High Resolution in Mass and Space: AP-MALDI Imaging Using Orbitrap-based InstrumentationBernhard Spengler1,3, Andreas Römpp1, Sabine Günther1, Oliver Schulz1, Klaus-Peter Hinz1, Alfons Hester1, Christian Schinz1, Christian Lotze1, Jörg-Ulrich Pötzl1, Kerstin Strupat2

1Institute of Inorganic and Analytical Chemistry, Justus Liebig University Giessen, Germany; 2Thermo Fisher Scientific (Bremen) GmbH, Germany; 3TransMIT GmbH, Giessen, Germany

2 High Resolution in Mass and Space: AP-MALDI Imaging Using Orbitrap-based Instrumentation

laser beam path = ion trajectory =observation path =base plate =

CCD Camera

mirror(centrally bored)

objective lens(centrally bored)

movable sample stage (x, y, z)

observation mirror(off axis)

aperture

lens mirror

static and variable attenuator

N2 Laser

Thermo ScientificQ ExactiveTM

mirror

Performance of AP-SMALDI MSImaging imagine10Spatial Resolution

ConclusionThe methods applied to Orbitrap-based instrumentation shows high reliability, validity and performance for routine histological analysis.

Imaging selectivity, dynamic range are significantly improved upon combining high spatial resolution (here down to 3 µm, typically 10 um) with high mass resolution and high mass accuracy obtained with Orbitrap-based instrumentation.

Such highly spatially resolved, high mass resolution MS/MS data are shown for the first time.

References[1] Spengler B, Hubert M, Kaufmann R, MALDI Ion Imaging and Biological Ion Imaging with a new Scanning UV-Laser

Microprobe, Proceedings of the 42nd Annual Conference on Mass Spectrometry and Allied Topics, Chicago, IL, May 29 - June 3, 1994, pp 1041.

[2] Römpp, A., S. Guenther, Y. Schober, O. Schulz, Z. Takats, W. Kummer, and B. Spengler (2010), Histology by Mass Spectrometry: Label-Free Tissue Characterization Obtained from High-Accuracy Bioanalytical Imaging. Angewandte ChemieInternational Edition. 49(22): p. 3834-3838.

[3] Guenther, S., Römpp, A., Kummer, W., Spengler B. (2011), AP-MALDI Imaging of neuropeptides in mouse pituitary gland with 5 µm spatial resolution and high mass accuracy. Int. J. Mass Spectrom. 305, 228-237.

[4] Römpp, A., Guenther, S., Takats, Z., Spengler, B. (2011), Mass spectrometry imaging with high resolution in mass and space (HR² MSI) for reliable investigation of drug compound distributions on the cellular level. Analytical and Bioanalytical Chemistry 401, 65-73.

[5] Schober, Y., Schramm, T., Spengler, B., Römpp, A. (2011), Protein identification by accurate mass MALDI imaging of trypticpeptides. Rapid Communications in Mass Spectrometry 25, 2475-2483.

AcknowledgementsThe authors acknowledge financial support by the Hessian Ministry of Science and Arts (HMWK) through LOEWE focus “AmbiProbe“.

imagine10 is trademark of TransMIT GmbH, Giessen, Germany. Orbitrap, Exactive and Q Exactive are trademarks of Thermo Fisher Scientific and its subsidiaries. Not all products are available in all countries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Presented at ASMS2012 (T436) Vancouver, Canada

OverviewPurpose: Short synopsis about the research approach.

Methods: Short synopsis of the experimental methods.

Results: Short synopsis of the results.

IntroductionMALDI Mass Spectrometry Imaging (MSI), 17 years after its first announcement [1], has turned into a routine method of highest performance for the molecular histology of biological tissue [2]. The method, providing high resolution and accuracy in mass and space, has been developed for a reliable identification and localization of individual tissue components, and has been applied recently to a number of research areas in pathology, cancer diagnostics, metabolic pathway analysis and plant research [2-5]. Targeted compound classes included phospholipids, peptides, proteins, drug compounds and metabolites. An atmospheric pressure (AP) ion source has been developed for these studies, based on a dedicated microoptical setup that provides highest spatial resolution of 3 to 5 micrometer on tissue in imaging mode on Orbitrap-based instrumentation.The advantages of Orbitrap detection are given here:1. High mass resolution allows to analyze complex tissue samples, sample complexity2. High mass accuracy enables for straightforward sum formula assignments and facilitates

substance identification, substance identificationAccurate MS and MS/MS imaging data allow to align images, confirmation

3. Irrelevance of sample morphology/flatness, ease of sample / matrix preparation4. Convenience of AP interface, compatibility to ambient analysis

