (isomeric

molecules at nominal mass 58 amu) utilizing NO+ as the precursor ion. According to Spanel et al. [4] NO+

interactions with aldehydes follow the reaction: NO+ + XH => X+ + NOH whereas ketones follow: NO+ + XH

=> XH+ + NO (and clustering). This means that we see isomeric compounds on different nominal masses and can

identify them unambiguously. On the other hand, by using O2+ precursor ions we are able to ionize molecules

that cannot be measured via hydronium proton transfer reaction (e.g. ethylene or acetylene). In conclusion it can

be said that the presented developments give great benefit to PTR-MS technology by improving the identification

capabilities to separate isomers, enhancing the analyzable substance classes and lowering the detection limit.

Neue Aspekte: For the first time it is possible to separate isomers and measure at ppqv levels with PTR-MS.

Referenzen: [1] A. Jordan, S. Haidacher, G. Hanel, E. Hartungen, J. Herbig, L. Märk, R. Schottkowsky, H.

Seehauser, P. Sulzer, T. D. Märk, Int. J. of Mass Spec., 286 (2009), 32-38.; [2] W. Lindinger, A. Hansel, A. Jordan;

Int. J. of Mass Spectrom. and Ion Processes, 173/3 (1998) 191-241.; [3] R. S. Blake, P. S. Monks, A. M. Ellis;

Chem. Rev., 109 (3) (2009), 861-896.; [4] P. Spanel, Y. Ji, D. Smith; Int.J. of Mass Spectrom. and Ion Processes,

165/166 (1997) 25-37.

Thema: Ionen-Molekül-Reaktionen, Umweltanalytik und Aerosole

Keywords: PTR-MS, SRI, isomer separation, low detection limit, high sensitivity


220

P124

Direct aqueous injection (DAI) analysis of trace compounds inwater with proton-transfer-reaction mass spectrometry (PTR-MS)

Jürschik, Simone (1); Sulzer, Philipp (1); Jaksch, Stefan (1); Haidacher, Stefan (1); Jordan, Alfons (1);Schottkowsky, Ralf (1); Hartungen, Eugen (1); Hanel, Gernot (1); Seehauser, Hans (1); Märk, Lukas (1);

Märk, Tilmann D. (1,2)

1: IONICON Analytik GmbH, Technikerstr. 21a, 6020 Innsbruck, Austria; 2: Institut für Ionenphysik undAngewandte Physik, Universität Innsbruck, Technikerstr. 25, 6020 Innsbruck, Austria

Einleitung: Proton Transfer Reaction- Mass Spectrometry (PTR-MS) is a well established technique in trace gasanalysis which offers many advantages, such as real-time analysis, no sample preparation, very low detection limits(ppqv region) and very short response time (about 100ms). However, PTR-MS requires the samples being presentin the gas phase for analysis. Here we report on a new instrumental development of high sensitivity PTR-MSthat allows direct analysis of liquid samples. Attempts to measure volatile organic compounds in water so far aremainly headspace analysis above the water surface or membrane inlet setups, which both are well suitable forcertain applications, but also suffer from significant disadvantages [1].Experimenteller Teil: Details about PTR-MS technique itself have been described elsewhere (e.g. [2], [3]). Inshort, a hollow cathode ion source produces H3O+ (or optionally NO+ and O+

2 with the novel switchable reagentions (SRI) feature [4]) from distilled water of very high purity (up to 99%), so that these reagent ions can beinjected directly, without a mass filter, into the drift tube, where the ionisation of the sample molecules takes placevia proton transfer (charge transfer with SRI). For a first proof-of-principle test of the DAI water solutions wereprepared with 1 to 1000 ppbw (part per billion weight) concentrations of methanol, acetonitrile, pyridine (in thiscase additional mixtures down to 125 pptw were prepared) and cyclohexanol in distilled water.Ergebnisse und Diskussion: After a series of tests with different injection speeds, carrier gas flow rates, syringesizes and temperatures in our direct aqueous injection system, we found the best working conditions to be 0.6µl/s liquid flow into 1.75 l/min air flow (at 70°C) to avoid droplet formation and achieve 100% vaporization ofthe liquid. With these conditions the detection of trace compounds in water was possible over several orders ofmagnitude down to a concentration level of about 100 pptw (for pyridine at protonated mass 80 m/z and for about 5min integration time) with great linearity, while only consuming about 100 µl of the sample. The response time ofthe setup is between 20 and 25 seconds. The DAI method is applicable to the analysis of all substances dissolvedin water and not limited by the permeability of a membrane. Therefore it will open completely new fields ofapplication for the PTR-MS technique (e.g. monitoring of water pollution in drinking water, rivers, ground water,etc.). In conclusion it can be said that the direct aqueous injection (DAI) technique which we present here turnsout to be an ideal solution for direct analysis of liquid samples with PTR-MS [5].Neue Aspekte: For the first time PTR-MS is coupled with a direct liquid injection system to analyze tracecompounds in water.Referenzen: [1] E. Boscaini, M. Alexander, P. Prazeller, T.D. Märk, Int. J. of Mass Spec. 239 (2004) 171-177.;[2] W. Lindinger, A. Hansel, A. Jordan; Int. J. of Mass Spectrom. and Ion Processes; 173/3 (1998) 191-241.; [3]J. de Gouw, C. Warneke, T. Karl, G. Eerdekens, C. van der Veen, R. Fall; Mass Spectrometry Reviews, 26 (2007),223-257.; [4] A. Jordan, S. Haidacher, G. Hanel, E. Hartungen, J. Herbig, L. Märk, R. Schottkowsky, H. Seehauser,P. Sulzer, T. D. Märk, Int. J. of Mass Spec., 286 (2009), 32-38.; [5] S. Jürschik, A. Tani, P. Sulzer, S. Haidacher, A.Jordan, R. Schottkowsky, E. Hartungen, G. Hanel, H. Seehauser, L. Märk, T.D. Märk; Int. J. of Mass Spec., 289(2009), 173-176.Thema: Ionen-Molekül-Reaktionen, Umweltanalytik und AerosoleKeywords: PTR-MS, direct injection, water analysis, proton transferKontakt: [email protected]

221

P125

MALDI Imaging zur Lokalisierung und Identifizierung vonPeptiden und Proteinen direkt von Gewebeschnitten

Pribil, Patrick (2); Booy, Aaron (2); Merkel, Dietrich (1); Glueckmann, Matthias (1)

1: AB SCIEX, Germany; 2: AB SCIEX, Canada

Einleitung: MALDI-Imaging ist eine neuartige Methode [1], welche die Anwendbarkeit der MALDI (Matrix-assisted laser desorption/ionization) - Massenspektrometrie bei der Analyse von dünnen Gewebeschnitten ermög-

licht. Dabei werden MALDI-MS Profile direkt vom Gewebe erstellt, wobei der Durchmesser des ionisierenden

Laserpulses in modernen Geräten wie dem 4800 Plus MALDI TOF/TOF Analyzer bei ca. 50 µm liegt, so dass

eine hohe räumliche Auflösung erreicht wird. Die Kopplung mit Tandem-Massenspektrometern erlaubt zusätzlich

eine weitere Charakterisierung der Biomoleküle mittels MSMS. Dies kann zu einer Identifizierung entweder der

nativen Peptide [2,3] oder nach tryptischem Verdau der tryptischen Peptide [4] direkt von der Gewebeoberfläche

führen.

Experimenteller Teil: Typischerweise können tryptische Peptide mittels MSMS einfacher identifiziert werden als

nicht-tryptische, native Peptide. Der tryptische Verdau von Peptiden/Proteinen direkt auf dem Gewebe erlaubt es,

ihre tryptischen Peptide mittels MALDI Imaging zu lokalisieren und mit MSMS zu identifizieren. Weiterhin er-

möglicht der tryptische Verdau den Nachweis von Proteinen, die auf Grund ihrer Masse nicht direkt mittels MSMS

fragmentiert werden können. Hierzu wird mittels eines Shimadzu ChIP-1000, nach Trocknung des Gewebes, eine

Trypsin Lösung aufgebracht. Nach Inkubation (12h, 37°C) wird eine MALDI matrix Lösung aufgetropft. Die Ge-

webeschnitte werden danach mittels MALDI MS und MSMS analysiert. Die Imaging und MSMS Daten werden

mit TissueView™ Software und MASCOT Software ausgewertet.

Ergebnisse und Diskussion:MS Daten wurden von Gewebeschnitten aus Rattenhirn aufgenommen. Verschiede-

ne native Peptide konnten in spezifischen Regionen des Rattenhirns nur von den Teilen des Gewebes identifiziert

werden, die nicht mit Trypsin behandelt worden waren. Dies ist darauf zurückzuführen, dass native Peptide von

Trypsin verdaut und daher nicht mehr im Massenbereich zugänglich sind. Jedoch konnten von den mit Trypsin

behandelten Gewebeschnitten verschiedene tryptische Peptide detektiert werden, welche mittels MSMS Massen-

spektrometrie und Datenbanksuche identifiziert werden konnten. Dies erlaubte die Identifikation verschiedener

Proteine direkt vom Gewebe. Die Kombination von MALDI Imaging mit MS und MSMS mit der zusätzlichen

Präparation mittels verschiedener Proteasen eröffnet die Möglichkeit Proteine im Gewebe zu identifizieren, was

in Zukunft evtl. die Prozesse der Identifizierung potentieller Peptid- oder Protein-Biomarker direkt im Gewebe

erlauben kann.

Neue Aspekte: Robotischer Verdau von Proteinen direkt auf Gewebeschnitten erlaubt die Identifizierung von

Peptiden und Proteinen mittels MALDI MS und MSMS Massenspektrometrie.

Referenzen: [1] Markus Stoeckli, Pierre Chaurand, Dennis E. Hallahan and Richard M. Caprioli, Nature Medicine

2001, 7, 493 - 496.; [2] Markus Stoeckli, Dieter Staab, Alain Schweitzer, James Gardiner and Dieter Seebach, J.

Am. Soc. Mass Spectrom. 2007, 18, 1921-1924.; [3] Stephanie S. DeKeyser, Kimberly K. Kutz-Naber, Joshua

J. Schmidt, Gregory A. Barrett-Wilt, and Lingjun Li, J. Proteome Res. 2007, 6 (5), pp. 1782-1791.; [4] M. Reid

Groseclose, Malin Andersson, William M. Hardesty, Richard M. Caprioli, J. Mass Spectrom. 2007, 42, 476-489.

Thema: Proteine und Peptide, Imaging

Keywords:MALDI, Imaging, tryptische Peptide, native Peptide, Proteine


222

P126

Imaging of endogenous peptides in pituitary gland with highlateral resolution and high mass accuracy by Matrix AssistedLaser Desorption/Ionization Mass Spectrometry (MALDI MS)

Guenther, Sabine (1); Roempp, Andreas (1); Kummer, Wolfgang (2); Spengler, Bernhard (1)

1: Justus Liebig Universität Gießen, Institut für anorganische und analytische Chemie, Germany; 2: Justus LiebigUniversity, Anatomy, Giessen, Germany

Einleitung: Mass Spectrometry Imaging is a useful tool to visualize the distribution of various endogenousbiomolecules such as lipids and peptides and exogenous substances like drugs and metabolites in tissues. Directidentification of analytes is often difficult due to the complexity of tissue. Until now imaging mass spectrometersexhibited a limitation in at least one of the following areas: lateral resolution, mass resolving power, mass accuracy,MSMS capability or sensitivity. A Linear Ion Trap (LTQ)-Orbitrap mass spectrometer combined with an in-housedeveloped atmospheric pressure MALDI imaging ion source for the first time combines all these parameters on ahigh level in one instrumental setup [1]. Imaging of neuropeptides in mouse pituitary gland with a lateral resolutionof 10 µm is presented.Experimenteller Teil: Adult Bl6/N mice were sacrificed and organs were immediately snap-frozen in liquidnitrogen. Tissue samples were cut in sections of 20 µm thickness. Sections were thaw-mounted on conductiveITO-coated glass slides. For measurements, tissue sections were covered with 2,5-dihydroxybenzoic acid by apneumatic sprayer without prior washing steps. The sample stage was moved with a step width of 10 µm. Ionswere generated under atmospheric pressure using a nitrogen laser and were transferred into the LTQ-Orbitrap(Thermo Scientific GmbH, Bremen). 30 laser pulses were accumulated in the linear ion trap for each Orbitrap massspectrum. MSMS measurements were performed in the linear ion trap. Control of the ion source and generationof mass spectral images were performed employing homebuilt software.Ergebnisse und Diskussion: Mass spectral images of peptides in pituitary gland were achieved in high qualitywith this setup. Sensitivity of the LTQ-Orbitrap was sufficient to allow for highly accurate mass measurementseven at 10 µm lateral resolution. Due to the high mass accuracy (< 2ppm) and mass resolving power (R=30000)of the LTQ-Orbitap ion images could be generated with a mass bin width (selectivity) of 0.01 u and therefore havea high probability to show solely the distribution of identified peptides without interferences. Eight neuropeptideswere imaged in the mass range up to 2000 u. The ion images of the peptides show on a cellular level that theirdistributions within the pituitary gland are restricted to accurately defined tissue types. This is in excellent agree-ment with the gland‘s structure and biological function. The identity of the peptides was not only confirmed byaccurate mass, but also by MSMS measurements from single 10 µm sample spots, directly from tissue. Postrans-lational modifications can be detected. Two peptides were alternately fragmented during scanning via CollisionInduced Dissociation and were measured in the LTQ. Fragment ion images were in good accordance with thedistributions of their parent peptides, confirming their identity and excluding analyte interferences. Fragment ionimages of almost the complete y-ion series could be generated for one peptide. The method appears to be ideallysuited for imaging peptide signatures on a cellular level with high confidence of identification and high sensitivity.In addition to peptide measurements, phospholipids signals were used to characterize structural features of theinvestigated tissue sections.Neue Aspekte: Accurate mass MALDI Mass Spectrometry Imaging of endogenous peptides at cellular resolution.Referenzen: [1] M. Koestler, D. Kirsch, A. Hester, A. Leisner, S. Guenther, B. Spengler, Rapid Communicationsin Mass Spectrometry 2008, 22, 3275Thema: Proteine und Peptide, ImagingKeywords: MALDI MS Imaging, Orbitrap, neuropeptides, high lateral resolutionKontakt: [email protected]

223

P127

Classification of HER2 Receptor Status in Breast Cancer Tissuesby MALDI Imaging Mass Spectrometry

Anders, Sandra Rauser (1); Marquardt, Claudio (1); Balluff, Benjamin (2); Albers, Christian (3); Belau, Eckhard

(3); Hartmer, Ralf (3); Suckau, Detlev (3); Specht, Katja (4); Ebert, Matthias P. (2); Schmitt, Manfred (5);

Aubele, Michaela (1); Höfler, Heinz (1); Anders, Ulrike (3); Walch, Axel (1)

1: Institute of Pathology, Helmholtz Zentrum München, Neuherberg, Germany; 2: Department of Medicine II,

Klinikum rechts der Isar, TUM, Munich, Germany; 3: Bruker Daltonik GmbH, Bremen, Germany; 4: Institute of

Pathology, TUM, Munich, Germany; 5: Department of Obstetrics and Gynecology, Klinikum rechts der Isar,

TUM, Munich, Germany

Einleitung: Clinical laboratory testing for HER2 status in newly diagnosed, primary breast cancer tissues is

critically important for therapeutic decision making. Matrix-assisted laser desorption/ionization (MALDI) imaging

mass spectrometry (IMS) is a powerful tool for investigating proteins through the direct and morphology-driven

analysis of tissue sections. Unlike immunohistochemistry (IHC), MALDI-IMS enables the acquisition of complex

protein expression profiles without any labeling. We hypothesized that MALDI-IMS may determine HER2 status

directly from breast cancer tissues.

Experimenteller Teil: Breast cancer tissues (n=48) predefined for HER2 status by IHC and fluorescence-in-situ-

hybridization (FISH) were subjected to MALDI-IMS and protein profiles were obtained through direct analysis

of tissue sections. Protein identification was performed by tissue micro-extraction and fractionation followed by

top-down tandem mass spectrometry on a spherical ion trap with ETD. A discovery and an independent validation

set were used to predict HER2 status by applying proteomic classification algorithms.

Ergebnisse und Diskussion: We found that specific protein/peptide expression changes strongly correlated with

the HER2 over expression (m/z 4740, 8404, 8419, 8455, 8570, 8607, 8626). Among these, we identified m/z 8404

as Cysteine-rich intestinal protein 1 (CRIP1). Of particular note, the proteomic signature was able to accurately

define HER2-positive from HER2-negative tissues achieving high values for sensitivity of 83%, for specificity of

92% and an overall accuracy of 89% (95% CI: 65% to 99%). Our results underscore the potential of MALDI-IMS

proteomic algorithms for morphology-driven tissue diagnostics such as HER2 testing and show that MALDI-

IMS can reveal biologically significant molecular details from tissues which are not limited to traditional high-

abundance proteins. CRIP1 is a cytosolic protein that is potentially useful for serum based diagnostics of HER2 if

tissue leakage can be demonstrated.

Neue Aspekte: A cytosolic Biomarker that classifies the HER2 status in breast cancer was detected and identified

by top-down MALDI tissue imaging

Thema: Proteine und Peptide, Imaging

Keywords: HER2, breast cancer, CRIP1, MALDI tissue imaging, biomarker


224

P128

Improved identification of proteins in high-mass accuracy MALDIImaging MS experiments via on-tissue MS/MS experiments and

LC/MS measurements

Schober, Yvonne (1); Strupat, Kerstin (2); Spengler, Bernhard (1); Römpp, Andreas (1)

1: Justus Liebig Universität Gießen, Institut für anorganische und analytische Chemie, Germany; 2: ThermoFisher Scientific, Hanna Kunath Str. 11, D-28199 Bremen

Einleitung: MALDI Imaging MS is an exciting, rapidly growing field in life science. It allows to visualize thespatial distribution of proteins, peptides, lipids, biomarkers or other chemicals in tissue samples. Identification ofcompounds directly from the tissue surface, however, is difficult due to limited sensitivity and MS2 capabilities.Additional analysis techniques such as LC/MS2 measurements are often necessary. We combined high-massaccuracy imaging measurements and LC/MS measurements in order to increase the number of identified proteinsin mouse brain sections.Experimenteller Teil: For all experiments a LTQ Orbitrap Discovery was used (1). For imaging measurements acommercial MALDI source (Thermo Fisher Scientific) and for the nanoLC/MS measurements a nanoLC system(Dionex) coupled with a nanospray interface was employed. Mouse brain samples for the imaging experiments(thickness of slices 20 um) were washed to remove phospholipids quantitatively. Trypsin droplets were depositedmanually on mouse brain sections in order to digest peptides and proteins. 2,5-DHB matrix was applied witha pneumatic sprayer. Mouse brain sections were homogenized and tryptically digested for LC/MS analysis. Adedicated fractionation protocol including ultracentrifugation was developed in order to improve the number ofidentified proteins.Ergebnisse und Diskussion: High accuracy peptide images with a bin size of m/z = 0.01 were generated. Peptideswere identified directly from tissue by data-dependant MS/MS measurements in the linear ion trap. This resultedin 15 identified peptides corresponding to 5 proteins. The interpretation of MS/MS spectra (manual or data-base)was complicated by the occurrence of matrix related fragment ion peaks. A series of intense peaks correspondingto the loss of m/z = 154 were observed when using 2,5-DHB as a matrix. Experiments with sprayed matrix onempty glass slides resulted in similar signals. Implications and possible mechanisms of this phenomenon will bediscussed. Comparison of the high mass accuracy imaging experiments with LC/MS measurements of mouse brainhomogenate increased the number of identified proteins by a factor of 4. The number of identified peptides andproteins could be significantly increased further by employing the fractionation protocol which was optimized forbrain tissue. More than 130 peptides corresponding to 90 proteins could be identified. The protocol was especiallysuccessful for unpolar membrane proteins. Compared to previous experiments the confidence by linking imagingand LC/MS data greatly increased employing sub-ppm accuracy of the LTQ Orbitrap used for both experiments.Neue Aspekte: Combination of MALDI MS Imaging measurements with LC/MS2 measurements for identifica-tion of tryptic peptides in tissue samplesReferenzen: [1] K. Strupat, V. Kovtoun, H. Bui, R. Viner, G. Stafford, S. Horning; MALDI Produced Ions In-spected with a Linear Ion Trap-Orbitrap Hybrid Mass Analyzer, J Am Soc Mass Spectrom 2009, 20, 1451-1463Thema: Proteine und Peptide, ImagingKeywords: MALDI MS Imaging, LC/MS2, triptic peptides, tissue sampels, high mass accuracyKontakt: [email protected]

225

P129 -Wolfgang Paul Studienpreis

C-H Bond activation with a non-metallic open-shell oxide cation

Dietl, Nicolas Paul Richard (1); Engeser, Marianne (2); Schwarz, Helmut (1)

1: Technische Universität Berlin, Germany; 2: Kekulé-Institut für Organische Chemie und Biochemie der

Universität Bonn; Germany

Einleitung: A large class of transition- and main groupmetal oxides have been investigatedtowards the activation

of methane and the role of oxygen-centered radicals in thermal hydrogen-atom abstraction has been discussed.[1]

Due to the structural similaritythe Vanadium oxide [V4O10]+, which is known to be highly active towards the

activation of methane at room temperature [2],we started to focus on the isostructuralphosphorous oxide [P4O10]+

to see, if a metal-free methane activationcould bepossible at room temperature.Furthermore, other Hydrocarbons

and substrateswill beinvestigated.

Experimenteller Teil: Experimental: A Bruker Apex IV FT-ICR Mass spectrometerwith 7 T-magnet, equipped

withanEI-source, was used for the Ion-Molecule reactions.Computational: All Calculations were performed at the

B3LYP level of theory, as implemented in the Gaussian03 program package, using the triple-ξ plus polarization

basis sets (TZVP) of Ahlrichs and co-workers.[3]

Ergebnisse und Diskussion: Our results show the activation of several hydrocarbons, including methane, ethane,

propane, and ethylene. Especially the room temperature activationof methane is of particular interest, since the

observed hydrogen-atom abstraction from CH4 to generate CH3 , is viewed as the decisive step in the oxidative

dehydrogenation and dimerization of methane.[4] By the use of different deuterated analogues, the mechanism

could be proved in all cases and isotopic effects could be defined, reflecting in excellent correlation the different

bond strength of the investigated hydrocarbons; all calculations are in perfect agreement with the experimental

results. The use of small C2 and C3hydrocarbons allows to present and discuss regioselectivities between primary

and secondary C - H-bonds, as well as the different appearing reaction channels. These insights into this new

aspect of saturated C - H-bond activation might help to improve for example heterogeneous catalyst by interaction

of mixed-oxo-frameworks, such as the already known VPO-catalysts.[5]

Neue Aspekte: C-H bond activation ofmethane and other small hydrocarbonsby polynuclearnon-metallic system

at room temperature

Referenzen: [1] S. Feyel, J. Döbler, R. Höckendorf, M. K. Beyer, J. Sauer, H. Schwarz, Angew. Chem. 2008, 120,

1972-1976.; [2] S. Feyel, J. Döbler, D. Schröder, J. Sauer, H. Schwarz, Angew. Chem. 2006, 118, 4797-4801.; [3]

A. Schafer, C. Hubers, R. Ahlrichs, J. Chem. Phys. 1994, 100, 5829 - 5835; [4] J. H. Lunsford, Catal. Today 2000,

63, 165-174.; [5] R. H. Crabtree, Nature Chemistry 2009, 1, 348.

