Field Desorption Mass Spectra of Gastrine Peptides and...

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Field Desorption Mass Spectra of Gastrine Peptides and Glutathione Derivatives

M i c h a e l P r z y b y l s k i * a n d I n g o L ü d e r w a l d

Institut für Organische Chemie, Universität Mainz, Johann-Joach im-Becher -Weg 18-20, D-6500 Mainz

E k k e h a r d K r a a s a n d W o l f g a n g V o e l t e r

Institut für Organische Chemie, Universität Tübingen, A u f der Morgenstelle 18, D -7400 Tübingen

S i d n e y D . N e l s o n

Department o f Medicinal Chemistry, University o f Washington, Seattle, Washington 98195, U . S . A .

Z . Naturforsch. 34b, 736-743 (1979) ; received December 28, 1978 Field Desorpt ion Mass Spectrometry , Oligopeptides, Gastrine Peptides, Glutathione Conjugates, Molecular Ions

Oligopeptides comprising the sequence of the C-terminal tetrapeptide o f gastrine, Trp-Met -Asp-Phe-NH2, and several derivatives o f glutathione, y -Glu-Cys (SR) -Gly , were characterized b y field desorption mass spectrometry. The field desorption mass spectra obtained at various field ion emitter temperatures reveal abundant molecular ions and fragmentation reactions that yield partial sequence information. I n the series o f g luta-thione derivatives investigated, characteristic ions formed b y cleavage o f the y-Glu-Cys peptide b o n d determine the substituent at the Cys residue and can therefore be used t o identi fy corresponding conjugat ion products o f drug metabolites with glutathione.

Introduction

A v a r i e t y o f m a s s s p e c t r o m e t r i c m e t h o d s f o r t h e

s t r u c t u r e a n a l y s i s o f o l i g o p e p t i d e s h a s b e e n d e v e l -

o p e d a n d t e s t e d i n t h e l a s t y e a r s [ 1 - 3 ] . P a r t i c u l a r l y ,

d e r i v a t i z a t i o n r e a c t i o n s t o e n h a n c e t h e n o t o r i o u s l y

l o w v o l a t i l i t y o f p e p t i d e s f o r m a s s s p e c t r a l a n a l y s i s

h a v e b e e n s t u d i e d e x t e n s i v e l y [4]. T h e m o s t a d -

v a n c e d a p p r o a c h d e v e l o p e d b y B i e m a n n et al. [5]

i n v o l v e s m u l t i p l e d e r i v a t i z a t i o n s t e p s t h a t e n a b l e

t h e a n a l y s i s o f c o m p l e x m i x t u r e s o f di- t o t e t r a / p e n t a -

p e p t i d e s b y g a s c h r o m a t o g r a p h y - m a s s s p e c t r o -

m e t r y (g. c . - m . s . ) u s i n g e l e c t r o n i m p a c t (e . i . ) i o n i z a -

t i o n . T h e v a l u e o f t h i s a p p r o a c h t o t h e s e q u e n c e

e l u c i d a t i o n o f p o l y p e p t i d e s c o n t a i n i n g u p t o 50

a m i n o a c i d r e s i d u e s h a s b e e n d e m o n s t r a t e d [5, 6].

H o w e v e r , m a j o r l i m i t a t i o n s o f d e r i v a t i z a t i o n p r o c e -

d u r e s f o r t h e m a s s s p e c t r a l a n a l y s i s o f p e p t i d e s a r e

t h a t t h e y r e q u i r e r e l a t i v e l y l a r g e a m o u n t s o f s a m p l e

a n d p r e s e n t d i f f i c u l t i e s w i t h c e r t a i n p o l a r a m i n o

a c i d r e s i d u e s . M o r e o v e r , i n f o r m a t i o n a b o u t c h a i n -

t e r m i n a l o r s i d e c h a i n s u b s t i t u e n t s t h a t f r e q u e n t l y

Abbreviations: g.c.-m.s., gas chromatography-mass spec t rometry ; e . i . , electron i m p a c t ; c . i . , chemical ionisation; f . i . , field ionizat ion; f .d . , field desorpt ion; G S H , g lutathione; B A T , best anode temperature.

* Reprint requests to Dr . M. Przybylski . 0340-5087/79/0500-0736/$ 01.00/0

d e t e r m i n e t h e b i o l o g i c a l a c t i v i t y o f p e p t i d e s w i l l b e

l o s t d u r i n g t h e n e c e s s a r y h y d r o l y s i s s t e p s . M o r e

r e c e n t l y , s t u d i e s b y M c L a f f e r t y et al. h a v e s h o w n

t h a t u s e f u l s p e c t r a a n d p a r t i a l s e q u e n c e i n f o r m a t i o n

o f l a r g e r o l i g o p e p t i d e s c a n b e o b t a i n e d b y c h e m i c a l

i o n i z a t i o n (c . i . ) m a s s s p e c t r o m e t r y [7], b u t s t i l l

s o m e d e g r e e o f ( N - , C - t e r m i n a l ) d e r i v a t i z a t i o n is

r e q u i r e d f o r a t t a i n i n g v a p o r i z a t i o n o f t h e p e p t i d e .