High Resolution in Mass and Space: AP-MALDI Imaging using Orbitrap-based InstrumentationBernhard Spengler1,3, Andreas Römpp1, Sabine Günther1, Oliver Schulz1, Klaus-Peter Hinz1, Alfons Hester1, Christian Schinz1, Christian Lotze1, Jörg-Ulrich Pötzl1, Kerstin Strupat2

1Institute of Inorganic and Analytical Chemistry, Justus Liebig University Giessen, Germany *bernhard.spengler@anorg.chemie.uni-giessen.de2Thermo Fisher Scientific (Bremen) GmbH, Germany, kerstin.strupat@thermofisher.com, 3TransMIT GmbH, Giessen, Germany, spengler@transmit.de

Open-cover view of the atmospheric pressure

imaging ion sourceimagine10

(TransMIT GmbH, Giessen) attached to

a Thermo Scientific Q ExactiveTM

Orbitrap instrument, Thermo Fisher

Scientific (Bremen) GmbH

Scheme of the atmospheric pressure

imaging ion sourceimagine10

(TransMIT GmbH, Giessen) attached to a

Thermo Scientific Q ExactiveTM Orbitrap

instrument (Thermo Fisher Scientific (Bremen) GmbH) Coronal mouse brain section

35 µm steps, 295 x 195 pixels

Left: Focus size measurements (most left) and ablation on

tissue reveal an effective pixel

resolution in the range of 5 µm, effective pixel size if laser irradiance

dependent.

Above: Un-blurred red/green pixels indicate

an effective spatial resolution on mouse brain tissue (lateral ventricle)

of 3 µm.

Left:Single pixel (3 µm) = single scan

Exactive MS Orbitrap mass spectrum

Lateral ventricle, 3 µm steps, 170 x 200 pixels

Experimental

RMS = 0.2 ppm

+/-1

ppm

30,000 consecutive scans of PC(34:1) at m/z = 798.5410 on tissue during

image acquisiton using internal lock mass operation.

Representative single pixel (10 x 10 µm2) mass spectrum upon automated internal

lock mass operation. imagine10 source attached to Q Exactive

Orbitrap instrument

MS and MS/MS Imaging

A coaxial ion source geometry was employed to irradiate, desorb, ionize and transport biological compounds under atmospheric pressure conditions. Ions are introduced into the mass spectrometer through an

extended heated capillary. Transfer efficiency was optimized using both, gas stream dynamics and electrostatic ion focusing. The

achievable mass accuracy during tissue imaging operation is typically better than 1 ppm RMS using lock mass calibration. Imaging speed is

up to 2 pixels per second, depending on mass range and mass resolution setting. Matrix preparation and on-tissue protein digestion

were performed using a new dedicated protocol.

50 µm100 µm

Red: backgroundGreen: PC(38:4)Blue: PC(34:1)

Healthy mouse brain tissue. Two phospholipids differing by 8 mmu (m/z

848.55 and m/z 848.63) are extracted into an MSimage.

10 µm, 400 x 400 pixels (= 4*4 mm2) Mass Resolution = 70000 @ m/z 400

imagine10 source attached to Q Exactive Orbitrap instrumentation

T007 CK75 MS-MSMS772 200x200 10um E17 #37003 RT: 805.25 AV: 1 NL: 9.03E4T: FTMS + p NSI Full ms [700.00-900.00]

700 720 740 760 780 800 820 840 860 880 900m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

798.55066

772.53479

896.61188

782.57678826.58234 870.59595

716.13373 760.59467734.57874 848.56702

820.53528

876.59943

S194 CK3 170x200 3um E170_RC798#26687 RT: 548.39 AV: 1 NL: 6.75E3

FTMS + p NSI Full ms [100.00-1000.00]

700 720 740 760 780 800 820 840 860 880 900 m/z0

50

100

Rel

ativ

e Ab

unda

nce

798.5412

772.5258

896.6012

769.5638 782.5681 870.5860739.4668 848.5556713.4514 826.5722756.5528844.5272804.5507

893.4771

Mass Resolution on Tissue

Mass Selectivity & Dynamic Range

Closely neighbored

signals, even of drastically

differing peak intensities, can be

clearly distiguished and

discriminated. Bin width Δm/z was

0.01 u.