Thema: Ionen-Molekül-Reaktionen

Keywords: C - H activation, cluster compounds, radicals,density functional calculations, gas-phase reactions


226

P130

Heteroleptic Ruthenium(II) complexes studied by ESI-MSMS

Starke, Ines; Kammer, Stefan; Holdt, Hans-Jürgen; Kleinpeter, Erich

Universität Potsdam, Germany

Einleitung: Ruthenium complexes containing intercalating ligands with DNA such as 1,12 diazaperylene (dap)possessing both a larger surface and increased π-delocalization as phenanthroline (phen) have been the focusof many studies.1 Recently Kammer et al2 synthesized the 2,11-dialkylated 1,12 diazaperylenes (alkyl= methyl:dmdap, ethyl: dedap and isopropyl: dipdap). The new ligands and the complexes [Ru(L)(bpy)2(PF6)2] and[Ru(L)(phen)2(PF6)2] with L=dmdap, dedap, dipdap and the 5,8-dimethoxy-substituted diazaperylene meodap

were investigated with ESI-MS and Collision Induced Decomposition (CID) experiments.Experimenteller Teil: Themass spectra were obtained in positive ion mode using a ESI-Q-TOFmass spectrometer(Micromass Manchester, UK). The cone voltages were optimized for maximum precursor ion abundance withinthe range of 20 - 30 V. The complex ion was selected and activated in CID mode. The collision energy was varied(5-50 eV) to identify the threshold for dissociations. The threshold activation voltage is defined in this study as thevoltage sufficient to produce fragments that constitute 10% of the total ion intensity.Ergebnisse und Diskussion: All the complexes formed both doubly charged ions [Ru(L)(bpy)2]2+ and [Ru(L)(phen)2]2+ and singly charged ions: [Ru(L)(bpy)2+H]+, [Ru(L)(phen)2+H]+, [Ru(L)(bpy)2(PF6)]+ and [Ru(L)(phen)2(PF6)]+. The general tendency for the fragmentation under the CID experiments of the doubly charged ions[Ru(L)(bpy)2]2+ and [Ru(L)(phen)2]2+ was the elimination of bpy and phen respectively, together with the lossof hydrogens. The singly charged complex ions [Ru(L)(bpy)2+H]+ decomposed easily through the eliminationof bpy however for the complexes [Ru(L)(phen)2+H]+ the ligand is lost. But the most important fragmentationpathway of the complex ions [Ru(L)(bpy)2(PF6)]+ and [Ru(L)(phen)2(PF6)]+ was the elimination of one ligandtogether with the anion PF−5 to build up the complexes [Ru(bpy)2+F]

+ and [Ru(phen)2+F]+.Neue Aspekte: New ruthenium complexes with different pyridyl ligands were investigated with ESI-MS andenergy variable collisionally activated dissociation measurements.Referenzen: [1] A. Chouai, S. E. Wicke, C. Turro, J. Bacsa, K. R. Dunbar, D. Wang, R. P. Thummel, Inorg. Chem.2005, 44, 5996; [2] S. Kammer, A. Kelling, H. Baier, W. Mickler, C. Dosche, K. Rurack, A. Knapp, F. Lisdat, H-J.Holdt, Eur. J. Inorg. Chem, 2009, 31, 4648Thema: Organische MassenspektrometrieKeywords: ruthenium(II) complexes, large surface ligands, esi-ms, CIDKontakt: [email protected]

227

P131

Untersuchung von Umlagerungen und Fragmentierungen anSteroidethern mittels ICR-MS III

Freudenhammer, Christoph; Grotemeyer, Jürgen

Institut für Physikalische Chemie, CAU Kiel, Germany

Einleitung: Elektrospray-Ionisation (ESI) ist zu einer potenten Technik in der Massenspektrometrie nicht nur

für Makromoleküle sondern auch für kleinere, mittelgroße Biomoleküle und Metabolite geworden. Um diese

Massenspektren und den Nutzen der ESI in der Identifikation von komplexen Mischungen zu verstehen, ist es

nötig, die ihr zugrunde liegenden Reaktionen genauer zu untersuchen. In diesem Beitrag berichten wir über einige

Ergebnisse der Untersuchung von verschiedenen Steroidethern und Benzyloxylindolen in einem ICR-MS. Diese

Messungen wurden dadurch motiviert, dass die Ether einenüberraschenden Wasserverlust aus der Ether- und den

freien Hydroxylgruppen aufweisen.

Experimenteller Teil: Die Experimente wurden mit einem APEX Qe FT-ICR Massenspektrometer von Bruker

Daltonics, Bremen, ausgeführt, bestehend aus zwei Hauptkomponenten, einem supraleitenden Kryomagneten. (9.4

T / 11 cm bore) und dem Vakuumcart, in dem sich die Ionenquelle, eine Apollo II Dual ESI/MALDI Version, ein

Quadrupolfilter zur Ionenisolierung mit Stoßzelle, die Ionentransferoptik und als Detektor eine ICR Infinity Cell

befinden. 120 µl/h der ESI-Probe wurden mittels einer Spritzenpumpe in das System injiziert und die gebildeten

Ionen in der Zelle durch On-resonance CID (meist bei Energien von 0.8% und Pulsdauern von 400 µs) bzw.IRMPD (häufig 30% Laserleistung und Pulsen von 150 ms) fragmentiert.

Ergebnisse und Diskussion: Wir beschäftigten uns in den letzten Monaten weiter mit der bereits diskutierten

[3.3]-sigmatropen Umlagerung in Steroidbenzyl- bzw. allylethern. Die mittels On-resonance-CID-Spektren erhal-

tenen Erkenntnisse über die Estr(ad)iolbenzyl, -phenetyl-, -propenyl-, -propyl-, -ethyl- und -methylether wurden

durch Deuterierung der freien Hydroxylgruppen der Benzylether und mit Hilfe der IR-Multiphotonendissoziation

verifiziert, um durch letztere auch energetische Aussagen zu ermöglichen. Generell wird bei den Ethern, die in

der Lage sind, zwischen Ring A und dem Etherrest ein ungesättigtes Sechsringsystem zu bilden, eine Wasser-

abspaltung mehr beobachtet als erwartet worden ist. Die Estradiolbenzyletherspektren bspw. zeigen ausgehend

vom MH+-Signal bei m/z 363.232 zwei Wasserabspaltungen bei 345.222 [MH+-(H2O)] und 327.212 [MH+-

(H2O)2] sowie weitere Signale im niedrigeren Massenbereich, wie denn Abspaltungen von C6H6 [285.187Da,

Benzol] bzw. C7H8 [271.172Da, Toluol]. Daher scheinen intensive interne Umlagerungsprozesse [1,2] stattgefun-

den zu haben, wir vermuten u.a. eine [3.3]-sigmatrope Claisen-Umlagerung. Um mit anderen Steroiden und Ben-

zylethern vergleichen zu können wurden daraufhin des Weiteren Spektren verschiedener Pregnanderivate [Preg-

nenolon, Hydroxy-Pregnenolon, Corticosteron] und der Estrogenvorstufe Dehydroepiandrosteron, sowie verschie-

dener Benzyloxy-Indole [5-Benzyloxy-indol, 5-Benzyloxyindol-3-essigsäure, 5-Benzyloxy-6-methoxy-indol, 6-

Benzyloxy-5-methoxy-2-carboxyindole] aufgenommen. Die Pregnane weisen dabei keine unerwarteten Wassere-

liminierungen aus nicharomatischen Hydroxylgruppen auf, während bei den beiden letztgenannten Indolen die

sigmatrope Umlagerung [3,4] des benzylischen Restes durch die Sperrung jeweils einer Nachbarposition am aro-

matischen Ring unterbleibt. Noch ist zu klären, welchen Einfluss dabei vermutlich das Heteroatom im benachbar-

ten Zyklus ausübt.

Neue Aspekte: Unerwartete Umlagerungsprozesse in Steroidbenzyl- und -allylethern

Referenzen: [1] P. Longevialle, R. Botter, Org. Mass Spectrom. 1983, 18 (1), 1.; [2] E. Hunter, S. Lias, J. Phys.

Chem. Ref. Data 1998, 27 (3), 413.; [3] L. Claisen, Ber. Dtsch. Chem. Ges. 1912, 45 , 3157.; [4] P. Traldi et al., J.

Mass Spectrom. 2007, 42, 1562.


Keywords: Fragmentierungsmechnismen/-wege, Ionencyclotronresonanz, Steroide


228

P132

Metastabile Zerfallsreaktionen von Steroiden und Steroidethern

Bannick, Heiko; Grotemeyer, Jürgen

Institut für Physikalische Chemie, Christian-Albrechts-Universität zu Kiel, Ludewig-Meyn-Str. 8, 24118 Kiel

Einleitung: Den biologisch wichtigen Steroiden Estradiol und Estriol wurde in der Vergangenheit viel Aufmerk-samkeit gewidmet. Hierbei wurde jedoch meist nur auf deren biologische Wirkung eingegangen, ebenso wur-den hauptsächlich biologisch aktive Derivate von Estradiol und Estriol untersucht. Ziel dieser Arbeit war es diemetastabilen Zerfallsreaktionen derMolekülionen von Estradiol und Estriol sowie deren an 3-Position veretherten

Derivaten zu untersuchen. Hierbei wurden die Methyl-, Propyl-, Propenyl- und Benzylether verwendet. Zudem

wurden ebenfalls die protonierten Molekülionen untersucht.

Experimenteller Teil: Verwendet wurde ein doppelt fokussierendes Sektorfeldgerät mit inverser Geometrie (VG,ZAB-2F), mit welchem mass analyzed ion kinetic energy (MIKE) Spektren aufgenommen wurden. Die Molekül-

ionen wurden unter EI-Bedingungen und die protonierten Molekülionen unter CI-Bedingungen, mit Ammoniak

als Reaktandgas, erzeugt.

Ergebnisse und Diskussion: Die MIKE-Spektren der Molekülionen von Estradiol, Estriol und deren Ethern

zeigen vom Verlust eines Wasserstoffradikals über Wasserabspaltungen bis hin zu Abspaltungen aus dem Steroid-

grundgerüst eine Vielzahl von Fragmenten. Betrachtet man im Vergleich dazu die MIKE-Spektren der protonierten

Molekülionen [MH]+ zeigt sich, dass diese meist von einigen wenigen Abspaltungen dominiert werden. Insbeson-

dere die Gerüstabspaltungen sind zwar durchaus erkennbar, treten aber nur mit geringer Intensität auf. Zu den in-tensiveren Fragmenten zählt die Abspaltung eines Wassermoleküls und beim Estriol bzw. den Estriolethern eineszusätzlichen zweiten Wassermoleküls. Der Estradiol-3-benzylether zeigt als Abspaltung aus dem [MH]+ den Ver-lust eines Toluolmoleküls und zudem die Abspaltung eines Benzolmoleküls. Diese Abspaltungen sind beide ausdem Molekülion nicht zu beobachten, hier tritt als intensivstes Signal die radikalisch abgespaltene Ethergruppeauf.Neue Aspekte: MIKE-Spektren der [MH]+ Ionen der Estradiol- und Estriolether.Thema: Organische Massenspektrometrie, NaturstoffeKeywords: Estradiol, Estriol, Metastabile IonenKontakt: [email protected]

229

P133

Therapeutisches Drug Monitoring des AntikmykotikumsPosaconazol mittels UPLC-MS/MS

Krüger, Ralf (1); Vogeser, Michael (2); Burghardt, Stephan (3); Vogelsberger, Rita (1); Lackner, Karl J. (1)

1: Institute für Klinische Chemie und Laboratoriumsmedizin, Universitätsmedizin Mainz; 2: Institut für KlinischeChemie, Klinikum der Universität München; 3: Chromsystems Instruments and Chemicals GmbH, München

Einleitung: Posaconazol (Noxafil®) (1) ist ein neuartiges Antimykotikum zur oralen Einnahme und dient zur

Therapie und Prophylaxe von systemischen und invasiven Mykosen, insbesondere bei beeinträchtigtem Immun-system (z.B. Einnahme von Immunsuppressiva, AIDS, Radiotherapie). Posaconazol ist besondere effektiv zurBehandlung von Candida und Aspergillus, aber wirkt auch bei seltenen Mykosen, im Gegensatz zum analogenMedikament Itraconazol. Aufgrund individuell unterschiedlicher Aufnahme undMetabolismus ist die regelmäßigeBestimmung der Plasma-Konzentrationen indiziert (therapeutisches drug monitoring, TDM). Dazu wurde eine LC-MS Methode zur Quantifizierung von Posaconazol in Serum etabliert.Experimenteller Teil: Die Methode erfordert nur eine minimale Probenvorbereitung, und die Messzeit beträgtunter 3 Minuten. Das zentrifugierte Serum wird zur Fällung der Proteine mit methanolischer ZnCl2-Lösung ver-setzt, in der auch der interne Standard enthalten ist. Nach Zentrifugation bei 13.000 rpmwird die Probe injiziert undmittels eines Step-Gradienten von der RPC18-Säule eluiert. Die Quantifizierung erfolgt mittels UPLC-MS/MS imMRM-Modus (multi reaction monitoring) an einem ESI-Triple-Quadrupol (Waters). Zur Kalibrierung und Qual-itätskontrolle werden matrixangepasste Standards und Kontrollen eingesetzt (Chromsystems). Nach erfolgreicherEvaluierung der Methode wurde zudem ein Vergleich mit drei unabhängigen Methoden durchgeführt (1x LC-MS(2), 2x HPLC-FLD).Ergebnisse und Diskussion: Die Validierung der Methode ergab eine sehr gute Performance mit folgendenErgebnissen: total imprecision 7-10%, recovery 96-107%, LOD 12 µg/L (S/N 3:1), LOQ 37 µg/L (S/N 9:1),linearer Bereich 25 - 3634 µg/L (r2 = 0.9948), keine ion suppression. Zwei zusätzliche Peaks in der MRM-Spur von Posaconazol, die nur bei Patienten detektiert wurden, waren nahezu basisliniengetrennt. Externe undinterne Vergleiche mit Patientenproben (n = 42) ergaben eine exzellente Übereinstimmung zwischen UPLC-MSund HPLC-FLD (r2 = 0.994, mittlere Abweichung 7%). Die Korrelation mit der LC-MS-Methode war ebenfallsgut (r2 = 0.977, n = 48). Die zunächst beobachteten systematischen Differenzen zwischen UPLC-MS und LC-MSkonnten auf interferierende Metabolite zurückgeführt werden. Nach Anpassung der LC-MS-Methode betrug dieAbweichung nur noch 10%. Weiterhin wurden Unterschiede bzgl. der verwendeten internen Standards und MRM-Übergänge beobachtet. Die UPLC-MS-Methode ist seit etwa einem Jahr im Routine-Einsatz und hat sich für dasTDM von Posaconazol bestens bewährt.Neue Aspekte: Die Methode auf Basis von UPLC-MS/MS erlaubt eine zuverlässige, einfache und schnelle Quan-tifizierung von Posaconazol in Serum für TDM.Referenzen: [1] 1. Torres HA, Hachem RY, Chemaly RF, Kontoyiannis DP, Raad II. Posaconazole: a broad-spectrum triazole antifungal. Lancet Infect Dis 2005;5:775-85.; [2] 2. Vogeser M, Rieger C, Ostermann H, Spöhrer

U. A routine method for the quantification of the novel antimycotic drug posaconazole in plasma using liquid

chromatography-tandem mass spectrometry. Clin Chem Lab Med 2009;47:579-584.

Thema: Organische Massenspektrometrie

Keywords: drug monitoring, Quantifizierung, UPLC-MS/MS, MRM


230

P134

FT-ICR-Massenspektrometrische Untersuchungen vonIsotopenaustauschreaktionen in der Gasphase

Losensky, Luisa; Winkler, Henrik D. F.; Springer, Andreas; Schalley, Christoph A.

Freie Universität Berlin, Germany

Einleitung:Gezeigt werden Isotopenaustauschreaktionen in der Hexapolregion eines FT-ICR-Massenspektrometers

(Gasphasen-H/D-Austausch). Mittels Isotopenaustausch kann die Wanderung von Kronenethern von Ammoni-

umgruppen zu Aminofunktionen auf unterschiedlichen Wirtkomplexen untersucht werden. Der Austausch von

protoniertem Ethylendiamin [EDA+H]+ mit MeOD erfolgt nach dem gut untersuchten Relay-Mechanismus.[1]

Im 1:1-Komplex des EDAs mit 18-Krone-6 sind die austauschbaren Protonen vom Kronenether geschützt, es

kann keine Zunahme des m/z beobachtet werden. Vorausgehende Arbeiten mit POPAM-Dendrimer-Kronenether-

Komplexen[2] bzw. Polylysin-Kronenetherkomplexen[3] zeigten den „Weltraumspaziergangs"des Kronenethers zwi-schen benachbarten Aminofunktionen. In diesem Beitrag wird zunächst der Einfluss der Alkankettenlänge auf

die Wanderungsgeschwindigkeit von 18-Krone-6 auf Diaminen dargestellt. Außerdem werden die Wanderungsei-

genschaften verschieden großer Kronenether auf EDA untersucht. Mit diesen an die oben zitierten Experimente

anknüpfenden Experimente können weitere mechanistische Einblicke in die Kronenetherwanderung gewonnen

werden.

Experimenteller Teil: Die Gasphasenexperimente wurden an einem Ionspec QFT-7 FT-ICR-Massenspektrometer

(Varian Inc., Walnut Creek, CA, USA), ausgestattet mit einem supraleitenden 7 T-Magneten und einer Micro-

mass Z-Spray-Elektrosprayionenquelle (Waters Co., Saint-Quentin, Frankreich), durchgeführt. Die Probenlösun-

gen (50 µM ; Methanol, 1% Ameisensäure) aller Kronenether und Diaminoalkane wurden mit Flussraten von 2 -

4 µL/min injiziert. Eine Optimierung der Ionisierungsbedingungen resultierte in konstanten Spraybedingungen

und das Erhalten der größtmöglichen Intensitäten für die jeweilig untersuchten Ionen. Ein gutes Signal-zu-Rausch-

Verhältnis wurde durch Akkumulation von bis zu 20 Scans pro Spektrum erreicht. Die H/D-Autauschreaktionen

der jeweiligen 1:1-Komplexe von Diaminoalkanen mit Kronenethern wurden in der Quadrupolregion des Massen-

spektrometers durch Austausch mit deuteriertem Methanol durchgeführt.

Ergebnisse und Diskussion: Im ersten Teil des Posters wird die Abhängigkeit der Geschwindigkeit der Isotopen-

austauschreaktion der austauschbaren Aminoprotonen von 1:1-Komplexen (1,n Diaminoalkan/18-Krone-6) mit

Methanol-OD in Abhängigkeit der Alkankettenlänge dargestellt. Die hierbei zu erkennende Tendenz lässt mögli-

cherweise auf einen Konformationseffekt der Alkankette schließen. Es sind weiterführende Experimente zu die-

sem Teil der Arbeit geplant, ebenso wie eine theoretische Abhandlung des Themas. Hierzu wurden vorbereitend

die Modellrechnungen mit CaChe durchgeführt. Der zweite Teil beschäftigt sich mit dem H/D-Austauschverhalten

verschieden großer Kronenether mit Ethylendiamin (EDA). Die untersuchten 1:1 Komplexe zeigten klare Tenden-

zen in ihrem H/D-Austauschverhalten. Während 18-Krone-6 effektiv vor einem Isotopenaustausch schützt, zeigtsich bei 15-Krone-5 und DB-21-Krone-7 bereits nach kürzerer Zeit, und bei 12 Krone-4 bzw. DB-24-Krone-8nach vergleichsweise sehr kurzer Zeit, ein Isotopenaustausch. Dabei ist die Frage zu klären, ob die Größe des

Kronenether-Ringes und die damit verbundene Anzahl der ausgebildeten Wasserstoffbrücken Einfluss auf die

Schutzfunktion des jeweiligen Kronenethers hat und ob zum Beispiel zusätzlich die symmetrische bzw. nicht-

symmetrische Ausrichtung der Wasserstoffbrücken eine Rolle spielt.

Neue Aspekte:DerMechanismus des „Weltraumspaziergangs"von Kronenethern auf Polyaminen[2,3] wurde durchVergleichsexperimente von verschiedenen Diaminoalkan-Kronenether-Komplexen untersucht.Referenzen: [1] S.-W. Lee, H. -N. Lee, H. S. Kim, J. L. Beauchamp: J. Am. Chem. Soc. 1998, 120, 5800-5805;[2] H.D.F. Winkler, D.P. Weimann, A. Springer, C.A.Schalley: Angew. Chem. 2009, 121, 7382-7386; [3] D.P.Weimann, H.D.F. Winkler, J.A. Falenski, B. Koksch, C.A.Schalley: Nature Chemistry 2009, 1, 573-577Thema: Isotopen-Massenspektrometrie, Organische MassenspektrometrieKeywords: H/D-Austausch, FT-ICR MS, Gasphase, Kronenether, SpacewalkKontakt: [email protected]

231

P135

Assessment of the new scoring function for protein identificationby PMF

Kaminska, Hanna (1,2); Rognvaldsson, Thorsteinn (3)

1: Wrocław University of Technology, Poland; 2: MedicWave, Sweden; 3: School of Information Science,

Computer and Electronic Engineering, Halmstad University, Sweden

Einleitung: Peptide Mass Fingerprinting (PMF) is a widely used protein identification method basing on MS data.