S i n c e i t s i n t r o d u c t i o n b y B e c k e y a n d S c h u l t e n

s e v e r a l y e a r s a g o , field d e s o r p t i o n ( f . d . ) m a s s

s p e c t r o m e t r y h a s b e c o m e a n e s t a b l i s h e d t o o l f o r t h e

m o l e c u l a r w e i g h t d e t e r m i n a t i o n o f l a r g e , p o l a r

a n d / o r t h e r m a l l y u n s t a b l e m o l e c u l e s [8, 9]. A b u n -

d a n t m o l e c u l a r i o n s w e r e o b t a i n e d i n f . d . m a s s

s p e c t r a o f underivatized o l i g o p e p t i d e s w i t h u p t o

n i n e a m i n o a c i d r e s i d u e s [10, 1 1 ] . H o w e v e r , l i t t l e

s y s t e m a t i c w o r k h a s y e t b e e n d o n e t o i n v e s t i g a t e

t h e q u e s t i o n w h e t h e r t h e fragmentation o b s e r v e d i n

f . d . s p e c t r a o f o l i g o p e p t i d e s c a n b e u s e d t o o b t a i n ,

b e y o n d t h e m o l e c u l a r i o n o f t h e p e p t i d e , a d d i t i o n a l

s t r u c t u r a l i n f o r m a t i o n t h a t m i g h t b e c o m p l e m e n t a r y

t o o t h e r i o n i z a t i o n t e c h n i q u e s . A p r e v i o u s l y p u b -

l i s h e d p a p e r o n o l i g o p e p t i d e s [ 1 1 ] a n d f . d . m a s s

s p e c t r a l w o r k o n t r i p e p t i d e s i n o u r l a b o r a t o r y [ 12]

i n d i c a t e d t h e p r e s e n c e o f s o m e s e q u e n c e - s p e c i f i c

f r a g m e n t i o n s u p o n f . d . i o n i z a t i o n , a t a p p r o p r i a t e

field i o n e m i t t e r t e m p e r a t u r e s . S u c h a d d i t i o n a l

i n f o r m a t i o n w o u l d b e e s p e c i a l l y u s e f u l i n t h e c a s e o f

M. Przybylski et al. • Field Desorption Mass Spectra of Oligopeptides 737

s u b s t i t u t e d or " b l o c k e d " p h y s i o l o g i c a l p e p t i d e s

(such as m a n y p e p t i d e h o r m o n e s ) t h a t c a n n o t b e

i so la ted in a m o u n t s s u f f i c i e n t f o r d e r i v a t i z a t i o n a n d

a l t e r n a t e m a s s s p e c t r a l t e c h n i q u e s . A s p a r t o f a

s y s t e m a t i c s t u d y of b i o l o g i c a l p e p t i d e s b y f . d . m a s s

s p e c t r o m e t r y , w e h a v e i n v e s t i g a t e d t h e f . d . m a s s

s p e c t r a o f t w o such e x a m p l e s : a) t h e t e t r a p e p t i d e -

a m i d e , T r p - M e t - A s p - P h e - N H 2 , w h i c h r e p r e s e n t s t h e

b i o l o g i c a l l y essent ia l C - t e r m i n a l s e q u e n c e o f h u m a n

g a s t r i n e I [13], u s i n g di- a n d t r i p e p t i d e s o f t h i s

s e q u e n c e as m o d e l s t o e v a l u a t e p o s s i b l e f r a g m e n t

ions u n d e r f . d . i o n i z a t i o n ; b) s e v e r a l d e r i v a t i v e s

( c o n j u g a t e s ) o f g l u t a t h i o n e ( y - G l u - C y s - G l y , G S H ) ,

a n i m p o r t a n t ce l lu lar " t r a p p i n g " a g e n t in m a n y

m e t a b o l i c d e t o x i c a t i o n p a t h w a y s o f drugs . T h e

p o t e n t i a l u t i l i t y of f . d . m a s s s p e c t r o m e t r y f o r t h e

d irect s t r u c t u r a l c h a r a c t e r i z a t i o n o f G S H d e r i v a -

t i v e s w a s o f p a r t i c u l a r i n t e r e s t , s ince s e l e c t i v e

h y d r o l y s i s r e a c t i o n s f o r t h e s e p a r a t e a n a l y s i s o f t h e

s u b s t r a t e m e t a b o l i t e a r e u s u a l l y n o t poss ib le f o r

t h i s t y p e o f c o n j u g a t e . C . i . m a s s s p e c t r o m e t r y o f

t h e G S H d e r i v a t i v e s i n v e s t i g a t e d in t h i s s t u d y d i d

n o t r e s u l t in t h e i r p o s i t i v e s t r u c t u r a l c h a r a c t e r i z a -

t i o n [14]. F . d . m a s s s p e c t r a o f s o m e G S H c o n j u g a t e s

w i t h r e a c t i v e m e t a b o l i t e s w e r e a l r e a d y b r i e f l y re-

p o r t e d [14].

Experimental T h e s y n t h e s i s o f t h e g a s t r i n e p e p t i d e s h a s b e e n de-

s c r i b e d p r e v i o u s l y [15]. T h e d i p e p t i d e M e t - A s p - O H w a s a c o m m e r c i a l p r o d u c t ( S e r v a , H e i d e l b e r g , W . -G e r m a n y ) . I t w a s h o m o g e n o u s o n t h i n - l a y e r c h r o m a t o g r a p h y in m e t h a n o l / w a t e r / a c e t i c a c i d , 4/4/1. G l u t a t h i o n e w a s p u r c h a s e d f r o m M e r c k , D a r m s t a d t , W . - G e r m a n y . T h e G S H d e r i v a t i v e s w e r e i s o l a t e d as c o n j u g a t i o n p r o d u c t s w i t h r e a c t i v e m e t a b o l i t e s o f t h e d r u g s a c e t y l h y d r a z i n e , i s o p r o p y l -h y d r a z i n e a n d o t h e r c o m p o u n d s a f t e r i n c u b a t i o n w i t h r a t l i v e r m i c r o s o m e s as d e s c r i b e d p r e v i o u s l y [16]. D e t a i l s o f t h e i s o l a t i o n p r o c e d u r e s , c h a r a c -t e r i z a t i o n a n d b i o l o g i c a l p r o p e r t i e s o f t h e s e c o m -p o u n d s h a v e b e e n r e p o r t e d [17] .