798.5416PC(34:1)+K+

798.5909PE(38:0)+Na+

797 798 799 800 801 802

798.5416

799.5453

800.5596801.5636

798.45 798.50 798.55 798.60m/z

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Rel

ativ

e A

bund

ance

798.5990

798.4895

12.5 ppm window (Δm/z=0.01)

m/z

Healthy mouse brain tissue, 10 um step size, 200x200 pixels (= 2 *2 mm2). MS and MS/MS scans on adjacent, neigboring pixels.Left: m/z 772 (MS Orbitrap, full scan) right: m/z 713 (MSMS of 772), loss

of trimethylamine (59 u).MS/MS experiment confirms the phospholipid classification as phosphatidylcholine or shingomyeline. imagine10 source attached to Q Exactive Orbitrap instrument.

m/z 772.523 m/z 713.450

3Thermo Scientific Poster Note • PN63602_E 06/12S

laser beam path = ion trajectory =observation path =base plate =

CCD Camera

mirror(centrally bored)

objective lens(centrally bored)

movable sample stage (x, y, z)

observation mirror(off axis)

aperture

lens mirror

static and variable attenuator

N2 Laser

Thermo ScientificQ ExactiveTM

mirror

Performance of AP-SMALDI MSImaging imagine10Spatial Resolution

ConclusionThe methods applied to Orbitrap-based instrumentation shows high reliability, validity and performance for routine histological analysis.

Imaging selectivity, dynamic range are significantly improved upon combining high spatial resolution (here down to 3 µm, typically 10 um) with high mass resolution and high mass accuracy obtained with Orbitrap-based instrumentation.

Such highly spatially resolved, high mass resolution MS/MS data are shown for the first time.

References[1] Spengler B, Hubert M, Kaufmann R, MALDI Ion Imaging and Biological Ion Imaging with a new Scanning UV-Laser

Microprobe, Proceedings of the 42nd Annual Conference on Mass Spectrometry and Allied Topics, Chicago, IL, May 29 - June 3, 1994, pp 1041.

[2] Römpp, A., S. Guenther, Y. Schober, O. Schulz, Z. Takats, W. Kummer, and B. Spengler (2010), Histology by Mass Spectrometry: Label-Free Tissue Characterization Obtained from High-Accuracy Bioanalytical Imaging. Angewandte ChemieInternational Edition. 49(22): p. 3834-3838.

[3] Guenther, S., Römpp, A., Kummer, W., Spengler B. (2011), AP-MALDI Imaging of neuropeptides in mouse pituitary gland with 5 µm spatial resolution and high mass accuracy. Int. J. Mass Spectrom. 305, 228-237.

[4] Römpp, A., Guenther, S., Takats, Z., Spengler, B. (2011), Mass spectrometry imaging with high resolution in mass and space (HR² MSI) for reliable investigation of drug compound distributions on the cellular level. Analytical and Bioanalytical Chemistry 401, 65-73.

[5] Schober, Y., Schramm, T., Spengler, B., Römpp, A. (2011), Protein identification by accurate mass MALDI imaging of trypticpeptides. Rapid Communications in Mass Spectrometry 25, 2475-2483.

AcknowledgementsThe authors acknowledge financial support by the Hessian Ministry of Science and Arts (HMWK) through LOEWE focus “AmbiProbe“.

imagine10 is trademark of TransMIT GmbH, Giessen, Germany. Orbitrap, Exactive and Q Exactive are trademarks of Thermo Fisher Scientific and its subsidiaries. Not all products are available in all countries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Presented at ASMS2012 (T436) Vancouver, Canada

OverviewPurpose: Short synopsis about the research approach.

Methods: Short synopsis of the experimental methods.

Results: Short synopsis of the results.

IntroductionMALDI Mass Spectrometry Imaging (MSI), 17 years after its first announcement [1], has turned into a routine method of highest performance for the molecular histology of biological tissue [2]. The method, providing high resolution and accuracy in mass and space, has been developed for a reliable identification and localization of individual tissue components, and has been applied recently to a number of research areas in pathology, cancer diagnostics, metabolic pathway analysis and plant research [2-5]. Targeted compound classes included phospholipids, peptides, proteins, drug compounds and metabolites. An atmospheric pressure (AP) ion source has been developed for these studies, based on a dedicated microoptical setup that provides highest spatial resolution of 3 to 5 micrometer on tissue in imaging mode on Orbitrap-based instrumentation.The advantages of Orbitrap detection are given here:1. High mass resolution allows to analyze complex tissue samples, sample complexity2. High mass accuracy enables for straightforward sum formula assignments and facilitates

substance identification, substance identificationAccurate MS and MS/MS imaging data allow to align images, confirmation

3. Irrelevance of sample morphology/flatness, ease of sample / matrix preparation4. Convenience of AP interface, compatibility to ambient analysis