A novel probability-based scoring scheme containing an innovative idea was developed. Our scoring scheme

assumes a different approach to modeling the distribution of protein masses in the database and considering

matches between experimental and theoretical peaks. The performance of the proposed probability-based scoring

method was assessed and compared with two popular scoring schemes, i.e. Mascot[4][5] and PBSF[1]. The

comparison includes scoring results obtained for simulated data. Different levels of samples contamination and the

different coverage of peptides sequences were considered in the simulations. Preliminary results indicate, that our

scoring scheme has a comparable or better performance to the well-known Mascot and PBSF scoring schemes.

Experimenteller Teil: The experiment is conducted on the basis of simulated peak-data - similar approach to

assessment of quality was presented in [2]: A fixed number of proteins is drawn randomly (without replacement)

from the proteins database. These proteins are then cleaved in silico and each peak list is modified using two

parameters. The first parameter (x) describes fraction of ’true’ peptides extracted from the drawn protein, the

second parameter (y) describes how big part of contaminated protein the ’true’ peaks constitute. Points (x,y) form

a grid, in which fractions spaces (varied from 0 to 1) were divided into the sections of length 0.05. In the next step,

contaminated proteins were identified using scoring algorithm being investigated, Mascot and PBSF.

Ergebnisse und Diskussion: The grid described above resulted in 20 points, both for x and y values. For each (x,y)

point 45 proteins were drawn randomly form the Swiss-Prot database and digested in silico using trypsin enzyme

- missed cleavages and proteins modifications were not considered. For such peaks, lists of matching masses for

each protein were found, and along with peptides masses were passed to the scoring functions. If the name of the

best scored protein agreed with the real name of protein, (from which peptide sequence was derived) - such event

was counted as a score hit. On the other hand, if the best scored protein was different, but was among top 20 results

from the BLAST[4] searching, with BLAST e-value less than 0.05 - it was also regarded as a hit. For both PBSF

and our scoring algorithm, for each grid point, a number of hits were counted and fraction of correctly identified

proteins was calculated. We can observe that for fraction values close to 0, both scoring algorithms perform rather

poorly. Note that, the large area (over 50% of points) was identified by scores with almost 100% of proper hits.

Results leads also to the conclusion, that new scoring method behaves noticeably better in the areas of (x,y) space

representing high contamination and where the number of true masses is small. An additional experiment was

performed using 5500 proteins from the area of (x,y) space with x varying from 0 to 0.2 and y from 0.15 to 0.45,

what represents masses being very likely to occur in the real experiment[2]. Contaminated proteins were submitted

to the Mascot and the new scoring function. The results indicated that Mascot identified correctly 47% while our

scoring approach identified properly 49% of proteins, what makes it more efficient in this simulation region.

Neue Aspekte: The proposed novel probabilistic approach to PMF scoring achieves comparable or better accuracy

to PBSF and Mascot (on simulated data).

Referenzen: [1] Song Z., Chen L., Ganapathy A., Wan X.F., Brechenmacher L., Tao N., Emerich D., Stacey G.,

Xu D., Electrophoresis, 2007 Mar;28(5):864-70.; [2] Samuelsson J., Dalevi D., Alm R., Rögnvaldsson T., PotthastF., "PIUMS®: A New Algorithm for Protein Identification Using Peptide Fingerprints", Piums handout poster; [3]BLAST tool: http://www.expasy.ch/tools/blast/; [4] MASCOT tool: http://www.matrixscience.com/; [5] Perkins,D. N.; Pappin, D. J. C.; Creasy, D. M.; Cottrell, J. S. Electrophoresis 1999, 20, 3551-3567Thema: Organische Massenspektrometrie, Proteine und PeptideKeywords: PMF, protein identification, scoring schemeKontakt: [email protected]

232

P136

Investigation of Ionic Liquid Matrices of the six Dihydroxybenzoicacid isomers

Hellwig, Nils; Gernert, Claus; Grotemeyer, Jürgen

Christian-Albrechts-Universität Kiel, Germany

Einleitung: The Suitability of the different Isomers of Dihydroxybenzoic acid (DHB) as solid-state matrices for

MALDI-MS has been subject of several earlier investigations.[1,2] There all came to the result, that the 2,5 isomer

is the best matrix at the standard laser wavelength of 337 nm. The applicability as MALDI-matrices of the isomers

in the solid-state can be explained as a combination of three properties: the absorption at the utilized wavelenght,

the acidity and crystallization behavior, e.g. the ability to include analyte ions in the matrix crystal. The use of Ionic

Liquid Matrices (ILM) negates the effect of the crystallisation, as the Analyte is solved in the ILM. Additionally

the absorption of the ILM can be varied by changing the counterion.

Experimenteller Teil: The MALDI mass spectra were recorded on an in-house build reflector time-of-flight mass

spectrometer based on a Scout ion source (Bruker Daltonics). The ablation laser used was a nitrogen laser. The

Measurement of the UV-Spectra was done on a XX UV/Vis Spectrometer (Perkin-Elmer). The Spectra were

recorded from the pure ILM, to obtain an absorption within the measurable range of the instrument a thin ILM film

was required. This thin film was made by pipetting∼ 3µl of a solution of the ILM solved in ethanol onto a suprasil

plate and subsequent evaporation of the solvent. The sample was then placed in the beam of the spectrometer.

Ergebnisse und Diskussion: The synthesis of ILMs of all six DHB isomers was attempted using the following

amines as counterions: Pyridine (py), aniline (an), triethylamine (tea), tributylamine (tba), butylamine (ba). All

combinations of these compounds that resulted in an Ionic Liquid were used as a Matrix for the analysis of

Gramicidine C. When using amines not absorbing at 337 nm (tea, tba, ba) the results are similar to the solid

state matrices. Usage of the 2,5 isomer leads to much better spectra compared to the other isomers. The UV

spectra of these ILMs also show that there is a significant difference in the absorption at the used wavelength

between the 2,5 isomer and all other isomers. The other amines can lead to a strong increase of the absorption at

337 nm, which can again also be seen in the mass spectra, where the analyte signal shows much higher intensities

compared to those of ILMs using the non-absorbing amines.

Neue Aspekte: The comparison of DHB isomers as MALDI matrices has up to now only been done in the solid

state.

Referenzen: [1] Schiller, J.; Süß, R.; Fuchs, B.; Müller, M.; Petkovic, M.; Zschörnig, O.; Waschipky, H. Eur.Biophys. J. 2007 36 517-527; [2] Horneffer, V.; Dreisewerd, K.; Ludemann, H.; Hillenkamp, F.; Lage, M.;Strupat, K. Int. J. Mass Spectrom. 1999 185/186/187 859-870Thema: Organische Massenspektrometrie, Proteine und PeptideKeywords: Dihydroxybenzoic acid, MALDI, Ionic Liquid Matrix, UV SpectroscopyKontakt: [email protected]

233

P137

Strukturaufklärung der thermischen Abbauprodukte derenzymatischen Kofaktoren NADH und NADPH mittels

ESI-FTICR-MS und HPLC-MS

Hofmann, Diana (1); Wirtz, Astrid (2); Santiago-Schübel, Beatrix (1); Disko, Ulrich (1); Pohl, Martina (1)

1: FZ Jülich, Germany; 2: Universität Düsseldorf

Einleitung: Generell akzeptiert ist die allgemein höhere Stabilität von Enzymen und ihren Kofaktoren im tro-ckenen Zustand gegenüber der Stabilität in Lösung. Fundierte Untersuchungen zur Stabilität von Redoxkofaktoren,

wie Nicotinamidadenindinukleotid (NADH) und Nicotinamidadenindinucleotidphosphat (NADPH) im trockenen

Zustand bei erhöhten Temperaturen sind bisher nicht bekannt. Diese Kofaktoren spielen insbesondere bei der Bio-

katalyse mit Oxidoreduktasen eine wichtige Rolle.Im Rahmen vergleichender Studien der Alkoholdehydrogenase

von Lactobacillus brevis (wtLbADH) mit einer NADH-abhängigen Variante wurde als ein Teilaspekt die thermi-

sche Stabilität der involvierten Kofaktoren NADH und NADPH im festen Zustand und in Lösung untersucht. Deren

Zersetzungsprodukte wurden mittels nano-ESI-FTICR-MS und HPLC-MS charakterisiert und ein möglicher Zer-

setzungsmechanismus abgeleitet.

Experimenteller Teil: Die Kofaktoren wurden im Temperaturbereich von 30 bis 95°Cthermisch behandelt. Die

entstandenen polaren, komplexen Substanzgemische wurden zunächst mit einer HPLC auf einer HILIC-Säule inihre Komponenten getrennt und mittels PDA und Fluoreszenzdetektor nachgewiesen. Die Strukturaufklärung derentstandenen Degradationsprodukte erfolgte in ausgewählten Ansätzen (Kofaktoren in fester Form bei 95°C; inWasser gelöst bei 50°C; je 16 h). Deren analytische HPLC-Läufe wurden fraktioniert. Die separierten Fraktionen

wurden anschließend zur Trockene eingeengt. Die aus jeweils 3-4 Läufen vereinigten Fraktionen wurden mit

der NanoMate (Fa. Advion) einem LTQ-FTICR-MS (Fa. ThermoFisher Scientific) per Fließinjektion zugeführt.

Die Untersuchungen erfolgten im positiven und negativen Modus.Zur Unterscheidung thermischer Spaltprodukte

von möglichen In-Source-CID-Produkten einerseits und empfindlicherer Detektion auch gering konzentrierter

Zersetzungsprodukte andererseits wurden analoge Ansätze mittels HPLC-MS untersucht.

Ergebnisse und Diskussion: Eine Reihe von erwarteten Bruchstücken wurde detektiert: Nicotinsäureamid, Ade-

nosinmonophosphat und Adenosindiphosphat (bzw. Adenosintriphosphat im Fall von NADPH). Ein Schlüssel-

fragment von NADH stellt die Neutralmasse 559 Da dar: im positiven Modus tritt neben dem (M+H)+-Peak auch

der (M+H,-H2O)+ auf, im negativen Modus wurde neben dem (M-H)− auch das doppelt geladene Ion (M-2H)2−

registriert. Die Massenanalyse ergibt unter entsprechenden ausgewählten Randbedingungen für die erwähnten vier

Ionen nur noch eine einzige mögliche Summenformel: C15H23O14N5P2. Der Vergleich mit der Ursprungsstruktur

ermöglicht keine direkt plausible Erklärung.In den anschließenden HPLC-MS-Untersuchungen mit einem emp-

findlicheren System (QTRAP4000, Fa. ABI) traten weitere Signale, zum Teil auch höherer Masse gegenüber

den Ausgangsverbindungen, auf. Charakteristisch waren die Massendifferenzen von 16 bzw. 32 Da, die auf ei-

ne einfache bzw. zweifache Oxidation der Kofaktoren hinweisen. Es wird vermutet, dass der eingeführte Sauer-

stoff (unter Ringöffnung und Wasserstoffwanderung) das C-1-Atom der Ribose zur Ketogruppe funktionalisiert.

Durch anschließenden Bruch der Bindung zwischen dem C-1-Atom der Ribose und dem Stickstoffatom des Ni-

cotinsäureamids entsteht unter Freisetzung von Nicotinsäureamid ein Molekül der Masse 559 Da. Das ebenfalls

registrierte Molekül mit der Masse 541 kann durch weitere Wasserabspaltung erklärt werden.Diese Oxidationen

treten bevorzugt bei thermischer Behandlung der Feststoffe auf, in Lösung wird nur die einfache Oxidation von

NADH registriert. Durch Vergleich der HPLC-MS-Massenchromatogramme konnte festgestellt werden, dass alle

unter zusätzlicher Ammoniak-Abspaltung gebildeten Ionen direkt in der ESI-Quelle aus den entsprechenden Frag-

mentionen entstehen (identische Retentionszeiten). Adenin selbst wird ebenfalls ausschließlich in der Ionenquelle

abgespalten. Im Gegensatz dazu erfolgen alle zusätzlichen Wasserabspaltungen bereits bei der Präparation - deren

Retentionszeiten unterscheiden sich entsprechend in den Massenchromatogrammen.

Neue Aspekte: · Vergleich In-Source-CID mit thermischer Präparation· strukturplausible doppelt geladene ESI-

Ionen rel. kleiner Massen (hier 558 Da)


Keywords: ESI-FTICR-MS, Kofaktor, NADH, NADPH, NanoMate


234

P138

Qualitative evaluation of the crossover reaction in ROMP withnon-charged monomers via ESI-TOF MS

Kurzhals, Steffen; Enders, Claudia; Binder, Wolfgang H.

Martin Luther Universität Halle Wittenberg, Germany

Einleitung: Ring opening metathesis polymerization (ROMP) is a living polymerization, proceeding via an in-

sertion of cyclic olefinic monomers into metal-carbene bond. Due to its broad range of polymerizable monomers,

this method is among the most valuable tools for the preparation of functionalized homo- and blockcopolymers

via sequential polymerization of monomers. We have demonstrated, that a deeper understanding of the crossover

mechanism in polymerization reactions (ROMP) can be gained byMALDI-TOFMS analysis after quenching of the

polymerization directly after the crossover-reaction from one monomer to the next nomomer. Subsequently, a quan-

titative evaluation of this crossover reaction can be achieved by integration of the respective polymer species.[1]

Experimenteller Teil: In the present account the active catalyst species of non-charged monomers directly at

the point of crossover was monitored by ESI-TOF MS in three different copolymerization systems. This re-

port therefore represents a detailed analysis of noncharged reactive species in ROMP, which overcomes the lim-

itation of previous works e.g. by Chen and coworkers[2], who needed charged comonomers (phosphonium or

ammonium-moieties) to achieve transfer of the active species (polymerization of norbornene with Grubbs cat-

alyst 1st-generation) into the gas phase. We have reacted Grubbs catalyst 1st or 3rd-generation with 1 eq.

of monomer A (endo,exo-bicyclo[2,2,1]-hept-5-ene-2,3-dicarboxylic acid-bis-O-methyl ester) and subsequently

with 1 eq. of the monomers D (exo-N-(4,4,5,5,6,6,7,7,7-nonafluoroheptyl)-10-oxa-4-azatricyclodec-8-ene-3,5-

dione), E[3](3-methyl-3-phenylcyclopropene) or T (endo,exo-bicyclo[2,2,1]-hept-5-ene-2,3-dicarboxylic acid-bis-

O-2,2,6,6-piperidinoxyl-ester).

Ergebnisse und Diskussion: The reaction solution was transferred to the ESI-TOF MS by direct injection, after

adding a small amount of isopropanol in order to improve the ionization. During the copolymerization reaction

with Grubbs catalyst 1st-generation we observed a monophosphine species with one monomer A unit. Neither

higher A-species nor copolymer species poly-A-E, poly-A-D or poly-A-T could be detected. The existence of the

monophosphine species supports a dissociative pathway in the mechanism of olefin metathesis.[4] Grubbs catalyst

3rd-generation showed higher activity in the ring opening polymerization of monomer A than Grubbs catalyst 1st-

generation. In this latter case, we could observe catalyst species bearing two, three or more units of monomer A.

Additionally, the crossover-reaction from monomer A to monomer D, E or T could be monitored successfully by

identifying copolymerspecies. From the detected species we conclude that both bromopyridine-ligands are labile

and can be cleaved off during the catalytic cycle.

Neue Aspekte: We monitored the crossover reaction in ROMP with noncharged monomers via ESI-TOF MS and

identified the intermediate catalyst species.

Referenzen: [1] Binder, W. H.; Pulamagatta, B.; Kir, O.; Kurzhals, S.; Barqawi, H.; Tanner, S. Macromolecules

2009, 42, 9457-9466.; [2] Adlhart, C.; Chen, P. Helv. Chim. Acta 2000, 83, 2192-2196.; [3] Binder, W. H.;

Kurzhals, S.; Pulamagatta, B.; Decker, U.; Manohar Pawar, G.; Wang, D.; Kühnel, C.; Buchmeiser, M. R.

Macromolecules 2008, 41, 8405-8412.; [4] Sanford, M. S.; Love, J. A.; Grubbs, R. H. J. Am. Chem. Soc.

2001, 123, 6543-6554.


Keywords: ROMP, ESI-TOF MS, Crossover reaction, Grubbs catalysts


235

P139

LC-ESI-TOF Mass Spectrometry of functionalizedPoly(3-Hexylthiophene)s

Enders, Claudia; Binder, Wolfgang H.

Martin-Luther Universität Halle-Wittenberg, Germany

Einleitung: Polythiophenes (PTPh) are important materials in solar cell-technology and molecular electronics.

Despite many efforts, the precise synthesis of PTPh’s with defined endgroups represent an important challenge

in organic synthesis and analytics. This report represents an analytical evaluation of the postfunctionalization of

poly(3-hexylthiophene)s via LC-ESI-TOF mass spectrometry. The current project aims in the synthesis of PTPhs

with a defined endgroup structure via Pd catalyzed C-C coupling reaction - called Sonogashira - to create functional

layer materials. These materials consist of PTPhs bearing H-bonding moieties introduced by azide/alkine "click"reaction which are held together into a sheet-structure via attractive supramolecular interactions. Over the pastyears, we have published about microphase-separated oligomers and polymers, in particular the generation ofsupramolecular pseudo-blockcopolymers via endgroup-functionalized, telechelic polymers1,2.Experimenteller Teil: Our effort is directed towards the synthesis and semi-quantitative analysis of telechelicPTPh’s, namely those with acetylene moieties to subsequently create a defined endgroup structures. Thus we haveexamined the influence of different solvents, Pd-catalysts and the influence of irradiation power on the compositionof the Sonogashira-reaction. Starting with bivalent and monovalent bromine PTPh we could observed differentend group structure after reaction (acetylene/H, acetylene/Br, acetylene/actelyene). The ionization of the HPLCseparated PTPhs was supported by postcolumn injection of methanol.Ergebnisse und Diskussion: In contrast to MALDI- TOF measurements the ionization of polymers higher than 4kDa molecular weight via ESI-TOF is more difficult because of their increasing chain length. Therefore the poly-merization of PTPhs was achieved up to a molecular weight of 2000 Da, up to which all species with increasingchain lengths could be easily separated by gradient elution and subsequent detection via ESI-TOF. The conversionof poly(3-hexylthiophene) with mono- and bivalent bromine endgroups into a mono- and bi-acetylene functional-ization was achieved by different microwave irradiation starting from 15 W to 100 W and different reaction times(10 min to 8 h). The reaction process was monitored by taking samples of different time intervals and measur-ing via ESI-TOF MS. During the reaction an exchange of bromines end groups into hydrogen end groups wasobserved, obviously by protonation of the intermediate metalated species. By use of increased reaction times andmicrowave-irradiation power, the relative amount of H/H end group functionalized polymer increased. By variationof the irradiation power the amount of acetylene functionalized polymer together with a decrease of the amount ofH/H end group functionalized polymer could be achieved, thus being able to create poly(3-hexylthiophene)s withdefined end group structure displaying mono- or bivalent acetylene units, respectively.Neue Aspekte: We introduced endgroups on polythiophenes via Sonogashira reaction and characterized the prod-ucts via ESI-TOF MS after separation by HPLC.Referenzen: [1] Binder, W. H.; Bernstorff,S.; Kluger, C.; Petraru, L.; Kunz, M.J., Tunable Materials fromHydrogen-Bonded Pseudo Block Copolymers. Advanced Materials 2005, 17,(23), 2824-2828.; [2] Binder, W.H.; Zirbs, R., Supramolecular Polymers and Networks with Hydrogen Bonds in the Main- and Side- Chain. Adv.Polym. Sci. 2007, 207.Thema: Organische MassenspektrometrieKeywords: ESI-TOF MS, conductive polymers, postfunctionalization of polymers, polythiophenesKontakt: [email protected]

236

P140

A Mass Spectrometry-based Multifactorial Analysis Strategy toDistinguish Patients with Preeclampsia from Matching Control

Individuals

Seidenspinner, Franka; Pecks, Ulrich; Röwer, Claudia; Coy, Cornelia; Reimer, Toralf; Rath, Werner;Glocker, Michael O.