T h e f. d . m a s s s p e c t r a w e r e r e c o r d e d w i t h a V a r i a n M A T C H 7 1 1 d o u b l e f o c u s i n g s p e c t r o m e t e r e q u i p p e d w i t h a n e.i ./f . i ./f .d. c o m b i n a t i o n i o n source . A c t i -v a t e d 10 jum t u n g s t e n f ie ld i o n e m i t t e r s w e r e u n i f o r m l y p r e p a r e d b y a h i g h t e m p e r a t u r e a c t i -v a t i o n p r o c e d u r e d e v e l o p e d b y B e c k e y a n d S c h u l -t e n [9] a n d w e r e u s e d o n c e f o r e a c h p e p t i d e . T h e p e p t i d e s w e r e d i s s o l v e d in D M S O a t c o n c e n t r a t i o n s o f 5 0 - 1 0 0 jug/20 ju\ a n d l o a d e d o n t h e e m i t t e r b y t h e d i p p i n g t e c h n i q u e [8]. T h e e m i t t e r w a s t h e n i n t r o -d u c e d in t h e ion s o u r c e b y a d i r e c t i n s e r t i o n l o c k a n d h e a t e d g r a d u a l l y ( 2 - 3 m i n ) f r o m a m b i e n t t o a

m a x i m u m h e a t i n g c u r r e n t o f a p p r o x i m a t e l y 20 m A . D u r i n g t h i s t i m e r e p e t i t i v e s p e c t r a w e r e r e c o r d e d o n a m a g n e t i c t a p e o f a c o m p u t e r ( V a r i a n S S 100). T h e e m i t t e r h e a t i n g c u r r e n t a t w h i c h t h e h i g h e s t r e l a t i v e a b u n d a n c e o f m o l e c u l a r ions o c c u r r e d is r e f e r r e d t o as " b e s t a n o d e t e m p e r a t u r e " ( B A T ) [8]. B o t h s p e c t r a r e c o r d e d a t B A T a n d f u r t h e r s p e c t r a a t g r e a t e r e m i t t e r t e m p e r a t u r e s w e r e e v a l u a t e d f o r f r a g m e n t ions . I n s t r u m e n t a l c o n d i t i o n s w e r e : 1000 ( 1 0 % ) r e s o l u t i o n , 11 k V p o t e n t i a l b e t w e e n field e m i t t e r a n o d e a n d c a t h o d e , 220 °C s o u r c e filament t e m p e r a t u r e , 2.8 k V v o l t a g e o f t h e e l e c t r o n m u l t i -pl ier u s e d f o r ion d e t e c t i o n . E . i . s p e c t r a o f h i g h boi l ing p e r f l u o r o k e r c s i n e (Merck, D a r m s t a d t , W . -G e r m a n y ) w e r e u s e d f o r m a s s c a l i b r a t i o n .

Results

Gastrine peptides A b u n d a n t m o l e c u l a r ions o f t h e C - t e r m i n a l t e t r a -

p e p t i d e o f g a s t r i n e I , T r p - M e t - A s p - P h e - N H 2 , w e r e

o b t a i n e d in s e v e r a l f . d . s p e c t r a a t e m i t t e r a n o d e

c u r r e n t s o f 10 m A or g r e a t e r . T h e s p e c t r u m a t

14 m A ( F i g . 1) t h a t c o n t a i n e d t h e m o s t u s e f u l

f r a g m e n t a t i o n p a t t e r n st i l l s h o w s t h e M+-ion

(m/e 596) as w e l l as t h e M H + - i o n (m/e 597) w i t h

r e l a t i v e intens i t ies o f m o r e t h a n 3 0 % . T h e a p p e a r -

a n c e o f " c l u s t e r " m o l e c u l a r ions ( m a i n l y M H + ) is

b e l i e v e d t o arise f r o m field e m i t t e r s u r f a c e r e a c t i o n s

a n d h a s b e e n c o n s i s t e n t l y o b s e r v e d in f . d . m a s s

s p e c t r o m e t r y [8]. A s a c o n s e q u e n c e , f . d . f r a g m e n t

ions f r e q u e n t l y a lso o c c u r as c l u s t e r ions, w i t h f o r m a l

a b s t r a c t i o n a n d a d d i t i o n o f h y d r o g e n . W i t h t h i s

c o n s i d e r a t i o n , a series o f f r a g m e n t a t i o n p r o d u c t s

l e a d i n g t o p a r t l y o v e r l a p p i n g s e q u e n c e i n f o r m a t i o n

c a n be i d e n t i f i e d f r o m t h e s p e c t r u m o f T r p - M e t - A s p -

P h e - N H 2 . I f ions ar i s ing b y d i r e c t c l e a v a g e o f

p e p t i d e b o n d s f r o m t h e m o l e c u l a r i o n a r e a s s i g n e d