High Resolution in Mass and Space: AP-MALDI Imaging using Orbitrap-based InstrumentationBernhard Spengler1,3, Andreas Römpp1, Sabine Günther1, Oliver Schulz1, Klaus-Peter Hinz1, Alfons Hester1, Christian Schinz1, Christian Lotze1, Jörg-Ulrich Pötzl1, Kerstin Strupat2

1Institute of Inorganic and Analytical Chemistry, Justus Liebig University Giessen, Germany *bernhard.spengler@anorg.chemie.uni-giessen.de2Thermo Fisher Scientific (Bremen) GmbH, Germany, kerstin.strupat@thermofisher.com, 3TransMIT GmbH, Giessen, Germany, spengler@transmit.de

Open-cover view of the atmospheric pressure

imaging ion sourceimagine10

(TransMIT GmbH, Giessen) attached to

a Thermo Scientific Q ExactiveTM

Orbitrap instrument, Thermo Fisher

Scientific (Bremen) GmbH

Scheme of the atmospheric pressure

imaging ion sourceimagine10

(TransMIT GmbH, Giessen) attached to a

Thermo Scientific Q ExactiveTM Orbitrap

instrument (Thermo Fisher Scientific (Bremen) GmbH) Coronal mouse brain section

35 µm steps, 295 x 195 pixels

Left: Focus size measurements (most left) and ablation on

tissue reveal an effective pixel

resolution in the range of 5 µm, effective pixel size if laser irradiance

dependent.

Above: Un-blurred red/green pixels indicate

an effective spatial resolution on mouse brain tissue (lateral ventricle)

of 3 µm.

Left:Single pixel (3 µm) = single scan

Exactive MS Orbitrap mass spectrum

Lateral ventricle, 3 µm steps, 170 x 200 pixels

Experimental

RMS = 0.2 ppm

+/-1

ppm

30,000 consecutive scans of PC(34:1) at m/z = 798.5410 on tissue during

image acquisiton using internal lock mass operation.

Representative single pixel (10 x 10 µm2) mass spectrum upon automated internal

lock mass operation. imagine10 source attached to Q Exactive

Orbitrap instrument

MS and MS/MS Imaging

A coaxial ion source geometry was employed to irradiate, desorb, ionize and transport biological compounds under atmospheric pressure conditions. Ions are introduced into the mass spectrometer through an

extended heated capillary. Transfer efficiency was optimized using both, gas stream dynamics and electrostatic ion focusing. The

achievable mass accuracy during tissue imaging operation is typically better than 1 ppm RMS using lock mass calibration. Imaging speed is

up to 2 pixels per second, depending on mass range and mass resolution setting. Matrix preparation and on-tissue protein digestion

were performed using a new dedicated protocol.

50 µm100 µm

Red: backgroundGreen: PC(38:4)Blue: PC(34:1)

Healthy mouse brain tissue. Two phospholipids differing by 8 mmu (m/z

848.55 and m/z 848.63) are extracted into an MSimage.

10 µm, 400 x 400 pixels (= 4*4 mm2) Mass Resolution = 70000 @ m/z 400

imagine10 source attached to Q Exactive Orbitrap instrumentation

T007 CK75 MS-MSMS772 200x200 10um E17 #37003 RT: 805.25 AV: 1 NL: 9.03E4T: FTMS + p NSI Full ms [700.00-900.00]

700 720 740 760 780 800 820 840 860 880 900m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

798.55066

772.53479

896.61188

782.57678826.58234 870.59595

716.13373 760.59467734.57874 848.56702

820.53528

876.59943

S194 CK3 170x200 3um E170_RC798#26687 RT: 548.39 AV: 1 NL: 6.75E3

FTMS + p NSI Full ms [100.00-1000.00]

700 720 740 760 780 800 820 840 860 880 900 m/z0

50

100

Rel

ativ

e Ab

unda

nce

798.5412

772.5258

896.6012

769.5638 782.5681 870.5860739.4668 848.5556713.4514 826.5722756.5528844.5272804.5507

893.4771

Mass Resolution on Tissue

Mass Selectivity & Dynamic Range

Closely neighbored

signals, even of drastically

differing peak intensities, can be

clearly distiguished and

discriminated. Bin width Δm/z was

0.01 u.