Proteom Zentrum Rostock, Germany

Einleitung: Preeclampsia is a major cause of maternal and neonatal morbidity and mortality. Because the onlydefinitive treatment available thus far is delivery, preeclampsia is one of the leading causes of a mandated prema-turity [1]. Preeclampsia usually manifest after the 20th week of gestation [2] and is unpredictable in onset andprogression. The disease is incurable except for termination of the pregnancy. There is no specific diagnostic testfor the clinic [3]. Early identification of the risk of developing preeclampsia would provide means for a bettersurveillance during pregnancy, and, hopefully, improve outcome of the disease. In order to develop a strategy fora molecular-based diagnostic assay, we applied a multifactorial differential analysis of serum proteins using massspectrometry [4].Experimenteller Teil: The developed assay distinguished samples from pregnant women with severe early-onsetpreeclampsia (n = 11) from those of control individuals with uneventful pregnancies (n = 13). Serum proteinswere fractionated by either their affinities to reversed-phase material coated magnetic beads or by fractionatedprecipitation. Serum samples were processed according to manufacturer´s instructions using the Profiling Kit 100MB-HIC8 (Bruker Daltonik, Bremen, Germany). Spectra were reproducible, rich of signals with a good signalto noise ratio. Spectra were processed with a Reflex III MALDI ToF MS (Bruker Daltonik) in linear positive ionmode in the mass ranges from 4-25 kDa and 20- 250 kDa, respectively. All spectra were further analyzed with theClinProt tools (Bruker Daltonik) software package.Ergebnisse und Diskussion: MS analysis of the serum samples showed similar spectra with approx. 70 distin-guishable ion signals. The on average most abundant ion signals were observed at m/z 9,390, m/z 9,103, andm/z 8,886. The best differentiating ion signals between the two sample groups were found at m/z 13,715, m/z13,834, and m/z 13,891. The normalized intensities of these ion signals were on average lower in the preeclamp-sia group than in the control group. The six ion signal intensities enabled sorting of the individual spectra withhigh accuracy. SDS-PAGE analysis showed that a protein band migrating just above the 14 kDa marker bandcontained transthyretin (P02766; Mr (avg.): 13,761). Densitometric analysis of the transthyretin bands showedlower intensities in the preeclampsia samples with respect to those of the controls. Nephelometric analysis of theserum samples determined the mean concentration of transthyretin in the preeclampsia group being lower (0.16mg/ml; range: 0.13 to 0.20; SD: 0.03) than that in the control group (0.19 mg/ml; range: 0.14 to 0.22; SD: 0.02),substantiating the role of transthyretin concentration differences in the comparison of the two groups. Altogether,our findings support the theory of preeclampsia being a heterogeneous disorder that might be sub-classified by adefined proteome signature in maternal blood using multifactorial analysis of affinity-fractionated serum samples.Neue Aspekte: We separated preeclampsia-patients from healthy controls by a defined proteome signature inmaternal serum using multifactorial analysis of affinity-fractionated samples.Referenzen: [1] [1] Roberts, J.; Cooper, D. Pathogenesis and genetics of pre-eclampsia. Lancet 2001, 357,53-56.; [2] [2] Heilmann L, Rath W, Pollow K. Hemostatic abnormalities in patients with severe preeclampsia.Clin Appl Thromb Hemost. 2007, 13, 285-291.; [3] [3] Rath W, Fischer T. The Diagnosis and Treatment ofHypertensive Disorders of Pregnancy: New Findings for Antenatal and Inpatient Care. Dtsch Arztebl Int 2009,106, 733-738.; [4] [4] Pecks, U.; Seidenspinner, F.; Röwer, C.; Reimer, T.; Rath, W.; Glocker, M.O. MultifactorialAnalysis of Affinity - Mass Spectrometry Data from Serum Protein Samples: A Strategy to Distinguish Patientswith Preeclampsia from Matching Control IndividualThema: Organische Massenspektrometrie, Proteine und PeptideKeywords: preeclampsia, affinity mass spectrometry, proteome signature, multifactorial analysis, transthyretinKontakt: [email protected]

237

P141

Schnelle Bestimmung von Melamin in Milchpulver mittelsMALDI-MS

Arnold, Anne

Goethe-Universität Frankfurt, Germany

Einleitung: Melamin rückte erstmals im Jahr 2007 in den Mittelpunkt des öffentlichen Interesses. 2008 wurdeMelamin in Milchpulver als Auslöser für Nierenschäden bei Kindern in China bekannt. Daraufhin erließ dieEuropäische Kommission Richtlinien, die einen Grenzwert von Melamin in Milcherzeugnissen von 2,5 ppm fes-tlegen.[1] Zum Nachweis dieses Stoffes wurden zahlreiche Analysenmethoden entwickelt. Dabei fanden vor allemLC/MS-Methoden Verwendung. [2-4] Trotz der Entwicklung neuer Methoden und Geräte ist die Analytik kleinerMoleküle mittels MALDI-MS noch immer unterrepräsentiert. Dabei ist diese Methode oft viel robuster was Verun-reinigungen betrifft, wodurch auf zeitaufwendige Aufreinigungsschritte verzichtet werden kann. Ziel dieser Studiewar die Entwicklung einer schnellen und direkten Methode zur Identifizierung und Quantifizierung von Melaminmittels MALDI-MS.Experimenteller Teil: Zur Methodenentwicklung wurde Melamin zunächst in wässriger Lösung quantifiziert.Anschließend wurde Milchpulver und Milch mit Melamin versetzt und eine Probenvorbereitung in Anlehnungan eine Methode der FDA durchgeführt. [3] Dabei dienten zunächst α-Cyano-4-hydroxyzimtsäure und Sinapin-säure als Matrices. Als interner Standard wurde Atrazin (6-Chlor-N-ethyl-N‘-isopropyl-1,3,5-triazin-2,4-diamin)verwendet. Bei Verwendung von Sinapinsäure konnte auf jegliche Probenaufreinigung verzichtet werden. ZurProbenauftragung wurde die Standard „dried droplet preparation" Methode verwendet. Sämtliche Messungen

wurden an einem MALDI-TOF/TOF (4800-Applied Biosystems) und einer MALDI-LTQ-Orbitrap (Thermo Sci-

entific) durchgeführt.

Ergebnisse und Diskussion: Die Bestimmung von Melamin mittels MALDI-MS bietet eine echte Alternative zu

bereits veröffentlichen LC/MS Methoden. [2-4] Eine Quantifizierung dieser Verbindung ist aufgrund der kurzenMessdauer bei MALDI (< 15 sec/Probe) mit einem höheren Probendurchsatz verbunden. Eine sichere Identi-fizierung wird dabei durch die hohe Massengenauigkeit und Auflösung moderner TOF-Geräte bzw. der LTQ-

Orbitrap gewährleistet. Melamin konnte in Wasser in einem Konzentrationsbereich von 5 µM bis 5 nM mit einer

guten Linearität (R2> 0,999), Präzision (RSD < 20%) und Genauigkeit (Error < 20%) quantifiziert werden. Auch

in Milchpulver und Milch konnte Melamin mit einer guten Linearität, Präzision und Genauigkeit quantifiziert

werden. Das Detektionslimit lag mit 0,5 ppm deutlich unter dem von der EU festgelegten Grenzwert von 2,5 ppm.

Neue Aspekte: Schnelle Quantifizierung von Melamin mittels MALDI-MS

Referenzen: [1] Amtsblatt der Europäischen Union, 2008/757/EG; [2] Tyan YC, Yang MH, Jong SB, Wang CK,

Shiea J, Anal Bioanal Chem. 2009 Oct;395(3):729-35; [3] Turnipseed S., Casey C., Nochetto C., Heller D. N.,

FDA-Lib No. 4421, 24, 2008; [4] Desmarchelier A, Guillamon Cuadra M, Delatour T, Mottier P., J Agric Food

Chem, 2009 Jul 24


Keywords: Melamin, MALDI-MS, Milchpulver, Atrazin


238

P142

LC-ESI-TOF analysis of azide/alkyne-"click"- reactions onamino-resins

Barqawi, Haitham; Binder, Wolfgang

Martin-Luther Universität Halle-Wittenberg, Germany

Einleitung: The copper-catalyzed cycloaddition of azides to alkynes is one of the most significant synthetic routes

to 1-substituted-(1,2,3)-triazoles1, resulting in the efficient attachment of functional additives to polymers and resin

materials by post-modification processes, which often leads quantitative yields. Technically, the azide/alkyne-

"click"-reaction also enables the efficient crosslinking of precondensates, yielding duroplastic materials with ahigh crosslinking density2,3.We here report on the identification and quantification of melamine-formaldehyderesins linked to terminal alkynes and their generated "click"-structures bearing various organic moieties via highperformance liquid chromatography coupled to electrospray ionization time-of-flight mass spectrometry (HPLC-ESI-TOF).Experimenteller Teil: Melamine-formaldehyde resins (MF-resins)4−5 linked to terminal alkynes were preparedby cocondensation of propargylic alcohol (1- 30 mol %) with the amino-resin, resulting in the formation of of di-,tri- and oligomeric melamine-species. The so prepared, alkyne-functional MF-resin structures were reacted withvarious organic azides such as 1-(3-azido hexyl)thymine, benzyl azide and octane azide in excess using variouscopper(I)-species as catalyst (Scheme 1). The alkyne-containing structures within the melamine-formaldehyderesins were characterized using high performance liquid chromatography coupled to electrospray ionization time-of-flight mass spectrometry (HPLC-ESI/TOF MS).Ergebnisse und Diskussion: MS experiments using an LC-MS system equipped with an ESI source were per-formed on the synthesized alkyne-terminated MF resins. The HPLC traces revealed a large series of new chro-matographic peaks, which were identified and a simultaneously characterized. The identification and quantifica-tion of at least nine different compounds with mono-, di-, tri-, acetylenic moieties bound to melamine-residuescould thus be unveiled and unambiguously assigned. Furthermore, the chemical structures after the azide/alkyne-"click"-reaction could be separated up to molecular weight of approximately 1000 g mol−1 and fully assigned.Due to overlap of various peaks, a simulation using Bruker data analysis software was achieved, enabling an ex-act assignment via the respective isotopic-pattern simulation of the m/z-values of the corresponding compounds.Additionally, an extensive mass spectrometry based quantification of both the alkyne-containing-species and thegenerated "clicked" structures could be achieved using LC-ESI-TOF methods, which yielded detailed informationon the reaction progress within this highly complex chemical system.Neue Aspekte: LC-ESI/TOF MS method is a suitable tool for analysis of the complex-alkyne-MF species and theresulting triazoles after the "click"-reaction.Referenzen: [1] Binder, W. H.; Sachsenhofer, R., Macromol. Rapid Commun. 2008, 29, (12-13), 952-981; [2]Liu, Y.; Diaz, D. D.; Accurso, A. A.; Sharpless, K. B.; Fokin, V. V.; Finn, M. G., J Polym Sci Part A: PolymChem 2007, (45), 5182-5189.; [3] Liu, Y.; Diaz, D. D.; Accurso, A. A.; Sharpless, K. B.; Fokin, V. V.; Finn, M.G., J Polym Sci Part A: Polym Chem 2004, (42), 4392-4403; [4] Binder, W. H.; Dunky, M.; Jahromi, S., Kirk-Othmer Encyclopedia of Chemical Technology (5th Edition) 2005, 15, 773-796.; [5] Binder, W. H.; Dunky, M.,Encyclopedia of Polymer Science and Technology 2004, 10, 369-384.Thema: Isotopen-Massenspektrometrie, Organische MassenspektrometrieKeywords: LC-ESI-TOF, click chemistry; amino resinKontakt: [email protected]

239

P143

Gaseous Multiply Charged Carbenes

Nachtigall, Fabiane (1,3); Corilo, Yuri (1); Abdelnur, Patricia (1); Dupont, Jairton (2); Eberlin, Marcos (1)

1: ThoMSon Mass Spectrometry Laboratory, UNICAMP, Brazil.; 2: Laboratory of Molecular Catalysis, UFRGS,

Brazil.; 3: Max-Planck Institut fuer Kohlenforschung, Germany

Einleitung: The continued search for new types of ionic liquids (ILs) that would broaden their vast range of

applications has recently led to the synthesis of ILs with several imidazolium (Im) ion sites. Deprotonation of

imidazolium cations at C-2 positions formally forms neutral carbon bases which are examples of nucleophilic

singlet carbenes strongly stabilized by the presence of two heteroatoms at the carbenic center. In our experiments

such carbenes has been generated from multiply charged ILs. The remaining charge sites function as handles

allowing these unprecedented species to be transferred to the gas phase and be studied via mass spectrometric

experiments.

Experimenteller Teil: ESI mass and tandem mass spectra in the positive ion mode were acquired using a Waters

Micromass (Manchester, UK) QTOF instrument. Typical ESI-MS conditions were as follow: source temperature

100 °C, desolvation temperature 100 °C, capillary voltage 3-3.5 kV, and cone voltage 40 V. ESI-MS/MS analyses

were performed using 20 eV collision-induced dissociation (CID) of mass-selected ions with argon. Reactions

were performed by selecting the ion of interest in the quadrupole (Q) and collisions (3 eV) with molecules (acetone

or acrolein) in the hexapolar collision cell (h). Liquid acetone or acrolein was inserted into the argon gas line of

the collision cell.

Ergebnisse und Diskussion: When di-, tri- and tetra-imidazolioum ILs are transferred to the gas phase, species

such as [Im + A]+ (A = singly charged anion), [Im + A]2+ and [Im + A]3+are observed. Via in-source CID, they

fragment with the loss of [AH] thus forming gaseous multiply charged carbenes. Depending on the imidazolium

ion used, different types of charged carbenes with multiple charges can be generated. These unprecedented species

were isolated and characterized by ESI-MS/MS experiments. Di-imidazolium IL generated singly charged carbene

whereas tri and tetra-imidazolium ILs generated doubly charged carbenes and triply charged carbenes, respectively.

All these ions were characterized by ESI-MS/MS experiments. To ensure that the species formed in the gas phase

were indeed carbenes, were performed reactions with two typical carbene reagents: acetone and acrolein. When the

singly charged carbene of m/z 191 was selected and reacted with acetone in the collision cell, an adduct product

of m/z 249 was observed. For the doubly charged carbene of m/z 143, the addition reactions with acetone and

acrolein led to adduct products of m/z 172 and 171, respectively. The triply charged carbene of m/z 136 generated

the respective triply charged adduct product of m/z 155 by reaction with acrolein.

Neue Aspekte: A new class of gaseous ions, multiply charged carbenes, has been formed and characterized.

Referenzen: [1] Nachtigall, F. M.; de Corilo, Y. E.; Cassol, C. C.; Ebeling, G.; Morgon, N. H.; Dupont, J.; Eberlin,

M. N. Angew. Chem. Int. Ed. 2008, 47, 151.; [2] Arduengo III, A. J. Acc. Chem. Res. 1999, 32, 913.


Keywords: ionic liquids, imidazolium ions, carbenes, addition reaction and mass spectrometry.


240

P144

Speciation Analysis of Gd-based MRI Contrast Agents andPotential Transmetalation Products

Telgmann, Lena (1); Künnemeyer, Jens (1); Tokmak, Faruk (2); Karst, Uwe (1)

1: University of Münster, Germany; 2: University of Bochum, Germany

Einleitung: Contrast agents for magnetic resonance imaging based on Gadolinium (Gd) have been used for

more than 20 years. For the application as contrast agent, Gd is complexed with polyaminocarboxylic acid

chelating agents. These complexes have very high thermodynamic stability constants, but a connection between

the medication with Gd-based contrast agents and a newly observed disease called Nephrogenic Systemic Fibrosis

(NSF) has been proposed. NSF only occurs for patients suffering from acute or chronic kidney disease. Its cause

is still unknown, but it has been postulated that transmetalation reactions with parental iron or oral chromium

supplements and the subsequent release of toxic Gd ions take part in its pathogenesis.

Experimenteller Teil: A separation technique for Gd chelates and potential transmetalation products is presented.

The separation efficiency of capillary electrophoresis for ionic compounds is combined with the high resolution of

time-of-flight mass spectrometry. The three ionic Gd-based contrast agents Gd-DTPA, Gd-BOPTA and Gd-DOTA

have each been added to different iron supplements or iron salts (iron citrate or iron gluconate) or chromium

supplements (chromium picolinate or chromium chloride), respectively. The samples were prepared in aqueous

solution and in blood plasma. After incubation at 37 °C for 5 days, the samples were analysed by CE/ESI-ToF-MS.

Based on this method, it is possible to conclude if a transmetalation reaction took place.

Ergebnisse und Diskussion: After 5 days of incubation, iron transmetalation products have been detected in the

samples that contained one of the iron salts (iron gluconate or iron citrate) and either of the contrast agents Gd-

DTPA or Gd-BOPTA. No transmetalation was found in the samples that contained iron supplements. On the one

hand, these results show that a transmetalation reaction is in general possible. On the other hand, it shows that

no transmetalation reaction takes place with iron supplements. A direct connection between the medication of

patients with acute or chronic kidney disease with parental iron supplements after the treatment with Gd-based

contrast agents and NSF cannot be proven based on these data. Gd-DOTA showed no transmetalation at all. This

can be explained with its macrocyclic structure which leads to a higher kinetic stability compared to complexes

based on linear ligands (Gd-DTPA and Gd-BOPTA). This is of particular importance, as NSF has until now only

been reported after medication with contrast agents based on linear ligands. It is thus obvious that the stability of

the complexes is one of the most important aspects in the development of NSF. The samples containing chromium

picolinate or chromium chloride did not lead to transmetalation as well. Most likely, the potential products of a

transmetalation are not as stable as the Gd complexes. A connection between a medication with chromium in any

binding form and NSF cannot be shown at all.

Neue Aspekte: New mass spectrometric methods were developed to investigate transmetalation effects of Gd-

based MRI contrast agents.

Thema: Element-Massenspektrometrie, Organische Massenspektrometrie

Keywords: MRI, contrast agent, gadolinium, NSF, CE/MS


241

P145

Imatinib plasma level determination with LC-MS/MS - Anoverview over biological variation in clinical samples

Thumfart, Jörg Oliver (1); Jäger, Evelyn (1); La Roseé, Paul (2); Findeisen, Peter (1); Neumaier, Michael (1)

1: Universitätsmedizin Mannheim, Institut f. Klinische Chemie, Germany; 2: Universitätsmedizin Mannheim, III.

Medizinische Klinik, Germany

Einleitung: Imatinib®is nowadays the treatment of choice for chronic myeloid leukemia (CML) with excellent

results. But a substantial proportion of patients does not achieve prognostically relevant mile-stones such as

complete cytogenetic response or major molecular response (CCR, MMR) (1).

Experimenteller Teil: Suboptimal Imatinib plasma levels may be a major cause for these therapy failures and

plasma level monitoring might become an important tool to guide treatment decisions in this regard. A plasma

concentration of around 1000 ng/mL Imatinib is described as lower limit for a promising therapy (2). In clinical

reality the standard daily dose of imatinib is leading to steady state plasma concentrations with remarkable in-

terindividual variability that might be related to pharmakocinetic/pharmacogenetic variations or compliance prob-

lems (3).

Ergebnisse und Diskussion: Here we present our analytical set-up sample preparation based on SPE, and quantifi-

cation of Imatinib via LC-MS. The results of over 50 routine measurements of Imatinib at the UMM over 9 months

are presented. Based on the current data, imatinib blood-level testing seems to be a useful aid when making clinical

decisions in CML treatment.

Neue Aspekte: Presentation of a truly routine application of LC-MS in clinical chemistry with high impact for

medical decisions.

Referenzen: [1] Baccarani M, Saglio G, Goldman J, Hochhaus A et al. Blood 2006; 108(6): 1809-1820; [2]

Picard S, Titier K, Etienne G, et al. Blood 2007; 109: 3496-99; [3] Larson RA, Druker BJ, Guilhot F, et al. Blood

2008; 111: 4022-28


Keywords: LC-MS, TDM in a clinical chemistry routine application, leukemia, Imatinib


242

P146

Characterization of different poly(oxazoline)s by ESI-MS andtandem MS

Altuntas, Esra (1); Kempe, Kristian (1); Crecelius, Anna (1,2); Schubert, Ulrich S. (1,2,3)

1: Friedrich-Schiller-University Jena, Germany; 2: Dutch Polymer Institute (DPI), The Netherlands; 3:

Eindhoven University of Technology, The Netherlands

Einleitung: Mass spectrometry has become an important tool for the characterization of different macromolecules

in recent years because of the development of electrospray ionization mass spectrometry (ESI-MS) and matrix-

assisted laser desorption ionization mass spectrometry (MALDI-MS). Although they involve different processes in

ion formation, both techniques generally allow ionization of different macromolecules with little or no fragmen-

tation. Structural information can be obtained by using tandem MS (MS/MS) analysis. In this study, an ESI-MS

instrument was used to elucidate the molar mass distributions of different poly(oxazoline)s. Tandem MS analysis

gave an understanding about the fragmentation mechanism of the poly(oxazoline)s.

Experimenteller Teil: ESI-(Q)-TOF-MS measurements were performed with micrOTOF II (Bruker Daltonics)

mass spectrometer equipped with an automatic syringe pump which is supplied from KD Scientific for sample

injection. The ESI-Q-TOF mass spectrometer was operated at 3.5 kV, at a desolvation temperature of 180 ºC.

The mass spectrometer was operating in the positive ion mode. For the MS/MS mode nitrogen was used as a

collision gas. The standard ESI source was used to generate the ions. The samples with concentrations ranging

from 1 µg/mL to 10 µg/mL were injected using a constant flow (3 µL/min) of sample solution. Chloroform and

acetonitrile mixture were used to dissolve the polymer samples. The instrument was calibrated with an internal

calibration standard solution which is supplied from Agilent.

Ergebnisse und Diskussion: Polyoxazolines are an important class of polymers with a wide range of applica-

tions because of their potential use as biomaterials, as thermoresponsive materials (such as in thermosensitive

materials), as sensors or as carrier systems for active agents in drug delivery. Moreover, they allow an easy

access to defined amphiphilic structures for self-assembly. Therefore, it is really important to identify these poly-

mers in a detailed way with different instrumentations to gain information about their molar mass distribution. A

detailed characterization of poly(oxazoline)s was performed by ESI-Q-TOF MS and MS/MS. In ESI-MS analy-

sis, poly(oxazoline)s synthesized with different end groups, with varying leaving group quality, side groups, i.e.

methyl, ethyl, pentyl, ethylpentyl, nonyl, phenyl or difluorophenyl group, and different molar masses were ana-

lyzed. Multiple charged species were obtained extending the molar mass range which could be analyzed. The

obtained spectra of poly(oxazoline)s resulted fitting mass distributions and equidistant peaks with a difference of

the mass of one monomer unit. In tandem MS analysis, endgroup is fragmenting first, then there is a step-wise de-

polymerization occurring during the fragmentation. MALDI-MS has also been employed for the characterization

of polyoxazolines and it provides similar results compared to ESI-MS, only in ESI-MS analysis multiply charged

species were obtained.

Neue Aspekte: Characterization of different new poly(oxazoline)s polymers with ESI-MS and MS/MS in order to

provide information about the fragmentation pathways.


Keywords: ESI-MS, tandem MS, polyoxazolines


243

P147

Schnelle Bestimmung kleiner Moleküle mittels MALDI-TOF-MS

Persike, Markus; Karas, Michael

Goethe Universität Frankfurt, Germany

Einleitung: Für die Untersuchung von kleinen Molekülen, wie z.B. von Arzneistoffen und ihren Metaboliten, wird

nach chromatographischer Trennung immer häufiger eine massenspektrometrische Detektion verwendet. Dabei

wird meist die Elektrospray-Ionisation (ESI) oder die chemische Ionisation bei Atmosphärendruck (APCI) einge-

setzt. Trotz der Entwicklung neuer Geräte und Techniken findet dieMatrix-unterstützte Laser-Desorption/Ionisation(MALDI) in diesem Massenbereich kaum Anwendung. Dabei besitzt MALDI einige Vorteile, wie eine höhereToleranz gegenüber Salzen und Verunreinigungen. Dies ermöglicht häufig eine direkte Messung ohne den Einsatzeiner LC. Dabei kann durch die kurzen Analysenzeiten (< 15 s pro Probe) ein sehr hoher Probendurchsatz realisiertwerden.Experimenteller Teil: Die Messungen erfolgten an einemMALDI-TOF und einen MALDI-TOF/TOF-Instrument(Voyager STR und 4800, beide Applied Biosystems). Zur Probenauftragung wurde die Standard „dried dropletpreparation" Methode mit α-cyano-4-hydroxyzimtsäure (CHCA) als Matrix verwendet.