C i , C2 etc., (C-terminus) a n d N i , N2 etc. ( N - t e r m i n u s ) ,

a l l poss ib le C - t e r m i n a l " s e q u e n c e " i o n s (m/e 164,

165, C i ; m/e 279, 280, C 2 ; m/e 4 1 1 , C 3 ) are f o u n d . O f

t h e t h r e e poss ib le N - t e r m i n a l ions, N3 (m/e 432, 433)

a n d N2 (m/e 3 1 7 , 318) are o b s e r v e d , w h i l e a f r a g m e n t

c o r r e s p o n d i n g t o N i (m/e 187) is m i s s i n g a n d w a s n o t

o b s e r v e d i n r e p e t i t i v e s c a n s a t e m i t t e r h e a t i n g

c u r r e n t s f r o m 12 t o a b o u t 18 m A . W h i l e t h e C-

t e r m i n a l ions c a n be r a t i o n a l i z e d w i t h a m i n e end-

g r o u p s , b o t h a c y l i u m (as k n o w n f r o m e.i. f r a g m e n -

t a t i o n p a t h w a y s ) a n d k e t e n are poss ib le e n d g r o u p s

f o r t h e N - t e r m i n a l ions b u t c a n n o t be d e t e r m i n e d

e x a c t l y d u e t o t h e u n c e r t a i n t y in t h e m a s s assign-

m e n t o f f . d . f r a g m e n t ions. H o w e v e r , a f a i r l y g o o d

c o n s i s t e n c e in t h e f o r m a t i o n o f t h e s e i o n s w a s f o u n d

738 M. Przybylski et al. • Field Desorption Mass Spectra of Oligopeptides 738

Table I . Fragment ions formed b y direct cleavage o f peptide bonds in the f .d . mass spectra of Trp-Met-Asp-Phe-N H 2 (I), Met -Asp -Phe -NH 2 (II) , A s p - P h e - N H 2 ( I I I ) and M e t - A s p - O H (IV) .

m/e Partial sequence

N- , C-terminal sequence ion a

( % relative intensity )b

I I I I I I I V

433 Trp-Met -Asp N 3 (8) 411 Met - A s p -Phe - N H 2 C3 (6) MH+ (100) 318 Trp-Met N 2 (35) 280 A s p - P h e - N H 2 C2 (6) C2 (13) MH+ (100) 247 Met -Asp N 2 (11) MH+ (100) c

187 T r p N i ( - ) 164 P h e - N H 2 Ci (20) Ci (15) Ci (16) 132 Met Ni ( — ) Ni (8)d 117 A s p Ni (20)

a Partial sequences starting f rom the N-terminus are assigned N i , N 2 , . . ., sequences starting f r o m the C-terminus Ci, C2 etc.

b See Table I I for emitter heating currents at which spectra were obtained, c MH+-ion, m/e 265 of Met -Asp -OH. d Same m/e-value as Ci-ion of Met -Asp-OH.

b y c o m p a r i s o n w i t h t h e f . d . m a s s s p e c t r a o f t h e

m o d e l p e p t i d e s , M e t - A s p - P h e - N H 2 , A s p - P h e - N H 2

( F i g . 2), a n d M e t - A s p - O H ( s p e c t r u m n o t s h o w n ) .

T h u s , a C 2 - i o n is a l s o o b s e r v e d i n t h e s p e c t r u m o f

M e t - A s p - P h e - N H 2 ( m o l e c u l a r i o n o f A s p - P h e - N H 2 ) ,

a n d t h e C i - i o n , P h e - N H 2 a p p e a r s i n t h e s p e c t r a o f

al l t h r e e h o m o l o g u e s p e p t i d e s . E x c e p t f o r t h e N i - i o n

t h a t is m i s s i n g i n t h e s p e c t r u m o f M e t - A s p - P h e - N H 2 ,

N - t e r m i n a l i o n s w e r e a l s o o b s e r v e d f o r t h e di- a n d

t r i p e p t i d e s ( T a b l e I ) .

L e s s c o n s i s t e n t l y , o t h e r f r a g m e n t a t i o n p r o d u c t s

w e r e o b s e r v e d . I n t h e s p e c t r a o f a l l p e p t i d e s ,

e l i m i n a t i o n o f w a t e r and/or N H 3 , a n d C 0 2 or C O O H

l e a d s t o a b u n d a n t i o n s a t e m i t t e r t e m p e r a t u r e s

a b o v e B A T , b u t c a n n o t be d i f f e r e n t i a t e d c l e a r l y

w i t h i n t h e c o r r e s p o n d i n g c l u s t e r i o n s . T h e s e f r a g -

m e n t s s e e m t o o c c u r u n s p e c i f i c a l l y i n f . d . m a s s

s p e c t r a o f p e p t i d e s [ 1 1 , 12], A m o n g o t h e r d i r e c t

f r a g m e n t a t i o n r e a c t i o n s o f t h e p e p t i d e b a c k -

b o n e , s e v e r a l i o n s o r i g i n a t e b y N - a l k y l c l e a v a g e ,

s u c h a s m/e 449 a n d 263 i n t h e s p e c t r u m o f

T r p - M e t - A s p - P h e - N H 2 a n d m/e 262/263 a n d 1 4 9 i n

t h e s p e c t r u m o f M e t - A s p - P h e - N H 2 ( T a b l e I I ) .

I n t h e f . d . s p e c t r a o f M e t - A s p - P h e - N H 2 a n d

A s p - P h e - N H 2 , s o m e f r a g m e n t s c o u l d a l s o b e as-

s i g n e d t o d i r e c t C - C c l e a v a g e r e a c t i o n s , f o r e x a m p l e

i o n s a t m/e 307/308 a n d m/e 1 9 1 / 1 9 2 (cf. F i g . 2). T h e

f o r m a t i o n o f t h e s e i o n s w a s q u i t e i n c o n s i s t e n t a t

- 100—1

CI 164,165

K9

HJN-CH-CO

CJ 409

CJ r27»

-263

NH-CH-CO+NH- -CH-COfNH

SCH,

311-Ni

COOH 433-1

Nj

M.W. 596

Nj 317,31«

Cl 263 219,790 J _ J

371 C, 411 433 449

~I 400

578,579

M*\ MH' 596,597

543

550

562

1 150 200

I 250

I— 300

~I— 350 600

Fig. 1. F . d . Mass spectrum of Trp-Met-Asp-Pl ie -NH 2 at 14 raA emitter heating current.