798.5416PC(34:1)+K+

798.5909PE(38:0)+Na+

797 798 799 800 801 802

798.5416

799.5453

800.5596801.5636

798.45 798.50 798.55 798.60m/z

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Rel

ativ

e A

bund

ance

798.5990

798.4895

12.5 ppm window (Δm/z=0.01)

m/z

Healthy mouse brain tissue, 10 um step size, 200x200 pixels (= 2 *2 mm2). MS and MS/MS scans on adjacent, neigboring pixels.Left: m/z 772 (MS Orbitrap, full scan) right: m/z 713 (MSMS of 772), loss

of trimethylamine (59 u).MS/MS experiment confirms the phospholipid classification as phosphatidylcholine or shingomyeline. imagine10 source attached to Q Exactive Orbitrap instrument.

m/z 772.523 m/z 713.450

4 High Resolution in Mass and Space: AP-MALDI Imaging Using Orbitrap-based Instrumentation

laser beam path = ion trajectory =observation path =base plate =

CCD Camera

mirror(centrally bored)

objective lens(centrally bored)

movable sample stage (x, y, z)

observation mirror(off axis)

aperture

lens mirror

static and variable attenuator

N2 Laser

Thermo ScientificQ ExactiveTM

mirror

Performance of AP-SMALDI MSImaging imagine10Spatial Resolution

ConclusionThe methods applied to Orbitrap-based instrumentation shows high reliability, validity and performance for routine histological analysis.

Imaging selectivity, dynamic range are significantly improved upon combining high spatial resolution (here down to 3 µm, typically 10 um) with high mass resolution and high mass accuracy obtained with Orbitrap-based instrumentation.

Such highly spatially resolved, high mass resolution MS/MS data are shown for the first time.

References[1] Spengler B, Hubert M, Kaufmann R, MALDI Ion Imaging and Biological Ion Imaging with a new Scanning UV-Laser

Microprobe, Proceedings of the 42nd Annual Conference on Mass Spectrometry and Allied Topics, Chicago, IL, May 29 - June 3, 1994, pp 1041.

[2] Römpp, A., S. Guenther, Y. Schober, O. Schulz, Z. Takats, W. Kummer, and B. Spengler (2010), Histology by Mass Spectrometry: Label-Free Tissue Characterization Obtained from High-Accuracy Bioanalytical Imaging. Angewandte ChemieInternational Edition. 49(22): p. 3834-3838.

[3] Guenther, S., Römpp, A., Kummer, W., Spengler B. (2011), AP-MALDI Imaging of neuropeptides in mouse pituitary gland with 5 µm spatial resolution and high mass accuracy. Int. J. Mass Spectrom. 305, 228-237.

[4] Römpp, A., Guenther, S., Takats, Z., Spengler, B. (2011), Mass spectrometry imaging with high resolution in mass and space (HR² MSI) for reliable investigation of drug compound distributions on the cellular level. Analytical and Bioanalytical Chemistry 401, 65-73.

[5] Schober, Y., Schramm, T., Spengler, B., Römpp, A. (2011), Protein identification by accurate mass MALDI imaging of trypticpeptides. Rapid Communications in Mass Spectrometry 25, 2475-2483.

AcknowledgementsThe authors acknowledge financial support by the Hessian Ministry of Science and Arts (HMWK) through LOEWE focus “AmbiProbe“.

imagine10 is trademark of TransMIT GmbH, Giessen, Germany. Orbitrap, Exactive and Q Exactive are trademarks of Thermo Fisher Scientific and its subsidiaries. Not all products are available in all countries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Presented at ASMS2012 (T436) Vancouver, Canada

OverviewPurpose: Short synopsis about the research approach.

Methods: Short synopsis of the experimental methods.

Results: Short synopsis of the results.

IntroductionMALDI Mass Spectrometry Imaging (MSI), 17 years after its first announcement [1], has turned into a routine method of highest performance for the molecular histology of biological tissue [2]. The method, providing high resolution and accuracy in mass and space, has been developed for a reliable identification and localization of individual tissue components, and has been applied recently to a number of research areas in pathology, cancer diagnostics, metabolic pathway analysis and plant research [2-5]. Targeted compound classes included phospholipids, peptides, proteins, drug compounds and metabolites. An atmospheric pressure (AP) ion source has been developed for these studies, based on a dedicated microoptical setup that provides highest spatial resolution of 3 to 5 micrometer on tissue in imaging mode on Orbitrap-based instrumentation.The advantages of Orbitrap detection are given here:1. High mass resolution allows to analyze complex tissue samples, sample complexity2. High mass accuracy enables for straightforward sum formula assignments and facilitates

substance identification, substance identificationAccurate MS and MS/MS imaging data allow to align images, confirmation

3. Irrelevance of sample morphology/flatness, ease of sample / matrix preparation4. Convenience of AP interface, compatibility to ambient analysis

High Resolution in Mass and Space: AP-MALDI Imaging using Orbitrap-based InstrumentationBernhard Spengler1,3, Andreas Römpp1, Sabine Günther1, Oliver Schulz1, Klaus-Peter Hinz1, Alfons Hester1, Christian Schinz1, Christian Lotze1, Jörg-Ulrich Pötzl1, Kerstin Strupat2