Ergebnisse und Diskussion: Bei dieser Studie wird anhand verschiedener Beispiele gezeigt, dass auch MALDI-

TOF-MS bei der Bestimmung kleiner Moleküle eingesetzt werden kann. Dabei ist keine spezielle Matrix oder

besondere Probenvorbereitung notwendig. Als erste Anwendung dient die gleichzeitige Quantifizierung ver-

schiedener pharmazeutischer Wirkstoffe in humanem Plasma. Dabei war als Probenvorbereitung lediglich eine

Flüssig-Flüssig Extraktion notwendig [1]. Ein weiterer Anwendungsbereich von MALDI-MS stellt die Bestim-

mung des Neurotransmitters Acetylcholin aus in-vivo Mäusehirndialysaten dar. Es zeigte sich, dass trotz des sehr

hohen Salzgehaltes der Proben (∼ 150 mM), eine direkte Messung möglich ist. Da keinerlei Probenaufreinigungnotwendig ist, kann ein viel höherer Probendurchsatz als mit LC/MS Systemen realisiert werden. Zudem ist eineerneute Messung zu einem späteren Zeitpunkt möglich. Durch die Verwendung eines MALDI-Spotter konnteaußerdem die zeitliche Auflösung gegenüber LC basierten Systemen erheblich verbessert werden [2]. Als letztesBeispiel für den Einsatz von MALDI-MS im niedrigen Massenbereich dient die Bestimmung von Melamin inMilchpulver. Dabei konnte auch hier ohne LC direkt und schnell quantifiziert werden.Neue Aspekte: Einsatz von MALDI-TOF-MS bei der schnellen Quantifizierung kleiner MoleküleReferenzen: [1] Persike, M.; Karas, M. Rapid Commun. Mass Spectrom. 2009, 23, 3555-3562; [2] Persike, M.;Zimmermann, M.; Klein, J.; Karas, M. Anal. Chem. 2010, 82, 922-929Thema: Organische MassenspektrometrieKeywords: Quantifizierung, Arzneistoffe, Acetylcholin, Melamin, MALDIKontakt: [email protected]

244

P148

LC-MS in der Lebensmittelüberwachung - Der Würfel Zucker imBodensee oder in der Sahnetorte

Stephan, Michael

Landesamt für Verbraucherschutz Sachsen-Anhalt, Germany

Einleitung: Jeder Verbraucher erwartet einwandfreie und sichere Lebensmittel. Dafür sind zunächst alle Un-

ternehmer in der Lebensmittelkette verantwortlich. Ziel der amtlichen Lebensmittelüberwachung ist es, Ver-

braucher vor gesundheitlichen Gefahren durch Lebensmittel, Tabakerzeugnisse, kosmetische Mittel und Bedarf-

sgegenstände sowie vor Irreführung und Täuschung zu schützen. Rechtsgrundlage dafür ist das Lebensmittel-

und Futtermittelgesetzbuch (LFGB). Insgesamt gibt es eine Fülle von gesetzlichen Vorschriften der Europäischen

Gemeinschaft und des Bundes. Die amtliche Lebensmittelüberwachung hat zur Aufgabe, für die Einhaltung und

Beachtung dieser Rechtsvorschriften zu sorgen.

Experimenteller Teil: Grundvoraussetzung für die Tätigkeit eines Überwachungsamtes ist eine Akkreditierungnach DIN EN ISO/IEC 17025, über die im Rahmen des internen Qualitätsmanagementsystems die Anforderungen

an die Kompetenz von Prüf- und Kalibrierlaboratorien umgesetzt werden. Notwendig sind auch die technischen

Bedingungen, um den Anforderungen (Bestimmungsgrenzen bei Nulltoleranz, Kontaminanten, zulässige Höchst-mengen) an die Überwachungstätigkeit gerecht zu werden.Ergebnisse und Diskussion: Für die Kontrolle der Grenzwerte nehmen LC-MS/MS-Methoden immer breiterenRaum ein. Die empfindliche und spezifische qualitative und quantitative Erfassung von Kontaminanten in un-terschiedlichsten Lebensmitteln wird allerdings von der Problematik der Matrixeffekte begleitet. Die Erzeugungvon gerichtsfesten Analysenergebnissen soll an ausgewählten Beispielen dargestellt werden: - Acrylamid in Back-und Röstwaren - Nitrosamine in Luftballons - Pestizide in Obst, Gemüse, Getreide - Mykotoxine in Getreide -Vitamin D in Margarine, Kindernahrung, Nahrungsergänzungsmitteln - Cereulid bei Lebensmittelvergiftungen -Melamin - China ist näher als man denkt Der Einsatz der LC-MS eröffnet auch in der Lebensmittelüberwachungseit etwa einem Jahrzehnt neue Möglichkeiten. Bei der Bearbeitung relevanter Fragestellungen ist die Analytikvon HPLC- oder auch aufwendigen GC-MS-Methoden auf spezifische, routinetaugliche und robuste LC-MS/MS-Methoden umgestellt worden (Acrylamid, Nitrosamine, Vitamin D) und neue Analyten konnten schnell und sichereingearbeitet werden (Cereulid, Melamin).Neue Aspekte: Der Trend geht eindeutig hin zu Multimethoden (Pestizide, Mykotoxine), wobei allerdings imVordergrund die Erzeugung sicherer Werte steht.Thema: Organische MassenspektrometrieKeywords: LC-MS, LebensmittelsicherheitKontakt: [email protected]

245

P149

Massenselektive resonante (1+1)-PhotodissoziationsSpektroskopie von C2H5I+ und C2D5I+

Schüttig, Hannes; Grotemeyer, Jürgen

Institut für physikalische Chemie, Universität Kiel, Germany

Einleitung: Für die Spektroskopie von Ionen existieren vielfältige Methoden [1]. Die Möglichkeit elektron-isch angeregte Zustände mittels Dissoziation nach der Absorption von einem oder meheren Photonen zu spek-troskopieren bietet allerdings einige Vorteile. Beispielsweise können somit auch dipolverbotene Übergänge Unter-sucht werden [2]. Die Multiphotonendissoziation bietet weiterhin die Möglichkeit neben prädissoziativen Zustän-den auch Zustände zu untersuchen die nach der Absorption eines Photons nicht dissozieren. Alkyliodide werdensehr oft als Modellverbindungen zum Studium der Photodissoziationsdynamik in angeregten Zuständen herange-zogen [3,4].Aller- dings beschränken sich diese Untersuchungen nur auf neutrale Moleküle. Detailiertes Kenntnissder elektronisch angeregte Ionen dienen damit als Grundlage auch die Dissoziationsdynamik von Ionen zu Unter-suchen was letzlich auch für die Massenspektrometrie von Bedeutung ist.Experimenteller Teil: Das von uns verwendete Photodissoziationsspektrometer ist mit einer Elektronenionisa-tionsquelle, zur Erzeugung der Vorläuferionen, und einem gepulsten Farbstofflaser für die eigentliche Photodis-soziation ausgestattet. Die Ionen werden in der ersten Stufe einer zwei stufigen Quelle erzeugt. Nach deren ex-traktionen in das zweite Beschleunigungsfeld erfolgt die Dissoziation in einem Bereich von 14600 - 15900 cm−1 .Durch die unterschiedlichen kinetischen Energien können die primar erzeugten Ionen und Photodissoziationspro-dukte mittels eines Reflektron-ToF-MS durch ein geeignetes Reflektronpotential separiert werden. Somit könnendie PD-Ionen nahezu ohne störende Fragmentsiganle detektiert werden.Ergebnisse und Diskussion: Der Ursprung des Ã Zustandes wurde für C2H5I+ zu 12968 ± 14 cm−1 und fürC2D5I+ zu 13085± 24 cm−1 bestimmt die harmonische Normalschwingungsfrequenz der C-C-I Biegeschwingungbeträgt ν(H) = 160cm−1 für C2H5I+und ν(H) = 166cm−1 für C2D5I+. Aufgrund von vergleichen mit den Io-nenspektren des CH3I+ wird der Übergang dem Ã←X2

1E12 Übergang zugeordnet.

Neue Aspekte: Schwingungsaufgelösten Spektren des Ethyliodid bzw. Ethyliodid-d5 Radikalkations des Ã-Zustandes.Referenzen: [1] Molecular Ions: Spectroscopy, Structure and Chemistry, ed T. Miller and V. Bondybey, North-Holland, 1983.; [2] K. Walter, R. Weinkauf, U. Boesl und E. W. Schlag, Chem. Phys. Lett., 1989, 162, 261-268.;[3] Y. Tang, W.-B. Lee, Z. Hu, B. Zhang und K. C. Lin, J. Chem. Phys., 2007, 126, 64302-64310.; [4] R. Zhang,A. Y. Ghazal, Y. Liu, Y. Zhang, B. Tang und B. Zhang, Optics Communications, 2009, 282, 2169-2173.Thema: Proteine und Peptide, MetabolomicsKeywords: Alkyliodide, IonenspektroskopieKontakt: [email protected]

246

P150

Analysis of Athabasca Oil Sands Process Water by ESI and APPIFourier Transform Ion Cyclotron Resonance Mass Spectrometry

Witt, Matthias (1); Barrow, Mark P. (2); Headley, John V. (3); Peru, Kerry M. (3)

1: Bruker Daltonik GmbH, Germany; 2: Department of Chemistry, University of Warwick, Coventry, United

Kingdom; 3: Environment Canada, Saskatoon, Canada

Einleitung: Pressure upon supplies of fossil fuels continues to grow, especially as sources of crude oil are in

demand due to their essential basis for fuels, plastics, solvents, waxes, lubricants, pesticides, and medicines.

Athabasca oil sands are a source of petroleum that has previously been considered non-viable, due to the inherent

costs associated with processing them; for example, approximately three barrels of fresh water per barrel of oil

produced. Crude oils are complex mixtures and "naphthenic acids," which are known to be toxic towards aquaticwildlife, are frequently amongst the classes of compounds present. There is a continuing need to develop methodsfor monitoring the environmental impact of the petroleum industry.Experimenteller Teil: The oil sands region of Alberta, Canada, was focused upon and a sample was obtained froma site within the area of the Athabasca River Basin. A resulting concentrate of extracted oil sands process waterwas used as the basis for mass spectrometric analysis.Mass spectra were obtained using a 12 T Fourier transformion cyclotron resonance (FTICR) mass spectrometer, using both electrospray ionization (ESI) and atmosphericpressure photoionization (APPI). Data visualization was performed using Aabel 3.0.1 (Gigawiz Ltd. Co., Tulsa,AZ, USA). Particular focus is placed upon the naphthenic acid content of the sample, with regards to toxicitytowards aquatic wildlife, and visualization of the data enables comparison of results obtained using the differentmethods.Ergebnisse und Diskussion: A 12 T Fourier transform ion cyclotron resonance mass spectrometer was used forthe analysis of the sample. FTICR mass spectrometry is well-known for its association with ultra-high resolutionand mass accuracy, which are prerequisites for characterization of highly complex mixtures, as it affords a highdegree of confidence in the assignments of species. The instrument was operated in both positive-ion and negative-ion modes, where the resulting mass spectra displayed clear differences in terms of general appearance and thenumber of peaks. Furthermore, two different ionization methods were used: ESI and APPI. The mass spectra,obtained using the different ionization techniques, displayed similarities but it could also be seen that the numberof species detected differed. Utilization of APPI led to the observation of more signals, and the ionization methodcan be applied to the investigation of compounds which are less polar than those typically studied using ESI.Following the acquisition of a mass spectrum, empirical formulae were assigned to the signals and the data wassorted according to various characteristics, including compound class, carbon content, and z series (otherwiseknown as the "hydrogen deficiency"). A range of data visualization methods were used to convey the resultsof analyses, including: plots based upon the Kendrick mass defect, van Krevelen diagrams, two dimensionalline plots, and heat maps. In this manner, the data sets obtained using the different ionization methods, andpositive/negative-ion modes, can be compared and "fingerprints" for the sample can be determined.Neue Aspekte: High field FTICR mass spectrometry for the study of environmental samples; a range of datavisualization methods used.Thema: Umweltanalytik und AerosoleKeywords: Naphthenic acids, FTMS, elemental formulaKontakt: [email protected]

247

P151

Anwendung der Massenspektrometrie inArzneimitteluntersuchungsstellen (OMCLs) zur Untersuchung von

Verdachtsproben am Beispiel von PDE-5-Hemmern

Raith, Klaus (1); Meyer, Matthias (1); Scherges, Michael (2); Heuermann, Matthias (2)

1: Landesamt für Verbraucherschutz Sachsen-Anhalt, Germany; 2: Landesinstitut für Gesundheit und Arbeit,

Nordrhein-Westfalen, Germany

Einleitung: Die Arzneimitteluntersuchungsstellen der Bundesländer (Official Medicines Control Labs, OMCLs)sind neben der regelmäßigen Untersuchung der im jeweiligen Bundesland hergestellten Arzneimittel auch für die

Untersuchung von Verdachtsproben zuständig. Dabei kann es sich um illegale Arzneimittel, Arzneimittelfälschun-

gen oder um sogenannte Borderline-Produkte handeln, bei denen erst nach der Begutachtung entschieden werden

kann, ob es sich um Arzneimittel, Lebensmittel oder andere Produktkategorien handelt. Wenn nicht bereits die

Kennzeichnung oder entsprechende Werbeaussagen zu einer Arzneimitteleinstufung führen, fordern die Gerichte

den Nachweis von Substanzen mit pharmakologischer Wirkung in einer Menge, die die Erheblichkeitsschwelle

überschreitet. Um dies leisten zu können, findet die Massenspektrometrie, insbesondere die LC/ESI-MS/MS, inOMCLs zunehmend Anwendung. Ein Beispiel ist die Untersuchung von PDE-5-Hemmern, deren bekanntesterVertreter Sildenafil häufig illegal hergestellt und abgewandelt wird.Experimenteller Teil:Am LIGA-NRW werden Verdachtsproben mittels LC/ESI-MS/MS an einer Linearen Ionen-falle vom Typ Thermo Finnigan LTQ untersucht. Eine Datenbank mit PDE5-Hemmern, in der Hauptsache Silden-afil, Tadalafil, Vardenafil und deren Derivate, befindet sich im Aufbau. Zur Ermittlung von relativen Retentionszei-ten wird bei der Aufnahme von Referenzsubstanzen Sildenafil-d8 als Interner Standard (ISTD) verwendet. Bei derIdentifizierung von Substanzen in Verdachtsproben werde alle im sogenannten Data-Dependent-Modus erhaltenenMS-, MS2- und MS3-Spektren bei der Bibliothekssuche mittels Software verwendet (Spectral Tree Search). AmLAV Sachsen-Anhalt wird ein QTRAP 3200 (Applied Biosystems) genutzt.Ergebnisse und Diskussion: In Verdachtsproben illegaler Arzneimittel wurden am LIGA-NRW bisher bereits dieSubstanzen Sildenafil, Vardenafil, Tadalafil, Dimethylsildenafil, Homosildenafil, Hydroxyhomosildenafil und Icari-in gefunden. Darüber hinaus wurden auch N-Desmethylsildenafil, Norneosildenafil, Acetildenafil, Aminotadalafil,

N-Desethylvardenafil und Pseudovardenafil in die Datenbank eingepflegt. Am LAV Sachsen-Anhalt wurden Paral-

lelimporte von Sildenafil- und Vardenafilhaltigen Arzneimitteln untersucht. Ebenso waren illegale Sildenafilhaltige

Arzneimittelproben eingesandt worden. Die Ergebnisse zeigen, dass mittels LC/ESI-MS/MS Sildenafil und ande-

re verwandte PDE-5-Hemmer eindeutig identifiziert und empfindlich quantifiziert werden können. Dadurch ist esmöglich, eine gerichtsfeste Einstufung entsprechender Produkte als illegale Arzneimittel vorzunehmen, was imInteresse des gesundheitlichen Verbraucherschutzes dringend geboten ist. Daneben wird das Problem der Arznei-mittelfälschungen immer bedrohlicher, was sich aus der außerordentlich hohen zu erzielenden Gewinnspanne er-klärt. Haupteinfallstor ist der illegale Arzneimittelversand via Internet.Neue Aspekte: Amtliche Arzneimitteluntersuchungsstellen bauen LC/ESI-MS/MS-gestützte Datenbanken auf,

mit deren Hilfe gefälschte bzw. illegale Arzneimittel identifiziert werden können.Thema: Umweltanalytik und AerosoleKeywords: PDE-5-Hemmer, illegale Arzneimittel, ArzneimittelfälschungenKontakt: [email protected]

248

P152

Studies of low-temperature combustion in a flow reactor atatmospheric pressure

Herrmann, Friederike; Struckmeier, Ulf; Kohse-Höinghaus, Katharina

Universität Bielefeld, Germany

Einleitung: For the next decade at least, energy conversion will continue to include combustion processes. Low-temperature combustion is a promising concept to minimize both soot- and NOx emissions at the same timewhile ensuring good efficiency. In this regime, temperature oscillations may occur which are thought to arisefrom switching between different combustion pathways that involve C1-flame chemistry on the one hand and C2-chemistry on the other [1]. Oscillations, including thermo-acoustic phenomena like gas turbine humming, must beprevented in practical applications. For fundamental studies of the relevant combustion chemistry, experimentalinvestigation of model systems and comparison with kinetic simulations are necessary.Experimenteller Teil: In this study, highly diluted atmospheric pressure oxidation of methane and ethane in a flowreactor are investigated by electron ionization time-of-flight molecular beam mass spectrometry. The simultaneouscharacterisation of important stable combustion intermediates is an important advantage of the chosen method.Ergebnisse und Diskussion: Mole fractions of species were quantified as a function of stoichiometry and res-idence time. Scans from 1170 to 870 K were performed to determine light-off temperatures and to observe thetemperature-dependent profiles of the stable combustion intermediates. Strong dependencies of species concen-trations on these parameters are observed. The combustion takes place in a homogeneous gas phase and catalyticwall effects are negligible. Simulations with a combustion model are in progress.Neue Aspekte: This study improves the detailed understanding of the oxidation process of small hydrocarbons inthe low-temperature regime.Referenzen: [1] M. de Joannon, A. Cavaliere, T. Faravelli, E. Ranzi, P. Sabia, A. Tregrossi, Proc. Combust. Inst.30 (2005), 2605-2612.Thema: Instrumentelle Entwicklungen, Umweltanalytik und AerosoleKeywords: low-temperature combustion, flow reactor, methane, ethaneKontakt: [email protected]

249

P153

Low-temperature combustion - a way to reduce emissions?Investigation of a highly diluted methane flame by molecular

beam mass spectrometry

Moshammer, Kai (1); Dreyer, Christoph (1); Struckmeier, Ulf (1); Lucassen, Arnas (1);

Kohse-Höinghaus, Katharina (1); Wada, Tomoya (2); Peters, Norbert (2)

1: Physikalische Chemie I, Universität Bielefeld, D-33615 Bielefeld; 2: Institut für Technische Verbrennung,RWTH Aachen, D-52506 Aachen

Einleitung: In conjunction with the reduction of NOx- and soot-emissions from practical combustion devices,low-temperature combustion attracts increasing interest in combustion research. The investigation of predomi-nantly fuel-lean, highly diluted flames becomes important [1]. Due to feedback loops between heat release andtemperature-sensitive reaction kinetics in such systems, temperature oscillations may occur. As a consequence,combustion follows different pathways involving C1- as well as C2-chemistry [2]. In view of technical applica-tions, these oscillations have to be prevented, using adequate control strategies. For this, low-temperature flamekinetics must be validated for such combustion conditions.Experimenteller Teil: In this study, low-temperature combustion at atmospheric pressure was achieved and in-vestigated in highly diluted methane flames with 90% argon as a diluent. Species mole fractions were quantifiedby electron ionization time-of-flight molecular beam mass spectrometry. To understand the influence of transportprocesses, measurements were performed under different environmental conditions.Ergebnisse und Diskussion: The results will be discussed, also in view of preliminary predictions of premixedone-dimensional flame chemistry with current combustion models.Neue Aspekte: The detailed investigation of low-temperature flames at atmospheric pressure was successful forthe first time.Referenzen: [1] A. Cavaliere, M. de Joannon, Prog. Energ. Combust. 30 (2004), 329-366.; [2] M. de Joannon, A.Cavaliere, T. Faravelli, E. Ranzi, P. Sabia, A. Tregrossi, Proc. Combust. Inst. 30 (2005), 2605-2612.Thema: Instrumentelle Entwicklungen, Umweltanalytik und AerosoleKeywords: Low-temperature combustion, flame, MBMSKontakt: [email protected]

250

P154

Einzelaerosolpartikel-LA-ToF-MS - Einfluss derAblationswellenlänge auf die Fragmentation

Klimach, Thomas; Roth, Anja; Drewnick, Frank; Borrmann, Stephan

Max-Planck Institut für Chemie, Germany

Einleitung: Zur direkten chemischen Analyse von einzelnen luftgetragenen Partikeln wird heute überwiegend

die Laser-Ablation mit UV-Lasern genutzt. Durch einen Laserpuls wird das Partikel verdampft und die freige-

setzten Moleküle ionisiert. Die Art und die Menge der entstehenden Ionen ist dabei abhängig von der Größedes Partikels, der Energie und Wellenlänge des Lasers, der Zusammensetzung und Form des Partikels und derArt und Weise wie das Partikel vom Laser getroffen wird. Da diese Größen von Schuss zu Schuss leicht vari-ieren liefern Partikel gleicher Zusammensetzung meist verschiedene Produkte. Untersucht werden soll nun derEinfluss der Ablationswellenlänge auf die Fragmetierung der Ausgangsverbindungen. Zu diesem Zweck werdendie durch Laser-Ablation von Partikeln entstandenen Ionen mit Hilfe der Massenspektrometrie, bei Verwendungverschiedener Ablationswellenlängen gemessen.Experimenteller Teil: Es wurden verschiedenen, insbesondere organische, Substanzen mit bekannten Ionisation-senergien ausgewählt. Die Analyten werden jeweils in Wasser gelöst und mit Hilfe einer Düse vernebelt. Derhergestellte Nebel wird getrocknet, bis nur noch feste Partikel neben der Gasphase vorkommen. Dieses Aerol-sol wird vom Messinstrument angesaugt und durch eine aerodynamische Linse fokussiert. Die Aerosolpartikelpassieren nach der Linse zwei Laserschranken (532 nm) durch die Flugzeit lässt sich so die Geschwindigkeit derPartikel bestimmen, die wiederum ein Maß für die Größe der Partikel ist. Ebenso lässt sich nun der Ablationslaserzur richtigen Zeit zünden. Es standen ein Excimer-Laser der mit einer Wellenlänge von 193 nm (ArF) oder 308nm (XeCl) arbeitet und ein Festkristall-Laser mit einer Wellenlänge von 248 nm zur Verfügung.Ergebnisse und Diskussion: Mit der Einzelpartikel Massenspektrometrie ist es möglich den Einfluss der Wellen-länge des Ablationslasers auf die Bildung von Ionen beim Beschuss von Partikeln zu untersuchen. Deutliche Un-terschiede lassen sich bei der Stärke der Fragmentierung von organischen Verbindungen finden. So bleibt bei 193nm selten mehr als die KohlenstoffclusterC1,C2 undC3 übrig. Während bei 248 nm und noch mehr bei 308 nm dieFragmetierung deutlich zurück gedrängt ist, sodass oft der Molekülpeak gefunden werden kann. Quantitative Aus-sagen lassen sich allerdings nicht treffen, da die Fragmentierung auch von der Art und Weise abhängig ist wie bzw.wo das Partikel vom Ablationslaser getroffen wird, diese Variationen lassen sich systembedingt nicht eliminieren,da der Partikelstrahl nicht beliebig fokussierbar ist. Desweiteren lässt sich feststellen dass der Ablations- /Ionisa-tions Prozess bei kleinenWellenlängen effizienter abläuft als mit energieärmeren Photonen, was sich positiv auf dieHitrate des Systems, dem Verhältniss von detektierten zu getroffenen Partikeln, auswirkt.Insgesamt fällt auf dassfür eine Substanz verschiedene Massenspektren gefunden werden. Diese verschiedenen Spektrentypen lassen sichzumindest zum Teil mit den Parametern (a, b) der Massenkalibration korrelieren. Die Umrechnung der Flugzeit(tToF ) in m/z Werte erfolgt mit der Gleichung m

z= a + b

√tToF . Die Parameter werden zum einen durch die

angelegten Spannungen bestimmt, allerdings werden sie durch den Ort der Ablation sowie dem Ionisationsprozessansich beeinflusst. Die Experimente zeigen deutlich die Abhängigkeit der gebildeten Ionen vom Ort der Ablation.Es wird gezeigt das vorallem die Spektrentypen der längerwelligen Ablation unterschiedliche Massenkalibratio-nen haben. Neben den Labormessungen wurden auch Daten verglichen, die während der Megapoli Kampange imSommer 2009 in Paris gesammelt wurden. Der Vergleich der Wellenlängen 193 nm und 308 nm zeigt, den im La-bor gefundenen Zusammenhang zwischen Fragmentierung organischer Molekülen und der Ablationswellenlänge,sowie zwischen der Hitrate des Instruments und der Ablationswellenlänge. Die Auswertung der Spektren konntezudem gut den Daten eines AMS (Aerosol-Massenspektrometer) verglichen werden.Neue Aspekte: Einfluss der Ablationswellenlänge auf die Massenspektren von Partikeln. Auswirkung desAblationsortes auf die Massenpektren mit der Massenkalibration als Indikator.Thema: Organische Massenspektrometrie, Umweltanalytik und AerosoleKeywords: Einzelpartikelmassenspektrometrie, LDI, AerosolanalytikKontakt: [email protected]

251

P155

The use of MRM and MRM3 mode for rapid analysis of iodinatedX-ray contrast media

Lembcke, Jan (1); von Oepen, Birgit (2)

1: Applied Biosystems, Germany; 2: Hamburger Wasserwerke GmbH, Germany

Einleitung: Iodinated X-ray contrast media (ICM) are applied in human diagnostics, used to aid visualization of

organs and vessels. Due to their high persistence and water solubility, ICM have been found in the effluent from

wastewater treatment plants, in groundwater and also in raw and treated drinking water. LC/MS/MS methods with

solid phase extraction for sample pre-concentration are mainly utilized for the quantitative analysis of ICM in the

ng/L range. We present a fast method for the quantitative determination of the major ICM using MRM and/or

MRM3mode for highest selectivity with direct injection of various filtrated water samples within an LC run time

of 10 min.