M. Przybylski et al. • Field Desorption Mass Spectra of Oligopeptides 739

1 0 0 - .

7 5 -

5 0 -

2 5 -

-191 C '

I T 1 6 3

H ^ c J c C N H j c H - C O - H H ,

/ 131 y COOH ( Q ) 117—' N,

N, 117

C, 88 " 7 1 3 1 1 9 1 1 9 2

L . Ii i 1 . h li

M4 ',MH* 279,280

262,263

-kiL

M.W. 279

( a )

UJ ar 100-,

7 5 -

5 0 -

2 5 -

k .1 L T 100

r-306 C2 C1 r278 r163

p263 j-148

H2N—CH-pCO- NHTCH—C0--NH-J-CH-C0-NH2 147 ^

COOH SCHj 2A7-'

N2

228

C, 164

N 2 246,247

C2

280

M.W. 410

( b )

M - , M H *

410,411

395

148 149 . 1 9 1 . 2 6 3 |

i r _ i 1 1 1I1 S t _ J ii i , l

307,308

366,367

I 381

150 200 250 r

300

1 1 I L 1

350

1 1 r~ 400

Fig. 2. F.d. mass spectra of Asp-Phe-NH2 (a) and Met-Asp-Phe-NH2 (b), both at 10 mA emitter heating current.

Table II. F.d. fragment ions of peptides I, II, III and IV formed by N-Alkyl cleavage or other direct frag-mentation of the peptide backbone.

Fragment ion, mje Peptidea mA" (% relative intensity)

I 14 449(8), 263(7), 149(8) II 10 308(17), 307(15), 263(8),

262(5), 149(10), 148(9) III 10 192(15), 191(18), 131(15),

88(19) IV 9 160(10), 148(8), 104(15)

a Cf. Table I. b Emitter heating current at which spectrum was

recorded.

d i f f e r e n t f ie ld e m i t t e r t e m p e r a t u r e s , a n d t h e y m a y

be o f l i t t l e v a l u e f o r o b t a i n i n g a d d i t i o n a l i n f o r m a -

t i o n o f t h e p e p t i d e s e q u e n c e . H o w e v e r , w i t h t h e

e x c e p t i o n o f t h e f r a g m e n t mje 228 i n t h e s p e c t r u m

o f M e t - A s p - P h e - N H a t h a t m i g h t a r i s e b y e l i m i n a t i o n

o f w a t e r f r o m m/e 246, t h e r e w a s a n o t a b l e l a c k o f

m o r e a b u n d a n t i o n s c o r r e s p o n d i n g t o m u l t i p l e

f r a g m e n t a t i o n o r f r a g m e n t a t i o n o f t h e p e p t i d e s ide

c h a i n .

Derivatives of glutathione G l u t a t h i o n e ( G S H ) a s w e l l a s s e v e r a l o f i t s

d e r i v a t i v e s i s o l a t e d a s c o n j u g a t e s w i t h v a r i o u s d r u g

740 M. Przybylski et al. • Field Desorpt ion Mass Spectra o f Oligopeptides

SR

COOH

1 O

S \ 130 0

1 " '

SR

h 2 N / - C H \ c o / - n h - ^ c o o h

c l e a v a g e o f t h e y - G l u - C y s p e p t i d e b o n d w a s f o u n d ,

w h e r e a s i n n o c a s e c l e a v a g e o f t h e C y s - G l y b o n d

w a s o b s e r v e d ( F i g . 3 a n d 4). T h e s p e c t r a o f a l l G S H

d e r i v a t i v e s i n v e s t i g a t e d s h o w e d c h a r a c t e r i s t i c f r a g -

m e n t s a t m/e 130 a n d m/e 84 o f h i g h a b u n d a n c e ,

w h i c h a r e l i k e l y t o e x p l a i n b y t h e f o r m a t i o n o f a

p y r o g l u t a m y l i o n . T h e t e n d e n c y f o r a p r e f e r e n t i a l

f o r m a t i o n o f p y r o g l u t a m y l h a s b e e n p r e v i o u s l y

o b s e r v e d i n f . d . m a s s s p e c t r a o f o l i g o p e p t i d e s

c o n t a i n i n g a - G l u a t t h e N - t e r m i n u s [ 1 8 ] :

C o n s i s t e n t w i t h t h e f o r m a t i o n o f t h e p y r o g l u t a m y l

i o n , t h e c o m p l e m e n t a r y f r a g m e n t i o n s o f t h e

C y s - G l y r e s i d u e w e r e f o u n d t h r o u g h o u t t h e s e r i e s o f

HN

M / T 84

/CHv^

II 0

m e t a b o l i t e s [ 1 7 ] g a v e m o l e c u l a r p e a k s o f h i g h

i n t e n s i t i e s i n t h e i r f . d . s p e c t r a , a t a r a n g e o f e m i t t e r

h e a t i n g c u r r e n t s f r o m 10 t o 1 5 m A . M o r e c o n s i s t e n t l y

t h a n i n t h e c a s e o f t h e g a s t r i n e p e p t i d e s , t h e M H + -

i o n w a s t h e p r e d o m i n a n t m o l e c u l a r i o n s p e c i e s

(m/e 308 f o r G S H , F i g . 3 , a ) . A t e m i t t e r t e m p e r a -

t u r e s w h i c h p r o d u c e d f r a g m e n t i o n s ( a b o u t 1 2 m A

h e a t i n g c u r r e n t ) , a l l G S H d e r i v a t i v e s s h o w e d

e l i m i n a t i o n o f H 2 0 a n d / o r CO2 {e.g., m/e 290 a n d

2 6 3 f o r G S H , m/e 3 3 2 a n d 305 f o r S - A c e t y l g l u t a -

t h i o n e ) , p r e s u m a b l y b y s i m p l e p y r o l y t i c p r o c e s s e s .