1Institute of Inorganic and Analytical Chemistry, Justus Liebig University Giessen, Germany *bernhard.spengler@anorg.chemie.uni-giessen.de2Thermo Fisher Scientific (Bremen) GmbH, Germany, kerstin.strupat@thermofisher.com, 3TransMIT GmbH, Giessen, Germany, spengler@transmit.de

Open-cover view of the atmospheric pressure

imaging ion sourceimagine10

(TransMIT GmbH, Giessen) attached to

a Thermo Scientific Q ExactiveTM

Orbitrap instrument, Thermo Fisher

Scientific (Bremen) GmbH

Scheme of the atmospheric pressure

imaging ion sourceimagine10

(TransMIT GmbH, Giessen) attached to a

Thermo Scientific Q ExactiveTM Orbitrap

instrument (Thermo Fisher Scientific (Bremen) GmbH) Coronal mouse brain section

35 µm steps, 295 x 195 pixels

Left: Focus size measurements (most left) and ablation on

tissue reveal an effective pixel

resolution in the range of 5 µm, effective pixel size if laser irradiance

dependent.

Above: Un-blurred red/green pixels indicate

an effective spatial resolution on mouse brain tissue (lateral ventricle)

of 3 µm.

Left:Single pixel (3 µm) = single scan

Exactive MS Orbitrap mass spectrum

Lateral ventricle, 3 µm steps, 170 x 200 pixels

Experimental

RMS = 0.2 ppm

+/-1

ppm

30,000 consecutive scans of PC(34:1) at m/z = 798.5410 on tissue during

image acquisiton using internal lock mass operation.

Representative single pixel (10 x 10 µm2) mass spectrum upon automated internal

lock mass operation. imagine10 source attached to Q Exactive

Orbitrap instrument

MS and MS/MS Imaging

A coaxial ion source geometry was employed to irradiate, desorb, ionize and transport biological compounds under atmospheric pressure conditions. Ions are introduced into the mass spectrometer through an

extended heated capillary. Transfer efficiency was optimized using both, gas stream dynamics and electrostatic ion focusing. The

achievable mass accuracy during tissue imaging operation is typically better than 1 ppm RMS using lock mass calibration. Imaging speed is

up to 2 pixels per second, depending on mass range and mass resolution setting. Matrix preparation and on-tissue protein digestion

were performed using a new dedicated protocol.

50 µm100 µm

Red: backgroundGreen: PC(38:4)Blue: PC(34:1)

Healthy mouse brain tissue. Two phospholipids differing by 8 mmu (m/z

848.55 and m/z 848.63) are extracted into an MSimage.

10 µm, 400 x 400 pixels (= 4*4 mm2) Mass Resolution = 70000 @ m/z 400

imagine10 source attached to Q Exactive Orbitrap instrumentation

T007 CK75 MS-MSMS772 200x200 10um E17 #37003 RT: 805.25 AV: 1 NL: 9.03E4T: FTMS + p NSI Full ms [700.00-900.00]

700 720 740 760 780 800 820 840 860 880 900m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

798.55066

772.53479

896.61188

782.57678826.58234 870.59595

716.13373 760.59467734.57874 848.56702

820.53528

876.59943

S194 CK3 170x200 3um E170_RC798#26687 RT: 548.39 AV: 1 NL: 6.75E3

FTMS + p NSI Full ms [100.00-1000.00]

700 720 740 760 780 800 820 840 860 880 900 m/z0

50

100

Rel

ativ

e Ab

unda

nce

798.5412

772.5258

896.6012

769.5638 782.5681 870.5860739.4668 848.5556713.4514 826.5722756.5528844.5272804.5507

893.4771

Mass Resolution on Tissue

Mass Selectivity & Dynamic Range

Closely neighbored

signals, even of drastically

differing peak intensities, can be

clearly distiguished and

discriminated. Bin width Δm/z was

0.01 u.