Experimenteller Teil: Water samples from various origins were filtrated (0.45 µm syringe filter) and 100 µLwere then injected onto an Agilent 1200 SL liquid chromatograph using the supplied autosampler. The ICM were

separated on an Agilent 1.8 µm ZORBAX Eclipse XDB C18 Rapid Resolution HT column (4.6 x 100 mm) using

a gradient of water/methanol with 5mM ammonium acetate and 0.1% formic acid. LC flow rate was 1.0 mL/min.

Mass spectrometric data was collected in MRM mode and MRM3 mode using an Applied Biosystems AB SCIEX

QTRAP®

5500 mass spectrometer.

Ergebnisse und Diskussion: For each of the seven ICM (Iopamidol, Amidotrizoic acid, Iopromide, Iohexol,

Iomeprol, Iopanoic acid, Iothalamic acid) the specific MRM transitions were optimized in positive ion mode,

MRM3 detection mode was used for compounds showing secondary fragmentation. The dynamic range of the

method covered at least 2.5 orders of magnitude. The LODs will fall below 50 ng/L. The CVs were below 15%

for 10 fold injections of various spiked water matrices, recoveries ranged from 85 to 115% depending on the ICM

and the matrix. MRM3 mode increases the selectivity to reduce the background without sacrificing sensitivity. For

wastewaters, highly selective MRM3 mode of a hybrid triple quadrupole / linear ion trap mass spectrometer could

be an option to obtain better quality data in quantitative analysis of trace contaminants.

Neue Aspekte: MRM3 mode for detection and quantitation of ICM in various water matrices.

Thema: Umweltanalytik und Aerosole

Keywords: MRM3, MS3, Roentgenkontrastmittel, Iodinated X-Ray Contrast Media, QTRAP 5500


252

P156

Effects of Sample Preparation on the FT-ICR Mass SpectrometryMeasurement of Short and Long Residues Deposit Samples from

Crude Oil

Lababidi, Sami; Schrader, Wolfgang

Max-Planck Institut fuer Kohlenforschung, Germany

Einleitung: Althoughonly a small percentage of non-hydrocarbon compounds are present in crude oil, they are

among the most deleterious to refining catalysts and confer adverse stability properties. Sulfur, Oxygen, and

Nitrogen compounds are concentrated mainly in the heavier fractions and in the residues, and they are responsible

for gum and color formation, beside other problems related to health and environmental issues. For instance,

high concentration of polar nitrogen species generally is responsible for fouling properties in crude oil because

of enhanced deposition, due to increased reactivity, or reduced solubility. Therefore, a detailed knowledge of the

distribution of the heteroatomic compounds in crude oils and their corresponding deposits is necessary to improve

their removal processes and to enhance coker performance.

Experimenteller Teil: Extraction is considered as one of the important steps in the sample preparation of residues

to be analyzed by mass spectrometry. Solid deposits were extracted by using different solvents with various polari-

ties, like toluene, dichloromethane, chloroform, methanol, and acetonitrile. Positive and negative ESI mass spectra

were obtained by 7T actively shielded magnet FT-ICR mass spectrometer. In order to investigate the possibility

of solvent selectivity in the dissolution, a subsequent extraction with different solvents was also performed on the

same residue sample. Extracted samples were diluted with methanol and spiked with different cationization agents

(like acetic acid) or anionization agents (like ammonium hydroxide).

Ergebnisse und Diskussion: Data analysis of various mass spectra obtained from extracted samples shows the

significant influence of solvents on extraction procedures. Different class distribution was observed for each sol-

vent, which indicates the difficulty of finding a complete solvent, which efficiently dissolves all the components

of the residues. Mass balance ratios of the dissolution process were calculated for each solvent. Silver triflate

cationization can convert nonpolar heteroatomic hydrocarbons into positive ions in the positive ESI mode. Dif-

ferent cationization properties were also observed by the acidification with either formic or acetic acid. NS is the

predominant class in the short residue, followed by N1 class. Long residue samples show the predominance of N1

class, with a notable presence of OS class. The determination of double bond equivalents of the OS class indicates

that it comprises of lower number of unsaturated compounds, whereas for N1 the DBE is higher. Kendrick plots of

N1 class of different extracted samples were not similar, which is an indication that some extraction solvents can

show selectivity towards certain compounds.

Neue Aspekte: Optimization of sample preparation methods in the analysis of crude oil solid deposit by FT-ICR

MS

Thema: Umweltanalytik und Aerosole

Keywords: FT-ICR MS, crude oil, Extraction


253

P157

Gasphasen-Reaktionen von laserdesorbierten Molekülen mitelektrogesprühten Ionen

Ganza, Viktoria; Gross, Jürgen H.

Universität Heidelberg, Germany

Einleitung: Ion-Molekülreaktionen sind in der Massenspektrometrie nicht neu.[1,2] Neuartig sind aber die Experi-

mente, welche die Dual Source MTP des Bruker ApexQe FT-ICR-Massenspektrometers ermöglicht.[3] Die Quel-le verfügt grundsätzlich über die Fähigkeit, gleichzeitig Ionen von der Elektrospray-Quelle und vom (MA)LDI-

Probenträger im Feinvakuum (3-4 mbar) eines Ion Funnels zu sammeln und gemeinsam in den Analysator zu trans-

ferieren. Es war daher interessant zu versuchen, ob laserdesorbierte Moleküle mit elektrogesprühten Ionen in dieser

Mischzone zur Reaktion gelangen. Die bei (MA)LDI oft im Verhältnis zu Ionen 105-fach größere Zahl desorbier-ter Neutralteilchen ließe eine ionisationsfreie Laserdesorption unterhalb der Schwellenenergie zu. Mit passendenBetriebsparametern der Quelle und einem hinreichend reaktiven Ion, sollten Gasphasenreaktionen grundsätzlichmöglich sein. Die Ergebnisse dieser Experimente stellen wir hier vor.[4]Experimenteller Teil: Die Experimente wurden an einem Bruker ApexQe FT-ICR-Gerät mit ESI-zu-MALDIumschaltbarer Ionenquelle (Dual Source MTP) und 9.4 TMagneten durchgeführt. Für gemeinsamen ESI/MALDI-

Betrieb sowie Ion-Molekül-Reaktionen wurden im auf 3 mbar evakuierten Ionenquellengehäuse die Spannungen

der Ablenkplatte und des Transferkapillarendes angepasst. Auf der ESI-Seite wurde Methanol:Wasser=1:2 mit

0.1% Ameisensäure bei 3 µlmin−1, 4,8 kV, Sprühgasfluss 1,0 lmin−1, Desolvationsgasfluss 2,0 lmin−1 bei

200°C gesprüht. Für die Silberadduktbildung wurde Silbernitratlösung verwendet. Zur Laserdesorption wurdendie Proben mit einem Nd:YAG Laser (355nm) mit je 15 Schuss pro Transient knapp an der Schwelle zur Ionisationdesorbiert. Es wurden positiv-Ionen Breitbandspektren aus 32 Transienten mit 1M Datenpunkten aufgenommen.Ergebnisse und Diskussion: Für die Experimente wurden 1-Aza[6]-helicen, 1,8-Dihydroxy-9-anthrone (Dithra-

nol) und 2-Mercaptobenzothiazol (MBT) aus ihren Lösungen auf ein MALDI-Target aufgetragen und laserdesor-biert. Die Spektren unter LDI-Bedingungen wurden dann mit denen verglichen, die unter gleichzeitiger Zufuhr vonReaktandionen über das ESI-Interface erhalten wurden. Dabei zeigte sich klar, dass Ion-Molekül-Reaktionen in der

Feinvakuumzone des Ion Funnels stattfinden. 1-Aza[6]-helicen und Dithranol ließen sich in der Gasphase proto-

nieren. Die Produkte wurden anhand ihrer exakten Massen identifiziert. Da die Intensitätszunahme der [M+H]+

Ionen insbesondere bei 1-Aza[6]-helicen trotz dessen hoher Protonenaffinität von 1000 kJmol−1 [5] nur einen Fak-tor 2,5 ausmachte, wurde auch deuteriertes Lösungsmittelgemisch gesprüht. Daraus resultierten [M+D]+ Ionen,

deren Auftreten die Laserdesorption/Ionisation als alleinige Quelle dieser Spezies ausschloss. Um die Überlage-rung des [M+D]+ Ions mit dem isobaren [13C-M+H]+ Ion aufzuheben (∆m=0,003u), wurden auch Messungen beieinem Auflösungsvermögen von ca. 230.000 durchgeführt. Damit konnten alle beteiligten Ionen getrennt und überihre exakte Masse charakterisiert werden. Bei Dithranol stieg die Intensität des [M+H]+ Ions um den Faktor 35ein, sobald die ESI-Spannnung eingeschaltet wurde. Auch hier wurde zusätzlich das Experiment mit deuteriertemLösungsmittel bei sehr hoher Auflösung durchgeführt, um die Interpretation abzusichern. 2-Mercaptobenzothiazolschließlich zeigte deutliche Adduktbildung mit Silberionen aus elektrogesprühter Silbernitratlösung. Damit ist ge-zeigt, das Ion-Molekül-Reaktionen, die letztlich eine Nachionisation laserdesorbierter Neutralteilchen bewirken,auch im Feinvakuum der Dual Source MTP grundsätzlich herbeigeführt werden können.Neue Aspekte: Erste Durchführung von Reaktionen elektrogesprühter Ionen mit laserdesorbierten Molekülen inder Feinvakuumzone einer Dual Source MTP am Bruker ApexQe FT-ICR-Massenspektrometer.Referenzen: [1] A. G. Harrison, Chemical Ionization Mass Spectrometry, 2nd ed. CRC Press, Boca Raton, 1992.;[2] W. Lindinger, A. Jordan, "Proton-Transfer-Reaction Mass Spectrometry (PTR-MS): Online Monitoring of Vo-latile Organic Compounds at Pptv Levels", Chemical Society Reviews 27, 347-354 (1998).; [3] G. Baykut, J.Fuchser, M. Witt, G. Weiss and C. Gosteli, Ä Combined Ion Source for Fast Switching Between Electrospray andMatrix-Assisted Laser Desorption/Ionization in Fourier Transform Ion Cyclotron Resonance Mass Spectrometry",Rapid Commun. Mass Spe; [4] V. Ganza, "Gasphasen-Reaktionen von laserdesorbierten Molekülen mit elektro-gesprühten Ionen", Bachelor-Arbeit, Universität Heidelberg, 2009.; [5] J. Roithová, D. Schröder, J. Míšek, I. G.Stará and I. Starý, "Chiral Superbases: the Proton Affinities of 1- and 2-Aza[6]Helicene in the Gas Phase", J. MassSpectrom. 42, 1233-1237 (2007).Thema: Organische Massenspektrometrie, Ionen-Molekül-ReaktionenKeywords: Ion-Molekülreaktion, Laserdesorption, Elektrospray, FT-ICR, Bruker Dual Source MTPKontakt: [email protected]

254

P158

Photodetachment-Photoelektronenspektroskopie (PD-PES) vonH2S2¯ und H2S2¯•(H2O)n (n = 1-3)

Boesl, Ulrich; Entfellner, Michaela

Technische Universität München, Germany

Einleitung: Nach neueren Simulationen entsteht H2S2 in größeren Mengen beim Claus-Prozess, einem industri-ellen Verfahren zur Gewinnung von Schwefel aus H2S. Deshalb wird neuerdings angenommen, dass H2S2 ammolekularen Wachstum des Schwefels in der Gasphase beteiligt ist [1]. Auch für die Thermolyse von H2S und dieSulfidierung von H2 wird ein Mechanismus vorgeschlagen, an dem maßgeblich diese Verbindung Anteil hat [2].Die H2S2•(H2O)n-Komplexe hingegen scheinen für die extreme Korrosion von Stahlpipelines bei der Förderungvon H2S-reichem Erdgas verantwortlich zu sein [3]. Hier wurden diese Verbindungen zum ersten Mal mittels PD-PES untersucht. Besondere Bedeutung kommt dem ersten angeregten Zustand, einem Triplettzustand des H2S2-Moleküls zu. Dieser wurde bisher nur durch theoretisch untersucht. Auch auf die Photochemie des H2S2-Anionsund der H2S2¯•(H2O)n-Komplexe wird eingegangen.Experimenteller Teil: Die Methode der Photodetachment-Photoelektronenspektroskopie (PD-PES) ermöglichtmassenselektive Spektroskopie - insbesondere die Bestimmung von Elektronenaffinitäten - an ausgewählten Mo-lekülen und Molekül-Komplexen. In schwingungsaufgelösten Spektren, lassen sich zusätzlich Schwingungen imneutralen Molekül sowie im Anion (hot bands) bestimmen und zuordnen. Bei der PD-PES steht am Anfang je-des Zyklus des gepulsten Experiments die Erzeugung von Anionen durch Anlagerung langsamer Elektronen inder Ionenquelle. Die Elektronenerzeugung erfolgt durch Laserbeschuss einer Metalloberfläche unter Ausnutzungdes Photoelektrischen Effekts. Im zweiten Schritt, dem Photodetachment, werden die zuvor angelagerten Elektro-nen mit einem festfrequenten (Detachment) Laser vom selektierten Molekül abgetrennt und in einem Elektronen-Flugzeitspektrometer nachgewiesen. Durch Variation der eingestrahlten Detachmentlaserwellenlänge ist es mög-lich, entweder kleinere Energiebereiche mit hoher Auflösung, oder große Bereiche mit geringerer Auflösung auf-zunehmenErgebnisse und Diskussion: H2S2

Hier konnten Photodetachment-Photoelektronenspektren des Grundzustands, sowie eines angeregten Zustands er-halten werden. Bei diesem angeregten Zustand kann es sich laut Berechnungen entweder um einen cis- oder trans-Triplettzustand handeln. Eine eindeutige Zuordnung ist hier nicht möglich, da sich die beiden Zustände wedergravierend in ihrer Struktur (bis auf den Diederwinkel), noch in ihrer energetischen Lage (der cis-Zustand liegtlaut einer Berechnung nur ca. 0,02 eV über dem trans-Zustand [4]) deutlich unterscheiden. Im Spektrum desH2S2-Grundzustands wird vermutlich die Torsionsschwingung beobachtet. Desweiteren wurde eine spektrosko-pische Struktur im Spektrum detektiert, die sich auf ein SH¯-Fragment zurückführen lässt. Vermutlich findet hierals Konkurrenzprozess zum Photodetachment eine Photodissoziation des H2S2-Anions in SH¯ und SH statt, mitnachfolgendem Photodetachment des SH-Anions. Aufgrund des großen strukturellen Unterschieds zwischen deranionischen und der neutralen Struktur des H2S2-Moleküls, lässt sich aus dem Spektrum nur die VDE zu ca.2,10 ± 0,02 eV ermitteln. Dieses Ergebnis wurde durch eine CCSD(T)-Rechnung bestätigt. H2S2•(H2O)n (n =

1-3) H2S2•(H2O): Hier wurden im Spektrum verschiedene Isomere beobachtet. Durch einen Vergleich der experi-mentell erhaltenen vertikalen Detachmentenergien mit denjenigen von berechneten isomeren Strukturen, konntendie verschiedenen Schwingungsprogressionen im Spektrum mindestens zwei Isomeren zugeordnet werden. Diesebeobachtete Feinstruktur lässt sich vermutlich auf Torsionsschwingungen des H2S2-Moleküls in den jeweiligenWasser-komplexen zurückführen. Genau wie im Spektrum des H2S2-Moleküls wurde auch in diesem Spektrumdas SH¯-Fragment detektiert.H2S2•(H2O)2 und H2S2•(H2O)3: Sowohl experimentell, als auch durch Rechnungen konnten hier mehrere Iso-mere dieser Komplexe nachgewiesen werden. In den zugehörigen Spektren wurden zusätzlich spektroskopischeStrukturen von Photodissoziationsprodukten (SH, H2S2•(H2O) bzw. H2S2•(H2O)2 und HS•(H2O)) detektiert. DerNachweis dieser Fragmente führt zu der Annahme, dass mehrere Zerfallskanäle bei der Photodissoziation vonH2S2¯•(H2O)n-Komplexen auftreten können. Für all diese Komplexe wurden ebenfalls die bisher unbekanntenverschiedenen isomeren Strukturen, die Ladungsverteilung in diesen Isomeren und ihre VDE‘s berechnetNeue Aspekte: Erstmalige spektroskopische Bestimmung des für chemische Reaktionen wichtigen Triplet-zutandesund der Wasser-Komplexe (n=1-3) von Disulfan.

255

Referenzen: [1] I. A. Gargurevich, Ind. Eng. Chem. Res., 2005, 44, 7706; [2] K. Sendt, M. Jazbec, B. S. Haynes,

Proc. Combust. Inst., 2002, 29, 2439; [3] R. Steudel, Top. Curr. Chem., 2003, 231, 99; [4] C. R. Zhou, K. Sendt,

B. S. Haynes, J. Phys. Chem. A, 2008, 112, 3239

Thema: Ionen-Molekül-Reaktionen

Keywords: Photoelektronenspektroskopie an Anionen, Disulfan, massenselektive Neutralen-Spektroskopie, H2S2-

Wasserkomplexe


256

P159

Effects of inherent receptor chirality on the recognition ofaminosugars

Letzel, Matthias; Fraschetti, Caterina; Paletta, Marlene; Mattay, Jochen


Einleitung: The remarkable enantioselectivity exhibited by most protein macromolecules is due to shape- and

size-specific non-covalent interactions with their chiral targets. The complexity of intrinsic factors governing

biomolecular recognition requires the synthesis of versatile pseudo receptors mimicking the complexity of the

enzymatic active sites. Resorc[4]arenes are able to form stable complexes with aminoacids and their derivatives,

discriminating their enantiomers if chiral sites are on the lateral chains.[1,2] The measured enantioselectivity of

inherently chiral resorc[4]arenes with quaternary amines carried out previously[3,4] unambiguously points to an

involvement of the ring frame in the coordination of these guests close to the chiral sites.

Experimenteller Teil: In the present study the recognition of three different dextrorotatory amino sugars by the

inherenty chiral rccc-2,8,14,20-tetra-n-decyl-4,10,16,22-tetra-O-methylresorc[4]arene was investigated by measur-

ing the rate constants of ligand exchange reactions with achiral and chiral amines. Following the Sawada method,

the solutions of equimolar pseudoenantiomer hosts (with the remote -CX3 of the decyl side chain x=H,D) were

prepared in the presence of the chiral guest and analyzed immediately after the preparation and after 24 hours. The

1:1 diastereomeric proton-bound complexes formed in the nano-ESI device were isolated and allowed to react with

the neutral gas inside the cell of the FT-ICR mass spectrometer.

Ergebnisse und Diskussion: The D-Mannosamine or D-Galactosamine complexes undergo a displacement re-

action by exhibiting a biexponential decay, thus suggesting the existence of at least two reactive regioisomers.

Furthermore the strong solution-aging effect found in the measured enantioselectivity points to a correlation be-

tween the gaseous reactivity and the solution thermodynamics. As a matter of fact the well known anomeric

equilibria in solution determine the relative distribution of the α- and β-anomers with time and are related to the

change in the kinetic effects observed in the ligand exchange reaction rate. On the contrary the guest displacement

reactions involving the D-Glucosamine complexes follows a pseudo first order decay and do not exhibits any time

dependence.

Neue Aspekte: Complexes of aminosugars with inherently chiral resorc[4]arenes are inventigated for the first time

by means of a FTICR ion-molecule-kinetics.