H o w e v e r , a s m a j o r f r a g m e n t a t i o n p a t h w a y , t h e

100-

7 5 -

^ 5 0 ->

TR 2 5 -10 z UJ I— z uj 100-, >

^ 7 5 -

130

,84

H 2 N - C H - ( C H 2 ) 2 -

COOH

r c o p

Table I I I . Fragment ions f o rmed b y cleavage of y-Glu-Cys peptide bonds in the f . d . mass spectra o f glutathione derivatives.

H 2 N - C H - C O - N H - C H 2 - C O O H © -

CH 2

S R R m A a m/e ( % relative intensity)

H 13 178 (11) CH 3 14 192 (22) C2H5 12 206(100) CO-CH3 14 220 (42) CH(CH 3 ) 2 15 220 (25) CH2-C6H5 14 268 (14) l - (2 -hydroxy ) -estradiol 18 446 (18)b

a Emitter heating current at which spectrum was recorded.

* Fragment ion (464-H 2 0)®- .

130

177

N H - C H - C O - N H - C H 2 - C O O H

CH2 1 SH

290 161

J J J L 178

50 -

2 5 -

130

78

84

T 100

308 , MH M.W. 307

( a )

19 H 2 N - C H - C H 2 - C H 2 - C O 4 - N H 4 C H - C O - N H - C H 2 - C O O H

COOH 130 146 9 H 2 M.W

220.221

M.W. 349

( b )

350, MH 332

146

I 150

T

200

"I r 250 300

m/.

305

L i _ 4 _

350

Fig. 3. F . d . mass spectra o f y -Glu-Cys-Gly at 13 m A (a) and y -Glu -Cys (CO-CH 3 ) -G ly at 14 m A (b) emitter heating current.

M. Przybylski et al. • Field Desorption Mass Spectra of Oligopeptides 741

G S H d e r i v a t i v e s . A l t h o u g h f o r G S H i t s e l f , t h e a p p e a r e d p a r t i a l l y w i t h h i g h a b u n d a n c e s ( T a b l e I I I ) ,

f r a g m e n t C y s - G l y (m/e 1 7 8 ) w a s r a t h e r w e a k , t h e T h e r e f o r e , t h e s e c h a r a c t e r i s t i c i o n s p r o v i d e a p o s -

c o r r e s p o n d i n g i o n s o f t h e S - s u b s t i t u t e d d e r i v a t i v e s s i b i l i t y f o r t h e d i r e c t s u b s t r a t e i d e n t i f i c a t i o n o f

100-,

75-

50-

25-

100 - 1

> 75-

50-

25-

50

84

78

78

84

191

K^N-CH-CH2-CH2-COINH-CH-CO- NH-CH2-C00H 130 COOH

co-pt

130 CH2 i s I CH3

M.W. 321

(a )

192 277

1__L

322 J MH *

i

100

T 205

H2N-CH-CH2-CH2-CO{NH-CH-CO-NH-CH2-COOH

COOH

206 130

130

CH2

S

C2H5

M.W. 335

336 ,MH*

(b)

317

150 ~T

200 - r ~ 250

- r ~ 300

—1 350

Fig. 4. F.d. mass spectra of y-Glu-Cys(CH3)-Gly at 14 mA (a) and y-Glu-Cys(C2H5)-Gly at 12 mA (b) emitter heating current.

100-

80-

- 6 0 -

> 40-

z 20 UJ z

UJ

% ioon < —i Ui 80 K

60-

40-

20-

110

H2N-CH-CH2-CH2-COTNH-CH-CO-NH-CH2-COOH

130

I COOH

288

• N r

LUj

130 I CH2

iCZ F - H O

2 8 7 HO

320

M.W. 593

OH

" I — 300

370 445,

I . .1 'l •• i

445,446

T 350 400 *50

(a)

(b)

501

550

500 —I— 550

MH 594

579 1

Fig. 5. F.d. mass spectra of y-Glu-Cys-(1 - (2 -hydroxyestra-diol))-Gly at 14 mA (a)

r and 18 mA (b) emitter 600 heating current.