798.5416PC(34:1)+K+

798.5909PE(38:0)+Na+

797 798 799 800 801 802

798.5416

799.5453

800.5596801.5636

798.45 798.50 798.55 798.60m/z

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Rel

ativ

e A

bund

ance

798.5990

798.4895

12.5 ppm window (Δm/z=0.01)

m/z

Healthy mouse brain tissue, 10 um step size, 200x200 pixels (= 2 *2 mm2). MS and MS/MS scans on adjacent, neigboring pixels.Left: m/z 772 (MS Orbitrap, full scan) right: m/z 713 (MSMS of 772), loss

of trimethylamine (59 u).MS/MS experiment confirms the phospholipid classification as phosphatidylcholine or shingomyeline. imagine10 source attached to Q Exactive Orbitrap instrument.

m/z 772.523 m/z 713.450

5Thermo Scientific Poster Note • PN63602_E 06/12S

laser beam path = ion trajectory =observation path =base plate =

CCD Camera

mirror(centrally bored)

objective lens(centrally bored)

movable sample stage (x, y, z)

observation mirror(off axis)

aperture

lens mirror

static and variable attenuator

N2 Laser

Thermo ScientificQ ExactiveTM

mirror

Performance of AP-SMALDI MSImaging imagine10Spatial Resolution

ConclusionThe methods applied to Orbitrap-based instrumentation shows high reliability, validity and performance for routine histological analysis.

Imaging selectivity, dynamic range are significantly improved upon combining high spatial resolution (here down to 3 µm, typically 10 um) with high mass resolution and high mass accuracy obtained with Orbitrap-based instrumentation.

Such highly spatially resolved, high mass resolution MS/MS data are shown for the first time.

References[1] Spengler B, Hubert M, Kaufmann R, MALDI Ion Imaging and Biological Ion Imaging with a new Scanning UV-Laser

Microprobe, Proceedings of the 42nd Annual Conference on Mass Spectrometry and Allied Topics, Chicago, IL, May 29 - June 3, 1994, pp 1041.

[2] Römpp, A., S. Guenther, Y. Schober, O. Schulz, Z. Takats, W. Kummer, and B. Spengler (2010), Histology by Mass Spectrometry: Label-Free Tissue Characterization Obtained from High-Accuracy Bioanalytical Imaging. Angewandte ChemieInternational Edition. 49(22): p. 3834-3838.

[3] Guenther, S., Römpp, A., Kummer, W., Spengler B. (2011), AP-MALDI Imaging of neuropeptides in mouse pituitary gland with 5 µm spatial resolution and high mass accuracy. Int. J. Mass Spectrom. 305, 228-237.

[4] Römpp, A., Guenther, S., Takats, Z., Spengler, B. (2011), Mass spectrometry imaging with high resolution in mass and space (HR² MSI) for reliable investigation of drug compound distributions on the cellular level. Analytical and Bioanalytical Chemistry 401, 65-73.

[5] Schober, Y., Schramm, T., Spengler, B., Römpp, A. (2011), Protein identification by accurate mass MALDI imaging of trypticpeptides. Rapid Communications in Mass Spectrometry 25, 2475-2483.

AcknowledgementsThe authors acknowledge financial support by the Hessian Ministry of Science and Arts (HMWK) through LOEWE focus “AmbiProbe“.

imagine10 is trademark of TransMIT GmbH, Giessen, Germany. Orbitrap, Exactive and Q Exactive are trademarks of Thermo Fisher Scientific and its subsidiaries. Not all products are available in all countries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Presented at ASMS2012 (T436) Vancouver, Canada

OverviewPurpose: Short synopsis about the research approach.

Methods: Short synopsis of the experimental methods.

Results: Short synopsis of the results.

IntroductionMALDI Mass Spectrometry Imaging (MSI), 17 years after its first announcement [1], has turned into a routine method of highest performance for the molecular histology of biological tissue [2]. The method, providing high resolution and accuracy in mass and space, has been developed for a reliable identification and localization of individual tissue components, and has been applied recently to a number of research areas in pathology, cancer diagnostics, metabolic pathway analysis and plant research [2-5]. Targeted compound classes included phospholipids, peptides, proteins, drug compounds and metabolites. An atmospheric pressure (AP) ion source has been developed for these studies, based on a dedicated microoptical setup that provides highest spatial resolution of 3 to 5 micrometer on tissue in imaging mode on Orbitrap-based instrumentation.The advantages of Orbitrap detection are given here:1. High mass resolution allows to analyze complex tissue samples, sample complexity2. High mass accuracy enables for straightforward sum formula assignments and facilitates

substance identification, substance identificationAccurate MS and MS/MS imaging data allow to align images, confirmation

3. Irrelevance of sample morphology/flatness, ease of sample / matrix preparation4. Convenience of AP interface, compatibility to ambient analysis

High Resolution in Mass and Space: AP-MALDI Imaging using Orbitrap-based InstrumentationBernhard Spengler1,3, Andreas Römpp1, Sabine Günther1, Oliver Schulz1, Klaus-Peter Hinz1, Alfons Hester1, Christian Schinz1, Christian Lotze1, Jörg-Ulrich Pötzl1, Kerstin Strupat2