Referenzen: [1] B. Botta , G. Delle Monache, C. Fraschetti, L. Nevola, D. Subissati, M. Speranza, Int. J. Mass

Spectrom. 267, 2007, 24.; [2] B. Botta, I. D’Acquarica, L. Nevola, F. Sacco, Z.B. Lopez, G. Zappia, C. Fraschetti,

M. Speranza, A. Tafi, F. Caporuscio, M.C. Letzel, J. Mattay, Eur. J. Org. Chem. 2007, 5995.; [3] A. Mehdizadeh,

M. C. Letzel, M. Klaes, C. Agena, and J. Mattay, Eur. J. Mass Spectrom. 2004, 10, 649.; [4] B. R. Buckley, P. C.

Bulman Page, Y. Chan, H. Heaney, M. Klaes, M. J. McIldowie, V. McKee, J. Mattay, M. Mocerino, E. Moreno, B.

W. Skelton, and A. H. White, Eur. J. Org. Chem. 2006, 5135.


Keywords: FTICR, Kinetic, rate constants, chiral recognition


257

P160

Do Simple N-Benzyl-alkylpyridinium Ions Fragment viaIon/Neutral Complexes?

Kuck, Dietmar; Barth, Dieter; Heitkamp, Sandra; Letzel, Matthias C.; Grützmacher, Hans-Friedrich


Einleitung: Gaseous N-benzylpyridinium ions are prominent as „thermometer ions" to estimate internal energydistributions in soft ionisation techniques (1,2). However, similar to benzylammonium ions in general, benzylpyri-dinium ions present interesting precursors for the study of gaseous ion/neutral complexes of the sort [C7H

+7 ···

amine]. The intra-complex chemical reactivity of such non-covalently bound species is governed by the fact thatthe benzyl cation reacts as a Lewis acid (but not as a Bronsted acid), and from examples in the literature it is evi-dent that hydride transfer reactions (3) indicate the intermediacy of such I/N complexes (4,5). In order to get moreinsight in this model, we have focussed on simple N-benzylpyridinium ions bearing potential hydride-donatinggroups at the heterocyclic moiety of the ion.Experimenteller Teil: A series of N-benzylpyridinium bromides bearing various alkyl substituents [CH3, C2H5,CH(CH3)2 and CH2C6H5] at the positions C-2, C-3 or C-4 of the heterocyclic nucleus were synthesised. Inaddition, site-specific deuterium labelling was performed in the latter case. ESI(+)/CID mass spectra of thepyridinium cations were measured by use of an Esquire 3000 ion trap ("ion cage") mass spectrometer equippedwith a standard nanoESI source. Thermochemical calculations on the dissociation and on the hydride transferprocesses were carried out on the B3LYP/6-311+G/3d,2p)//B3LYP/6-31+G(d) level of theory.Ergebnisse und Diskussion: The salts derived from the three N-benzylpicolines were studied first. However,neither of the N-benzyl-methylpyridinium ions were found to undergo any fragmentation other than the simpleheterolytic C-N bond cleavage. i.e., the formation of C7H

+7 ions. The same holds true for the corresponding ethyl

homologues: Loss of the ethylpyridine was observed as the sole reaction channel of the N-benzyl-ethylpyridiniumions. However, the corresponding N-benzyl-isopropylpyridinium ions react differently: Elimination of toluenerepresents an additional, albeit less prominent fragmentation of the ortho-isomer, whereas this reaction is againalmost absent with the other two isomers. Similarly, among the three N-benzyl-benzylpyridinium ions, only theortho-isomer undergoes significant loss of toluene. The experimental effects of the alkyl substituents on thefragmentation reactions were found to be mirrored by the structures and the thermochemistry obtained by thetheoretical calculations. However, the observed ortho-specificity was unexpected and points to the intermediacyof I/N complexes that cannot rotate freely prior to fragmentation. Rather, it is assumed that the benzyl cationreleased is trapped within the vicinity of the nitrogen centre, which would prevent the abstraction of a hydridefrom a remote position. This is corroborated by the finding that loss of benzene occurs exclusively from the N-benzyl-(2-benzylpyridinium) ion but not from its isomers. The deuterium labelling experiments shed additionallight on the mechanism of this unexpected competing fragmentation channel.Neue Aspekte: The unimolecular formation of ion/neutral complexes consisting of a benzyl cation and an alkyl-substituted pyridine has been probed.Referenzen: [1] F. Derwa, E. de Pauw, P. Natalis, Org. Mass Spectrom. 1991, 26, 117.; [2] K. V. Barylyuk, K.Chingin, R. M. Balabin, R. Zenobi, J. Am. Soc. Mass Spectrom. 2010, 21, 172.; [3] C. Matthias, D. Kuck, Croat.Chem. Acta 2009, 82, 7, and references cited therein.; [4] H. E. Audier, F. Dahhani, A. Milliet, D. Kuck, J. Chem.Soc. Chem. Commun. 1997, 429.; [5] J. Bialecki, J. Ruzicka, A. B. Attygalle, J. Mass Spectrom. 2006, 41, 1195.Thema: Organische Massenspektrometrie, Ionen-Molekül-Reaktionen

Keywords: Ion/neutral complexes, benzylpyridinium ions, hydride transfer, rearrangement, benzyl cation


258

P161

Quantifizierung von antikörperangereicherten in vivoPlatin-DNA-Addukten per HPLC/ICP-MS und CE/ICP-MS

Ziehe, Matthias (1); Hochkirch, Ulrike (1); Thomale, Jürgen (2); Linscheid, Michael W. (1)

1: Humboldt-Universität zu Berlin, Germany; 2: Universitätsklinikum Essen, Germany

Einleitung: In der Chemotherapie spielen platinhaltige Pharmazeutika immer noch eine Hauptrolle bei der Bekämp-fung von Krebs. Trotz ihres teils jahrzehntelangen Einsatzes sind Cisplatin und seine Analoga immer noch Gegen-stand intensiver Untersuchungen, da die genauen molekularen Mechanismen ihrer Wirkungsweise immer nochweitestgehend ungeklärt sind. Zum Nachweis der Platinaddukte sowohl von Proteinen als auch von DNA in in

vivo Proben hat sich in letzter Zeit die ICP-MS als Methode der Wahl herausgestellt.[1] Die Nachweisgrenzen imunteren fmol-Bereich bei einem Platinierungsgrad von bis zu 108-1010 Nukleotiden pro Addukt wurden bisher nurvon Methoden wie dem 32P-Labeling erreicht. Um die in vivo Addukte zur Charakterisierung auch einer massen-spektrometrischen Analyse zugänglich zu machen, wird eine Anreicherung über monoklonale Antikörper (MAK)entwickelt.Experimenteller Teil: Zur Identifikation und Quantifizierung der DNA-Addukte wird zur MethodenetablierungKalbsthymus-DNA verwendet. Die DNA wird nach Inkubation enzymatisch durch Benzonase, Alkaline Phos-phatase und Nuclease S1 auf Dinukleotid-Level verdaut und mittels HPLC/ICP-MS und CE/ICP-MS analysiertund quantifiziert. Die in vivo Proben stammen von mit Cisplatin behandelteten Ratten. Die isolierte DNA wirddurch Ultraschall in Fragmente mit einer Größe von ca. 500 Basenpaaren fragmentiert. Die Anreicherung derAdduktspezies erfolgt durch einen MAK.[2] Nach enzymatischem Verdau erfolgt die Analyse der Addukte analogder Kalbsthymus-DNA.Ergebnisse und Diskussion: Nach erfolgreicher Charakterisierung des Cisplatin-DNA-Adduktsprektrums mitHilfe der HPLC/ESI-MS/MS erfolgte eine Übertragung der Methoden auf in vivo Proben von mit Cisplatin be-handelten Ratten. Erste Ergebnisse zeigen, dass die in vivo Adduktverteilung vergleichbar ist mit der des in vitroKalbsthymus-DNA Systems. Die Anreicherung der in vivo Adduktspezies erfogte dabei über einen monoklonalenAntikörper. Dieser zeigte bei Anwendung in Cryoschnitten des Innenohrs von mit Cisplatin behandelten Mäuseneine spezifische Anreicherung des Pt-GG Adduktes in den äußeren Haarzellen und Marginalzellen. Dies kann alsentscheidender Beweis für eine der Hauptnebenwirkungen des Cisplatins - der Ototoxizität - gedeutet werden. EineCharakterisierung der in vivo DNA-Adduktspezies ist wegen des geringen Platinierungsgrades nur mit Hilfe derAnreicherung über denMAK zu erzielen. Erste Versuche mit Kalbsthymus-DNA ergaben für das Pt-GG intrastrangAddukt eine nahezu vollständige Anreicherung auf dem MAK. Somit sollte die Untersuchung der Sequenzspezi-fität des in vivo Adduktes auch über ESI-MS/MSMessungen möglich sein. Für die Quantifizierung werden sowohlHPLC/ICP-MS als auch CE/ICP-MS genutzt, um die äußerst komplexen Produktgemische aufzutrennen.Neue Aspekte: Selektive antikörperangreicherte Quantifizierung des Pt-GG intrastrang crosslink-Adduktes in invivo Proben mittels HPLC/ICP-MS und CE/ICP-MS.Referenzen: [1] Esteban-Fernández, D. et al. Metallomics, accepted.; [2] Liedert, B.; Pluim, D.; Schellens, J.;

Thomale, J. Nucleic Acids Research 2006, 34, e47.

Thema: Element-Massenspektrometrie

Keywords: Quantifizierung, ICP, monoklonale Antikörper, Cisplatin, DNAKontakt: [email protected]

259

P162

Determination of Ochratoxins in Wine by an ICP-MS-basedEnzyme-Linked Immunosorbent Assay

Giesen, Charlotte; Weller, Michael G.; Jakubowski, Norbert; Panne, Ulrich

BAM Federal Institute for Materials Research and Testing, Germany

Einleitung: We report the use of inductively coupled plasma mass spectrometry (ICP-MS) for the quantitative

determination of ochratoxins in wine. Ochratoxins are mycotoxins naturally occurring in different types of foods

and beverages [1] with Ochratoxin A (OTA) as the most prevalent and potentially carcinogenic species. ICP-

MS is a powerful tool for trace element analysis. Recently, it has been applied for bio molecule detection by

chemically labelling these bio molecules with elemental tags [2]. Enzyme-linked immunosorbent assay (ELISA)

is well established to detect antigens and small molecules in various biological matrices with only minimal need

for sample clean-up. It is employed for many applications such as HIV testing [3], vaccine studies [4], or the

quantification of caffeine in surface waters [5].

Experimenteller Teil: ICP-MS measurements were performed on a sector field instrument (Element 2, Thermo

Fisher Scientific, Bremen, Germany). A Spectramax 384 plus (Molecular Devices, Sunnyvale / USA) was used

for absorption spectrometry. The assay format was based on an indirect ELISA using well-plates coated with an

OTA-BSA conjugate. OTA standard solutions and wine samples were incubated with mouse monoclonal anti OTA

antibody. A second incubation step followed for 48 wells with gold labelled anti-mouse IgG and with anti-mouse

IgG Peroxidase, respectively. Detection was conducted by means of absorption spectrometry. After digestion with

aqua regia and addition of 1 µg/L Ir as an internal standard, samples were subjected to ICP-MS measurements.

Ergebnisse und Diskussion: For quantification and detection of antigens in biological samples, ELISA combined

with absorption spectroscopy has been the method of choice. However, ELISA suffers from several drawbacks such

as a low linear dynamic range and poor precision. The method developed in this work combines the advantages of

an ELISA with the sensitivity and precision of ICP-MS by means of element tagging. ICP-MS measurements were

more precise than the results obtained by absorption spectrometry. By measuring gold as the element used to tag

the antibody and iridium as an internal standard, a very precise standard curve for OTA was determined. The assay

sensitivity was 0.036 ± 0.002 µg/L OTA in water. The ICP-MS-based ELISA was then evaluated in wine matrix

and similar results to the standard curve in water were obtained. As for the wine samples, the precision achieved

by ICP-MS was superior to a measurement by absorption spectrometry. We designed an ICP-MS-based ELISA for

OTA quantification in wine. The described detection method is very sensitive, precise and in these terms superior

to the classical absorption spectrometry. For the future, it is desirable to exploit the multi element capacity of

ICP-MS and to perform an assay with several analytes in one sample by tagging the respective antibodies with

different elements.

Neue Aspekte: We combined both the advantages of ICP-MS and ELISA for OTA detection.

Referenzen: [1] H. A. Clark and S. M. Snedeker, Journal of Toxicology and Environmental Health-Part B-Critical

Reviews, 2006, 9, 265-296.; [2] A. Prange and D. Proefrock, Journal of Analytical Atomic Spectrometry, 2008,

23, 432-459.; [3] J. Schneider et al., Medical Microbiology and Immunology, 1987, 176, 47-51.; [4] C. P. Quinn

et al., Emerging Infectious Diseases, 2002, 8, 1103-1110.; [5] J. J. Carvalho et al., Analytical and Bioanalytical

Chemistry, in revision.


Keywords: ICP-MS, ELISA, OTA, immunoassay, gold label


260

P163

Application of RISQ proteins for quantitative proteomics

Tittebrandt, Sebastian

DKFZ, Germany

Einleitung: Quantification of proteins is a fast growing field in applied proteomic research. Relative quantification

is fast and gives evidence about the regulation of the protein of interest. Absolute quantification, in contrast, offers

a conclusion about the total amount of proteins of interest within a sample. Additionally it provides comparabil-

ity independent of applied quantification methods. Approaches using stable isotope labeled peptide standards are

widely known but there are only few methods for production of quantified and isotope labeled proteins. Here a

novel method, using recombinant isotope labeled selenium quantified (RISQ) proteins, is applied to the quantifi-

cation of MAPK11 which plays an important role in cancer related signaling pathways.

Experimenteller Teil: The production of the RISQ protein consists of three steps. (i) The target protein (MAPK11)

DNA obtained from a clone repository is transferred into an expression vector using the gateway cloning technol-

ogy in a one step procedure. (ii) The generated vector is used as a template in a cell-free protein expression system.

During the expression of the His-tagged protein both [13C6,15N4]-arginine and [13C6]-lysine are introduced, se-

lenomethionine instead of methionine is incorporated and the protein is purified by cobalt(II) immobilized metal

ion affinity chromatography. (iii) Protein quantification is performed via element mass spectrometry and selenium

detection. The MAPK11 RISQ protein can be used as reference in downstream absolute quantitative proteomics.

Ergebnisse und Diskussion: MAPK11 is synthesized using cell-free expression with stable isotope labeled amino

acids and selenomethionine incorporation. The synthesis and purification procedure suited for high throughput

protein production is described. The synthesized MAPK11 is characterized towards its identity and completeness

of the labeling. The absolute quantification by ICP-MS of MAPK11 is compared with corresponding data obtained

from a quantitative microspot immunoassay.

Neue Aspekte: Preparation of stable isotope labeled protein suited for absolute quantitative proteomics.


Keywords: standardprotein, quantitative proteomics, stable isotope.


261

P164

Screening of selenium in different organs of African catfisch(Clarias gariepinus) by LA-ICP-MS

Feldmann, Ingo (1); Pedrero, Zoyne (2); Madrid, Yolanda (2); Schram, E. (3); Luten, J.B. (4);

Jakubowski, Norbert (1); Camara, Carmen (3)

1: Leibniz-Institut für Analytische Wissenschaften - ISAS, Dortmund, Germany; 2: Complutense University of

Madrid, Ciudad Universitaria s/n, 2804 Madrid, Spain; 3: Wageningen IMARES, PO box 68, 1970 AB Ijmuiden,

The Netherlands; 4: Fiskeriforskning, PO box 6122, N-9291 Tromso, Norway

Einleitung: Speciation of Se in fish could contribute to elucidate the metabolism of this element in the animals.

The previously developed methodology [1] was applied to measure the selenium distribution in several organs

and tissues (liver, gills, kidney and gastrointestinal tract) of African catfish fed with selenized garlic based diet.

Screening of selenium in proteins in the Tris-buffer soluble fraction of different tissues was carried out by laser

ablation-ICP-MS after SDS-PAGE and electroblotting onto membranes. Cu and Zn were detected in a parallel

measurement to obtain additional information. The method enables the localization of Se containing proteins

bands on a gel, so that the specific band could be cut out, digested and the proteins identified by means of ESI-MS.

Experimenteller Teil: The lyophilized tissue was mixed for 30 s in 50mM Tris-HCl buffer at pH 7.5 with a

dispersing device. The extracts were centrifuged at 15000 g and 4°C for 40 min. A Flatbed Electrophoresis System

was used for electrophoretic separation. The gel was divided into two parts. One part was stained with Coomassie

Brilliant Blue, for visualization of proteins bands. A second part was transferred onto a nitrocellulose membrane

by electro blotting processes. A double focusing sector field ICP-MS instrument was used for determination of Se,

Cu and Zn by coupling to an LA system. For laser ablation on the membrane, a Nd:YAG laser was utilized.

Ergebnisse und Diskussion: While we observed not any difference in the protein pattern on the stained gel the

Se distribution showed an increasing intensity in some of the protein bands of the Se treated animals. This holds

true for all organs with the only exception of liver. Nevertheless this organ shows the highest Se intensity even

for the control group. The general increase of the Se intensity in some of the bands shows that selenium coming

from a supplemented diet is at least partially incorporated to proteins. The organ with the highest Se accumulation

according to the Se supplementation was kidney. Since the applied Tris buffer extraction mobilize only a part of

the proteins the results reflect not fully the total change of Se content in the tissues, but the general trend is similar.

Two other essential trace elements - zinc and copper - were also measured in kidney of both groups of animals. The

resulting metal distribution was quite similar for both groups. However, the most remarkable difference between

both groups was the increase of Se in 4-5 positions. Since one of the protein bands at about 80 kDa correlates with

signals of all three elements, the allocation of the other metal containing protein bands on the gel is not easy and

needs more effort. In previous work we tried to identify the belonging selenoproteins in fillet sample. However,

the very low Se concentration compared to sulphur circumvented the detection of Se-containing peptides by ESI-

MS/MS. Since the Se concentration in kidney and liver is much higher compared to fillet, it looks promising to

realize their identification in these organs.

Neue Aspekte: For the first time we detected Protein bound selenium in four different organs and tissues of African

catfish.

Referenzen: [1] Pedrero, Z.; Madrid, Y.; Camara, C.; Schram, E.; Luten, J.; Feldmann, I.; Waentig, L.; Hayen,

H.; Jakubowski, N.: J. Anal. Atom. Spectrom. 24 (2009) 775-784


Keywords: Selenium speciation, metalloproteins, LA-ICP-MS


262

P165

Trace element profiles in a multicrystalline silicon ingot

Meyer, Sylke; Hagendorf, Christian

Fraunhofer Gesellschaft, Germany

Einleitung: The level of impurities is one of the key factors for cost control and yield optimization in photovoltaic

industry. Prevalent contaminants in solar silicon are metallic impurities which can severely degrade the perfor-

mance of the final devices either by forming metal silicides and/or by acting as recombination centres. ICP-MS is

currently the most powerful and useful trace analysis tool capable of quantitatively measuring the total elemental

impurity concentrations in PV Si down to ppbw level. Coupling of ICP-MS with laser ablation can be used for

direct sampling of solid silicon. The purpose of this work is to study the concentration profiles of some common

metallic impurities and of the dopants along the length of a solidified multicrystalline silicon ingot.

Experimenteller Teil: The multicrystalline ingot analyzed was a standard ingot from a commercial process for

solar cell production. Crystallization occurred from bottom to top, resulting in a vertical, columnar grain structure.

A slice of 4 mm thickness sawn vertically from the centre of the ingot was taken for analysis by laser ablation-

ICP-MS. The slice was divided into 14 smaller parts fitting into the sample cell. Laserablation was performed with

a Nd:YAG laser (wavelength 213 nm) using the following conditions: Energy 100%, pulse rate 20 Hz, spot size

200 µm, scan speed 100 µm/s, scan modus single line, He flow 0.7 L/min. Simultaneous analysis of the ablated

material was done with a high resolution sector field ICP-MS using the mean resolution modus.

Ergebnisse und Diskussion: The profiles of several metals as well as B and P along the vertical axis of the Si-

ingot are shown. The total concentrations of P, B, Fe, Co and Cu along the length of the Si-ingot appear to be

determined by segregation from the liquid to the solid phase in the central region of the ingot. Near the bottom

the concentrations are higher due to diffusion in the solid state from the crucible. Near the top the segregated

impurities are enriched in the ingot during solidification. These results confirm similar measurements done with

Neutron Activation Analysis (NAA) and prove the power of high resolution ICP-MS with laser ablation for trace

element analysis.

Neue Aspekte: The coupling of laserablation with high-resolution ICP-MS is shown to be a suitable tool to

characterize silicon materials for PV.


Keywords: solar silicon, trace elements


263

P166

Characterization of surface contaminations on solar silicon wafersusing ICP-MS and ToF-SIMS

Richter, Susanne; Meyer, Sylke; Hagendorf, Christian

Fraunhofer-Center für Silizium-Photovoltaik, Germany

Einleitung: For the chemical analysis of contaminants on the surface of Si-wafers, treatment with HF vapour,

called vapour phase decomposition (VPD), has been widely used in semiconductor industry. Ultra-pure HF permits

an improved method using a direct HF droplet decomposition (HF-DD) without requirement of HF vapour. We

show here that a combination of HF-DD with high resolution ICP-MS is an excellent tool for analyzing surface

metallic contaminations. This analysis can provide valuable information on type, source and levels of inorganic

contaminations. In addition, ToF-SIMS measurements were performed on the same wafer surfaces to evaluate the

data and to check for organic contaminations. ToF-SIMS is a suitable analysis method for surface contaminations

including organic molecules due to the available high mass range.

Experimenteller Teil: Direct HF drop decomposition (HF-DD) is a technique to collect contaminants on the

surface of a Si wafer. The contaminants and the native oxide layer are dissolved and concentrated in one step

by placing a drop of 500 µl HF/HNO3/H2O on the wafer and rolling it around the entire surface several times.

This was done twice; the drops were combined and analyzed with high resolution ICP-MS equipped with a HF

compatible nebulizer and spray chamber. For the ToF-SIMS analysis each time a 300x300 µm2 target area was

chosen. To detect organic components, alkali metals and metals the measurements were performed in the positive

ion mode with 150 scans/spectrum using a pulsed 25keV Bi sputter source.