742 M. Przybylski et al. • Field Desorption Mass Spectra of Oligopeptides 742

c o r r e s p o n d i n g c o n j u g a t i o n p r o d u c t s o f d r u g s a n d

t h e i r m e t a b o l i t e s b y f . d . m a s s s p e c t r o m e t r y , i n

a d d i t i o n t o t h e m o l e c u l a r i o n . A s a n e x a m p l e f o r t h e

u t i l i t y o f f . d . m a s s s p e c t r o m e t r y t o i d e n t i f y cor-

r e s p o n d i n g m e t a b o l i t e c o n j u g a t e s w i t h G S H , w e

i n v e s t i g a t e d a G S H c o n j u g a t i o n p r o d u c t o f 2 - h y d r o -

x y e s t r a d i o l w h i c h w a s i s o l a t e d a s a m e t a b o l i t e

d u r i n g s t u d i e s o f t h e h e p a t i c m i c r o s o m a l m e t a b o l i s m

o f e s t r a d i o l [14] . T h e f . d . s p e c t r u m o f t h i s c o m p o u n d

( F i g . 5) s h o w e d a t 1 4 m A e m i t t e r h e a t i n g c u r r e n t

a l m o s t e x c l u s i v e l y t h e m o l e c u l a r i o n M H + (m/e 594)

c o n s i s t e n t w i t h a G S H c o n j u g a t e o f h y d r o x v l a t e d

e s t r a d i o l . I n t h e s p e c t r u m r e c o r d e d a t 18 m A

h e a t i n g c u r r e n t , n o m o l e c u l a r i o n b u t s e v e r a l

f r a g m e n t a t i o n p r o d u c t s p r o v i d i n g s t r u c t u r a l in-

f o r m a t i o n a r e o b t a i n e d ( F i g . 5 , b ) . B e s i d e s t h e i n t e n -

s i v e i o n o f t h e p y r o g l u t a m y l r e s i d u e (m/e 130), a

c o m p l e m e n t a r y i o n a t m/e 4 6 4 is n o t f o u n d , b u t a

c l u s t e r o f p e a k s c e n t e r e d a t m/e 4 4 6 i n s t e a d , w h i c h

i s l i k e l y t o b e f o r m e d b y c l e a v a g e o f t h e G l y - C y s

b o n d a n d a d d i t i o n a l e l i m i n a t i o n o f w a t e r f r o m t h e

s t e r o i d s k e l e t o n . I n a d d i t i o n , t h e t w o a b u n d a n t

f r a g m e n t i o n s a t m/e 320 a n d m/e 288 p r o v i d e d i r e c t

e v i d e n c e t o i d e n t i f y t h e c o n j u g a t i o n p o s i t i o n o f t h e

m e t a b o l i t e a t t h e C y s r e s i d u e (see f r a g m e n t a t i o n

s c h e m e d e p i c t e d i n F i g . 5) .

Discussion C o n s i s t e n t w i t h t h e p r e v i o u s s t u d i e s o f o l i g o -

p e p t i d e s b y f . d . m a s s s p e c t r o m e t r y , a b u n d a n t

m o l e c u l a r i o n s w e r e r e a d i l y o b t a i n e d i n t h e f . d .

s p e c t r a o f t h e g a s t r i n e p e p t i d e s a n d g l u t a t h i o n e

d e r i v a t i v e s w h i c h , w i t h o u t a n y d e r i v a t i z a t i o n , d i d

n o t g i v e u s e f u l s t r u c t u r e i n f o r m a t i o n b y e . i . o r e . i .

m a s s s p e c t r o m e t r y [14] . T h u s , t h e e . i . m a s s s p e c t r a

o f G S H a n d i t s d e r i v a t i v e s w e r e d o m i n a t e d b y a

m u l t i p l i c i t y o f p y r o l y t i c f r a g m e n t s i n t h e l o w m a s s

r e g i o n (M. P r z y b y l s k i , u n p u b l i s h e d o b s e r v a t i o n ) .

D e r i v a t i z a t i o n p r o c e d u r e s o f p e p t i d e s c o n t a i n i n g

a m i n o a c i d s w i t h m u l t i p l e f u n c t i o n a l g r o u p s a s

t h o s e s t u d i e d h e r e w o u l d l e a d t o a d r a s t i c a l i n c r e a s e

i n m o l e c u l a r w e i g h t a l r e a d y f o r s m a l l e r o l igo-

p e p t i d e s , i n t o a m a s s r a n g e t h a t m i g h t b e d i f f i c u l t

t o d e t e r m i n e e v e n i f s u f f i c i e n t v o l a t i l i t y f o r e . i . o r

e . i . m a s s s p e c t r a l a n a l y s i s is o b t a i n e d . T h u s , t h e f . d .

m a s s s p e c t r o m e t r i c m o l e c u l a r w e i g h t d e t e r m i n a t i o n

m i g h t b e a v a l u a b l e i n i t i a l s t e p i n t h e p r o b l e m o f

e l u c i d a t i n g t h e s t r u c t u r e o f o l i g o p e p t i d e s i s o l a t e d i n

s m a l l a m o u n t s f r o m b i o l o g i c a l m a t e r i a l . M o l e c u l a r

i o n s h a v e b e e n o b t a i n e d i n f . d . m a s s s p e c t r a o f

p e p t i d e s c o n t a i n i n g a m i n o a c i d s s u c h a s A r g [10]

a n d H i s [18] t h a t a r e k n o w n t o p r e s e n t d i f f i c u l t i e s

w i t h o t h e r m a s s s p e c t r o m e t r i c m e t h o d s . H o w e v e r ,

m o r e s y s t e m a t i c w o r k is n e e d e d t o s h o w w h e t h e r a

m o l e c u l a r i o n d e t e r m i n a t i o n o f o l i g o p e p t i d e s c a n b e

o b t a i n e d w i t h c e r t a i n t y b y f . d . m a s s s p e c t r o m e t r y .

A c r i t i c a l e x a m p l e h a s b e e n o b s e r v e d p r e v i o u s l y i n

f . d . m a s s s p e c t r a o f t r i p e p t i d e s w i t h a - G l u a t t h e

N - t e r m i n u s b e c a u s e o f t h e t e n d e n c y t o f o r m p y r o -

g l u t a m y l [12], y i e l d i n g a b u n d a n t ( M - H 2 0 ) ® - i o n s .