1Institute of Inorganic and Analytical Chemistry, Justus Liebig University Giessen, Germany *bernhard.spengler@anorg.chemie.uni-giessen.de2Thermo Fisher Scientific (Bremen) GmbH, Germany, kerstin.strupat@thermofisher.com, 3TransMIT GmbH, Giessen, Germany, spengler@transmit.de

Open-cover view of the atmospheric pressure

imaging ion sourceimagine10

(TransMIT GmbH, Giessen) attached to

a Thermo Scientific Q ExactiveTM

Orbitrap instrument, Thermo Fisher

Scientific (Bremen) GmbH

Scheme of the atmospheric pressure

imaging ion sourceimagine10

(TransMIT GmbH, Giessen) attached to a

Thermo Scientific Q ExactiveTM Orbitrap

instrument (Thermo Fisher Scientific (Bremen) GmbH) Coronal mouse brain section

35 µm steps, 295 x 195 pixels

Left: Focus size measurements (most left) and ablation on

tissue reveal an effective pixel

resolution in the range of 5 µm, effective pixel size if laser irradiance

dependent.

Above: Un-blurred red/green pixels indicate

an effective spatial resolution on mouse brain tissue (lateral ventricle)

of 3 µm.

Left:Single pixel (3 µm) = single scan

Exactive MS Orbitrap mass spectrum

Lateral ventricle, 3 µm steps, 170 x 200 pixels

Experimental

RMS = 0.2 ppm

+/-1

ppm

30,000 consecutive scans of PC(34:1) at m/z = 798.5410 on tissue during

image acquisiton using internal lock mass operation.

Representative single pixel (10 x 10 µm2) mass spectrum upon automated internal

lock mass operation. imagine10 source attached to Q Exactive

Orbitrap instrument

MS and MS/MS Imaging

A coaxial ion source geometry was employed to irradiate, desorb, ionize and transport biological compounds under atmospheric pressure conditions. Ions are introduced into the mass spectrometer through an

extended heated capillary. Transfer efficiency was optimized using both, gas stream dynamics and electrostatic ion focusing. The

achievable mass accuracy during tissue imaging operation is typically better than 1 ppm RMS using lock mass calibration. Imaging speed is

up to 2 pixels per second, depending on mass range and mass resolution setting. Matrix preparation and on-tissue protein digestion

were performed using a new dedicated protocol.

50 µm100 µm

Red: backgroundGreen: PC(38:4)Blue: PC(34:1)

Healthy mouse brain tissue. Two phospholipids differing by 8 mmu (m/z

848.55 and m/z 848.63) are extracted into an MSimage.

10 µm, 400 x 400 pixels (= 4*4 mm2) Mass Resolution = 70000 @ m/z 400

imagine10 source attached to Q Exactive Orbitrap instrumentation

T007 CK75 MS-MSMS772 200x200 10um E17 #37003 RT: 805.25 AV: 1 NL: 9.03E4T: FTMS + p NSI Full ms [700.00-900.00]

700 720 740 760 780 800 820 840 860 880 900m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

798.55066

772.53479

896.61188

782.57678826.58234 870.59595

716.13373 760.59467734.57874 848.56702

820.53528

876.59943

S194 CK3 170x200 3um E170_RC798#26687 RT: 548.39 AV: 1 NL: 6.75E3

FTMS + p NSI Full ms [100.00-1000.00]

700 720 740 760 780 800 820 840 860 880 900 m/z0

50

100

Rel

ativ

e Ab

unda

nce

798.5412

772.5258

896.6012

769.5638 782.5681 870.5860739.4668 848.5556713.4514 826.5722756.5528844.5272804.5507

893.4771

Mass Resolution on Tissue

Mass Selectivity & Dynamic Range

Closely neighbored

signals, even of drastically

differing peak intensities, can be

clearly distiguished and

discriminated. Bin width Δm/z was

0.01 u.

798.5416PC(34:1)+K+

798.5909PE(38:0)+Na+

797 798 799 800 801 802

798.5416

799.5453

800.5596801.5636

798.45 798.50 798.55 798.60m/z

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Rel

ativ

e A

bund

ance

798.5990

798.4895

12.5 ppm window (Δm/z=0.01)

m/z

Healthy mouse brain tissue, 10 um step size, 200x200 pixels (= 2 *2 mm2). MS and MS/MS scans on adjacent, neigboring pixels.Left: m/z 772 (MS Orbitrap, full scan) right: m/z 713 (MSMS of 772), loss

of trimethylamine (59 u).MS/MS experiment confirms the phospholipid classification as phosphatidylcholine or shingomyeline. imagine10 source attached to Q Exactive Orbitrap instrument.

m/z 772.523 m/z 713.450

PN63602_E 06/12S

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