Ergebnisse und Diskussion: Trace analyses of 18 elements were conducted for wafers from several steps of

industrial process. Detection limits varied between 5E06 (Ag) and 1E10 (Fe) atoms per cm2 depending on different

background levels of individual elements. By testing wafers from different steps of industrial process, severe

differences in the quality of wafer cleaning bathes were recovered. The evaluation of the integrated ToF-SIMS

spectra yields characteristic fingerprints due to organic contaminations, which however did not change along

the cleaning process. The measured intensities of some alkali metals and metals for the different Si wafers are

shown by comparison. The results from ICP-MS analysis and ToF-SIMS are in good agreement for most of the

investigated elements. Some elements like Ti or Zn were below the ToF-SIMS detection limit.

Neue Aspekte: ICP-MS and ToF-SIMS for surface analysis on silicon wafers is shown to complete each other due

to their specific advantages.


Keywords: Spurenanalyse, ICP-MS, ToF-SIMS, Oberflächenkontaminationen, Si-WaferKontakt: [email protected]

264

P167

Entwicklung und Validierung einerIsotopenverdünnungs-Referenzmethode für freies Cortisol im

Serum basierend auf Equilibrium Dialyse mit on-lineFestphasenextraction und LC-MS/MS.

Kirchhoff, Fabian; Vogeser, Michael

Klinikum der Universität München-Großhadern, Institut für Klinische Chemie, Germany

Einleitung: Nur freies, nicht Protein-gebundenes Cortisol ist im Körper biologisch wirksam; dennoch wird inder labormedizinischen Analytik bislang das gesamte Cortisol quantifiziert. Insbesondere bei kritisch krankenPatienten unterscheiden sich die Bindungsverhältnisse von Cortisol drastisch vomNormalzustand und die Messungdes freies Cortisols ist von überlegener Aussagekraft. Unser Ziel war die Entwicklung einer gut praktikablenReferenzmethode zur Messung von freiem Serum-Cortisol.Experimenteller Teil: Freies Cortisol konnte mittels Equilibrium Dialyse (Verhältnis Probe zu Puffer 1:1, MW-CO ∼ 8000 Da, 16h, 37°C) in einem proteinfreien Dialysatpuffer aus Serum gewonnen werden. Die Bestim-

mung des Cortisols im Dialysat erfolgte mit on-line Festphasenextraktion gekoppelt mit HPLC und Tandem-

Massenspektrometrie. Quantifiziert wurde im positiven Modus mit Multiple Reaction Monitoring ( Übergang363>121 ).Ergebnisse und Diskussion: In bisherigen Experimenten erwies sich die Methode als sehr gut reproduzierbar.Linearität (R>0.99) konnte im relevanten Konzentrationsbereich von 1 µg/L bis 50 µg/L aufgezeigt werden. Das

S/N Verhältnis des kleinsten Standards (0.8 µg/L) lag bei 13:1. Cortisol konnte mittels LC-MS/MS von möglichenisobaren Molekülen abgetrennt werden.

Neue Aspekte: Auf Grundlage der Equilibrium Dialyse und der LC-MS/MS konnte eine Referenzmethode zur

Messung von freiem Serum-Cortisol entwickelt und validiert werden.

Referenzen: [1] Briegel J, Vogeser M, Keh D, Marik P. Kortikosteroidinsuffizienz bei kritisch Kranken. Patho-

mechanismen und Empfehlungen zur Diagnose und Behandlung. Anaesthesist 2009;58:122-133; [2] Yue B, Rock-

wood AL, Sandrock T, La´ulu SL, Kushnir MM,Meikle AW. Free Thyroid Hormones in Serum by Direct Equilibri-

um Dialysis and Online Solid-Phase Extraction-Liquid Chromatography/Tandem Mass Spectrometry. Clin Chem

2008;54:642-51

Thema: Isotopen-Massenspektrometrie

Keywords: Freies SerumCortisol, Hochleistungsflüsssigkeitschromatographie-Tandem-Massenspektrometrie (LC-

MS/MS), Equilibrium Dialyse, On-line Festphasenextraktion


265

Keyword-Index

266

Index

Aβ-antibodies, 128

Aß-antibodies, 153

Absolute quantitation, 98

Acetylcholin, 244

Acylcarnitines, 182

addition reaction and mass spectrometry., 240

ADP-ribosyltransferase, 92

Aerosolanalytik, 251

Affinity, 130

Affinity Chromatography, 91

affinity chromatography, 133

affinity enrichment, 37

affinity mass spectrometry, 124, 237

affinity proteomics, 112

Affinity-MS, 128, 157

aggregates, 140

akute klinische Toxikologie, 74

Alkyliodide, 246

alpha Synuclein, 127

Amino Acids, 182

amino resin, 239

amyloid β (Aβ), 124amyloid beta, 128

AngiotensinII, 152

Anionen, 213

Anregungsenergie, 63

anti-Aβ antibodies, 124

antibiotics, 219

Antibiotika, 135

antibody profiling, 133

APCI, 177

AQUA, 151

Arabidopsis, 155, 186

arachidonic acid metabolites, 193

arginine catalysis, 119

arginine methylation, 121

Aromaten, 66

Arzneimittelfälschungen, 248

Arzneistoffe, 244

ATM, 137

atmospheric pressure ionization, 216

Atrazin, 238

Authenticity, 214

automated SPE, 160

axialization, 207

Azadinium spinosum, 167

Bacillus subtilis, 135

Background Reduction, 91

bacterial toxins, 92, 109

barley, 61, 99

benzyl cation, 258

benzyl phenyl ether, 171

benzylpyridinium ions, 258

Betäubungsmittel, 54

Biglycan, 202

bioactivity, 181

Bioaffinity, 153

biofluids, 177

Biofuel, 45

Bioinformatics, 103, 106, 111, 175, 192

biomarker, 224

Biomassepyrolyse, 66

bottom-up, 97

Brassica napus, 184

Breast Cancer, 137

Breast cancer, 224

breast carcinoma, 62

Bruker Dual Source MTP, 254

C - H activation, 226

Calmodulin, 154

calmodulin, 48

carbenes, 240

Catechin derivatives, 171

CDR peptide, 128

CE/MS, 241

cell wall phenolics, 185

charge reduction, 156

Chemical Cross-Linking, 158

Chemical Cross-linking, 154

Chemical cross-linking, 119

chemical proteomics, 37

chiral recognition, 257

Chirale Moleküle, 206

Chlamydomonas, 126

Chondroitin/dermatan sulfate, 202

Chondroitinase ABC, 143

Chondroitinsulfat, 143, 198

Chromatography, 120

CID, 68, 212, 227

Circulardichroismus, 206

Cisplatin, 178, 259

cleavage site specificities, 117

click chemistry, 239

clinical applications, 70

Clinical Metabolomics, 182

Clinical Study, 175

Clostridiopeptidase A, 143

Clostridium difficile, 95

Clostridum difficile, 109

cluster compounds, 226

Clusters, 22

COFRADIC, 106

coherent ion motion, 204

collision induced dissociation, 156

collision-induced dissociation, 202

colorectal carcinoma, 133

Combinatorial libraries, 41

Combustion, 45

267

Complex Mixture, 172

composition-based sequencing, 40

conductive polymers, 236

constant neutral loss, 125

contrast agent, 77, 241

correlation analysis, 181

Corydalis, 173

CRD, 157

CREDEX-MS, 157

CRIP1, 224

Cross-Linker, 64

cross-linking, 48, 125, 159

Crossover reaction, 235

crude oil, 253

cyclization, 134

cytosolic expression, 129

data analysis, 47

data dependant acquisition, 194

DBnovo, 39

de-novo sequencing, 122

dealkylation tool, 179

degradation of xenobiotics, 101

density functional calculations, 226

dephosphorylation, 144

Derivatisierung, 73

DESI, 215

detection, 52

Deutsch, 80

DIGE, 95, 108, 135, 137

Dihydroxybenzoic acid, 233

direct analysis, 215

direct injection, 221

Disufidbruecken, 131

Disulfan, 256

disulfide bonds, 123

disulfide shuffling, 123

DNA, 259

DnaB split mini-intein, 134

Dorstenia gigas, 162

Drosophila melanogaster, 216

drug monitoring, 230

dynamic range, 106

ECD, 152, 203

Echtzeitdetektion, 66

edible oils, 168

Effect of Chromatography, 188

eicosanoids, 193

Einzelpartikelmassenspektrometrie, 251

Einzelphotonenionisation, 54

Elastase, 110

Elastasen, 139

Elastin, 139

Electrochemistry, 55

Electrospray, 76

Electrospray ionization., 46

Elektrospray, 254

elemental formula, 247

ELISA, 260

embryogenesis, 180

enantiomeric excess determination, 170

Endothelin, 174

Englisch, 80

ENL, 136

enrichment, 141

enzyme immobilization, 211

EPR, 140

Equilibrium Dialyse, 265

Erythropoietin-receptor, 116

ESI, 111, 174

ESI Imaging, 76

ESI mass spectrometry, 171

ESI-FTICR-MS, 234

ESI-LTQ-Orbitrap MS, 158

ESI-MS, 23, 130, 211, 227, 243

ESI-MS/MS, 126

ESI-TOF MS, 235, 236

essential oil, 160

Estradiol, 229

Estriol, 229

ETD, 146, 147, 212

ethane, 249

explosives, 52

extracellular, 180

Extraction, 76, 253

Fachbegriffe, 80

field-supported ionization, 216

Fingerabdruck, 44

flame, 250

flow reactor, 249

Flugzeimassenspektrometrie, 66

Fluorescein, 152

Food analysis, 161, 172

Forensik, 57

Fragmentation, 120

fragmentation, 114, 125

Fragmentierungsmechnismen/-wege, 228

Fragmentionen Chromatogramme, 131

Freies Serum Cortisol, 265

frequency shift, 204

frog peptides, 40

fruit fly, 216

FT-ICR ion detection, 204

FTICR-MS, 157, 203, 231, 253, 254, 257

FTMS, 161, 172, 247

Fully automated chip-based nanoelectrospray, 202

Fumaria, 173

furanocoumarins, 162

gadolinium, 241

gangliosides, 190

gas phase fractionation, 145

gas-phase reactions, 226

Gasphase, 84, 231

GC-MS, 42, 160, 173, 177

GCAP-2, 158

268

Gel Protein Recovery (GPR-800), 148

Genetics, 71

Geometric Structure, 22

Gerätesteuerung, 39Gewebeextraktion, 139Glimmentladung, 210GLP, 214Glucosinolate biosynthesis, 165glycopeptides, 141glycoprotein, 199glycosphingolipid, 187Glycyrrhizin, 135Glykane, 200gold label, 260Grubbs catalysts, 235GxxPG, 117

H/D Exchange Mass Spectrometry, 164H/D-Austausch, 231H2S2-Wasserkomplexe, 256half decimal place rule, 101HCD, 72HEK293T, 113HER2, 224high lateral resolution, 223high mass accuracy, 225High Resolution, 183high resolution UPLC/TOF MS, 179high sensitivity, 220high throughput screening, 170high-resolution mass spectrometry, 163high-throughput, 190HILIC, 122, 141, 178Hochauflösung, 147, 176, 209homology-driven proteomics, 102HPLC, 150HPLC/Chip - mass spectrometry, 199HPTLC, 219HtrA1, 94human γB crystallin, 134Human FFPE tissue, 98human neutrophil elastase, 117human serum, 187Humanmilcholigosaccharide (HMOS), 73Hyaluronsäure, 198hybrid triple quadrupole, 193hydride transfer, 258

ICP, 259ICP-MS, 260, 264ICPL, 93ICR MS, 207IgG, 122illegale Arzneimittel, 248illicit substances, 52Imaging, 82, 222Imatinib, 242imidazolium ions, 240immunoassay, 260

IMS, 209ImzML, 214in-gel, 96In-situ-MS, 45in-vitro-Diagnostik, 218Inorganic Compounds, 23intact protein analysis, 150intact protein mass, 148Intelligent Met ID workflow, 179interaction, 159inverse turn conformation, 121Iodinated X-Ray Contrast Media, 252ion funnel, 208Ion Mobility MS, 140ion mobility spectrometry, 156ion storage, 204, 207Ion trap, 146, 208Ion trap mass spectrometry, 202Ion-Molekülreaktion, 254

Ion/neutral complexes, 258

Ionencyclotronresonanz, 228

Ionenfalle, 74

Ionenfallenmassenspektrometer, 54

Ionenmobilität, 209, 212Ionenmobilitätsspektrometer, 54Ionenmobilitätsspektrometrie, 49, 213Ionenspektroskopie, 63, 246Ionic Liquid Matrix, 233ionic liquids, 240Ionisation, 84Ionisationsenergie, 63IR spectroscopy, 81IR-LDI-MS, 43IR-MALDI MS, 219IR-MPD, 22IRMPD, 194, 203ISD, 79isomer separation, 220Isopropylmalate isomerase, 165isoquinoline alkaloids, 173isotope dilution, 116

K63, 130kD, 153Kinetic, 257Klinische Chemie, 218Kofaktor, 234Kollagen, 143Kollisionsinduzierte Dissoziation (CID), 64KRAB, 91Kronenether, 231

LA-ICP-MS, 262label-free quantification, 62label-free quantitation, 112laboratory automation, 170Labormedizin, 218laminin, 123large surface ligands, 227

269

laser-induced cleavage and fragmentation, 41

Laserdesorption, 254

Lasermassenspektrometrie, 206

LC-ESI-MS/MS, 162

LC-ESI-TOF, 239

LC-MALDI, 111

LC-MS, 38, 105, 109, 242, 245

LC-MS/MS, 70, 194, 218

LC-MS/MS), 265

LC/ESI-MS, 77

LC/ICP-MS, 77

LC/MS2, 225

LDI, 251

Lebensmittel, 44

Lebensmittelsicherheit, 245

Leucine metabolism, 165

leukemia, 242

Ligan-target Non-covalent Interaction, 164

linear ion trap mass spectrometry, 193

linear trap-orbitrap, 107

Lipide, 191

Lipidomics, 71

liquid chromatography, 55

low abundant species, 98

low detection limit, 220

Low molecular weight proteins, 105

Low-temperature combustion, 249, 250

LTQ Orbitrap, 72

Lucky-Survivor, 84

Lysophospholipide, 69

macromolecular protein complexes, 47

macrophage elastase, 117

MALDI, 51, 110, 131, 159, 168, 174, 191, 200, 222,

233, 244

MALDI MS Imaging, 223, 225

MALDI tissue imaging, 224

MALDI-FTMS, 41

MALDI-imaging MS, 58, 61

MALDI-MS, 79, 119, 196, 211, 238

MALDI-TOF, 74, 141, 175, 198

MALDI-TOF MS, 69, 143, 148, 188, 189

MALDI-TOF/TOF, 68, 73, 158

mars, 42

MASCOT, 96

mass accuracy, 217

Mass defect Filter (MDF), 179

mass resolution, 146, 208

mass spectrometry, 46, 55, 62, 122, 133, 170

Massenauflösung, 82

Massengenauigkeit, 82

massenselektive Neutralen-Spektroskopie, 256

Matrices, 189

matrikines, 117

MBMS, 45, 250

mechanism, 81

Mechanistic study, 46

medizinische Diagnostik, 218

Melamin, 238, 244

membrane proteins, 106

MES, 51

Messgeschwindikeit, 147

metabolite profile, 181

metabolome, 163

Metabolomics, 43, 70, 183

Metal, 38

metal labeling, 114

metalloproteins, 262

metamorphic standard pairs, 144

Metastabile Ionen, 229

methane, 249

method development, 160

microbial consortia, 101

microdissection, 99

microscale emitter, 216

Mikroorganismen-Identifizierung, 74

Milchpulver, 238

miR-155, 113

mitochondrial dysfunction, 100

monoklonale Antikörper, 259

monolith, 211

Moraceae, 162

MRI, 241

MRM, 138, 230

MRM3, 252

ms data mining automation, 102

MS-Blast, 96

MS/MS, 191

MS3, 252

MSn, 146, 208

multifactorial analysis, 237

Multiphotonenionisation, 206

multiple reaction monitoring, 184

multiple reaction monitoring (MRM), 115

multiple reaction monitoring cubed (MRM3), 115

multiplexed LC-MS, 61, 99

multistage collision-induced dissociation, 190

multivariate Datenanalyse, 196

myxobacteria, 163

N-glycopeptides, 122

NADH, 234

NADPH, 234

Nano-ESI, 139

nano-HPLC, 187, 211

NanoMate, 190, 234

Naphthenic acids, 247

native Peptide, 222

natural products, 163

Neoepitope screening, 133

Neurodegeneration, 71

neuropeptides, 223

Neutralverlust (CNL), 64

NHS esters, 119

Nicotiana, 176

Non-alcoholic steatohepatitis, 100

270

North Sea, 167

novel hybrid mass spectrometer, 107

NSF, 241

nuclear proteome, 92

Nucleation, 23

O-glycosylation, 60

Oberflächenkontaminationen, 264Oligosaccharide, 73, 198, 203On-line Festphasenextraktion, 265Open Format, 214Optimierte Sequenzierung, 39optimized sample preparation, 105Orbitrap, 118, 145, 150, 183, 200, 217, 223Organocatalysis, 46Ortsauflösung, 82OTA, 260overcoming undersampling, 107oxidative metabolism, 55oxidative modifications, 145oxygenates, 42

P-TEFb Positive Transcription Elongation Factor b,136

PABPN1, 121pancreatic cancer, 187Pancreatic Carcinoma, 175parallel kinetic resolution, 170Pathogen-Plant interaction, 43PCA, 43, 168, 176PDE-5-Hemmer, 248Penning trap, 204PepNovo, 96Peptide, 64Peptide fragmentation, 40, 81peptide synthesis, 48peptide-protein interactions, 50Peptidomics, 174, 175Peptidsequenz, 79Pflanzenöle, 196pharmacokinetics, 215Phosphohistidine, 120Phospholipide, 69, 188, 189Phosphopeptide, 38, 138phosphorylation, 103, 138, 144Photoaffinity labeling, 48photocatalysis, 42Photodetachment, 213Photoelektronenspektroskopie an Anionen, 256Photoionisierung, 66phycotoxins, 167Phytochelatins, 126phytoplankton, 167PIPx, 194plasma proteins, 97Plazenta, 94PMF, 232Pollen, 186pollen, 180

poly(e-caprolactone), 68poly(styrene), 58Polyaminkonjugate, 186polymer films, 58polyoxazolines, 243Polyphenol flavonoids, 164Polyphenols, 172polythiophenes, 236postfunctionalization of polymers, 236Präformierung, 84Preanalytical Protocol, 182precursor ion scanning, 60preeclampsia, 237Protease, 51, 94, 174Protein, 49, 111, 118, 222protein and peptide identification, 217protein biomarkers, 115protein complexes, 156protein identification, 232protein kinases, 37Protein phosphorylation, 120protein sequencing, 149protein standards, 59protein-protein interactions, 155Protein-SIP, 101Proteinbiosynthese, 135Proteinkomplex, 49Proteinstruktur, 131Proteinstrukturaufklärung, 64proteome signature, 62, 237proteomics, 57, 60, 97, 100, 103, 104, 107, 108, 137,

146proton transfer, 221Protonierung, 84PTR-MS, 52, 220, 221PTR-TOF, 52pyro Aβ peptides, 124

Q-TOF mass spectrometer, 212Q-Trap-MS, 185QC, 111QTOF, 147, 176, 177, 209qTOF-MS, 139QTRAP, 57, 186, 252Quantifizierung, 57, 230, 244, 259quantitative mass spectrometry, 37, 50Quantitative proteomics, 59quantitative proteomics, 112, 114, 116, 261

radicals, 226rate constants, 257Raumstruktur, 49reactivity of hydroxyl amino acids, 119realtime, 75rearrangement, 258Recovery, 38REIMS, 75relative quantification, 51resonante Multiphotonenionisation, 54

271

retro-Diels-Alder reaction, 171

Reverse metabolomics, 181

Rho proteins, 92

RISQ, 59

RNA, 49

Roentgenkontrastmittel, 252

ROMP, 235

ruthenium(II) complexes, 227

SAW, 153

SAW-bioaffinity, 157

SAW-ESI-MS, 153

Schwefeldioxid, 210

Schwingungsstruktur, 63

scoring scheme, 232

Screening, 44

SDS-PAGE, 148

secretome, 109

seed development, 61, 99

Selektivität, 82Selenium speciation, 262SF6, 213Shotgun lipidomics, 72, 192Si-Wafer, 264sialylierte Zucker, 200SILAC, 50, 108, 113Silicate Species, 23simulation, 47sinapate ester metabolism, 184SMN, 151sodium cation adducts, 171solar silicon, 263Spacewalk, 231speciation analysis, 77Spektrenbibliothek, 74Spermien, 69SPME-GC-MS, 196SPPS, 127Spurenanalyse, 264SRI, 220SRM, 51, 151stable isotope, 144, 261standardprotein, 261star-shaped polymers, 68Steroide, 228Steroidhormone, 166stoichiometry, 151stomach cancer, 187Stosskühlung, 79stress induced protein, 155structural characterization, 127Substrat-Suche, 94SUMO-fusion, 129surface analysis, 58Surface Sampling, 76synapse, 48

T cell signaling, 50TAG, 168

tandem MS, 46, 60, 103, 167, 182, 185, 186, 243TAP, 155Targeted data acquisition, 37targeted metabolomics, 184, 185targeted proteomics, 115TCEP, 126TDM in a clinical chemistry routine application, 242tetracycline, 219theory, 81therapeutic drug monitoring, 215Thiole, 178thiols, 126tissue analysis, 75tissue sampels, 225TLC, 69, 188, 191, 198TMT, 110TNT, 213tobacco, 155TOF, 45, 159, 163, 191TOF MSE functionality, 179ToF-SIMS, 264Top-down, 149top-down glycolipidomics, 190Top-Down Sequencing (TDS), 148Toxicoproteomics, 93Toxin A, 95trace elements, 263Tracer, 210transthyretin, 237TRIM28, 91triptic peptides, 225Tryptic peptides, 212tryptische Peptide, 222tumor cells, 116tumor necrosis factor alpha, 129

Ubiquitin, 130, 152Ultra-high performance nanoLC, 104undersampling, 106unintended ion ejection, 207unsequenced genomes, 102UPLC, 146, 166UPLC-MS/MS, 230UV Spectroscopy, 233

Verdau, 152Vibrational Spectroscopy, 22

Wörterbuch, 80Wasserstofftransfer, 79wastewater, 77water analysis, 221

xenobiotic, 55

Zeolites, 23Zweiphotonenabsorption, 206

272

Documents

DGMS2010 abstracts com€¦ · Reviews, kurzen Trends oder von Originalarbeiten eingereicht werden, ... V6-V10 Seite:42-46 V25-V28 Seite:63-68 InstrumentelleEntwicklungen: V16-V20