I n t h e ser ies o f G S H d e r i v a t i v e s ( N - t e r m i n a l y - G l u ) ,

p y r o g l u t a m y l f o r m a t i o n d i d n o t a f f e c t m o l e c u l a r

w e i g h t d e t e r m i n a t i o n b u t e v e n l e a d s t o f r a g m e n t

i o n s t h a t a l l o w t o d i f f e r e n t i a t e s p e c i f i c a l l y t h e

b i n d i n g s i te o f c o r r e s p o n d i n g d r u g m e t a b o l i t e - G S H

c o n j u g a t e s [14] (c . f . T a b l e I I I ) . A p r o b l e m i n t h e

d e f i n i t e m o l e c u l a r w e i g h t d e t e r m i n a t i o n o f p e p t i d e s

b y f . d . m a s s s p e c t r o m e t r y c o u l d b e t h e f o r m a t i o n

o f c l u s t e r i o n s [8, 19, 20]. B e s i d e s t h e a d d i t i o n o f a

p r o t o n (MH®-ion), p e a k s d u e t o t h e a d d i t i o n o f H 2 O

or o t h e r s m a l l m o l e c u l e s s u c h a s e t h a n o l p r e s e n t in

t h e s a m p l e s o l u t e h a v e b e e n o b s e r v e d [ 1 1 ] , a l t h o u g h

t h e s e i o n s a r e g e n e r a l l y o f l o w e r i n t e n s i t y t h a n t h e

m o l e c u l a r i o n . I n t h e c a s e o f a c o m p l e t e l y u n k n o w n

s a m p l e , a c e r t a i n m o l e c u l a r w e i g h t d e t e r m i n a t i o n

m i g h t t h e r e f o r e b e s t b e o b t a i n e d i n c o n n e c t i o n w i t h

a n i n d e p e n d a n t a m i n o a c i d a n a l y s i s w h i c h , l i k e w i s e ,

w i l l b e v e r y u s e f u l f o r t h e i n t e r p r e t a t i o n o f f r a g m e n t

i o n s i n f . d . m a s s s p e c t r a .

I n a d d i t i o n t o t h e m o l e c u l a r i o n i n f o r m a t i o n

a v a i l a b l e , t h e p r e s e n t f . d . m a s s s p e c t r a l d a t a o f

p e p t i d e s s e e m t o i n d i c a t e t h a t a t l e a s t s o m e d e g r e e

o f u s e f u l s t r u c t u r e i n f o r m a t i o n c a n b e o b t a i n e d

f r o m f r a g m e n t i o n s a r i s i n g a b o v e B A T . I n t h e

ser ies o f t h e g a s t r i n e p e p t i d e s , a n a l m o s t c o m p l e t e

s e q u e n c e o f p e p t i d e b o n d c l e a v a g e p r o d u c t s

( " s e q u e n c e i o n s " [21]) w a s f o u n d . A l t h o u g h t h e

m e c h a n i s m s o f f . d . f r a g m e n t a t i o n p a t h w a y s a r e

l a r g e l y u n k n o w n , f r a g m e n t i o n f o r m a t i o n s e e m s t o

o r i g i n a t e m a i n l y f r o m t h e m o l e c u l a r i o n d i r e c t l y

w h i c h , i n t h e c a s e o f p e p t i d e s , f a c i l i t a t e s g r e a t l y

t h e i r a s s i g n m e n t t o p a r t s o f t h e m o l e c u l a r s t r u c t u r e .

T h e o b s e r v a t i o n f r o m t h i s a n d p r e v i o u s i n v e s t i g a -

t i o n s t h a t f . d . f r a g m e n t a t i o n o f t e n y i e l d s c o m p l e -

m e n t a r y b r e a k d o w n p r o d u c t s [22] m i g h t b e a

f u r t h e r a d v a n t a g e t h a t f a c i l i t a t e s t h e a n a l y s i s o f

s e q u e n c e i o n s . T h e q u e s t i o n o f d i f f e r e n t i a t i n g

t h e r m a l a n d f i e l d - i n d u c e d f r a g m e n t a t i o n r e a c t i o n s

h a s r e c e n t l y b e e n i n v e s t i g a t e d i n a d e t a i l e d m o d e l

s t u d y o n t h e f . d . f r a g m e n t a t i o n o f m e t h i o n i n e [23],

M. Przybylski et al. • Field Desorption Mass Spectra of Oligopeptides 743

u s i n g t e c h n i q u e s s u c h a s i s o t o p e l a b e l l i n g a n d h i g h

r e s o l u t i o n d a t a . F o r o l i g o p e p t i d e s , a n i m p o r t a n t

f a c t o r m i g h t b e t h e d r a s t i c a l c h a n g e s i n v o l a t i l i t y

a n d c o r r e s p o n d i n g d e s o r p t i o n r a t e s w i t h i n t h e

m o l e c u l a r s i ze r a n g e t h a t s e e m s p r a c t i c a b l e a t

p r e s e n t f o r f . d . a n a l y s i s ( p e p t i d e s w i t h u p t o 1 5 t o

20 a m i n o a c i d r e s i d u e s a s s u g g e s t e d b y W i n k l e r et al. f r o m a f . d . m a s s s p e c t r o m e t r i c s t u d y o f g l u c a g o n

[24]). T o b e t t e r u n d e r s t a n d t h e s e k e y q u e s t i o n s , f . d .

m a s s s p e c t r o m e t r i c s t u d i e s w i t h m o d e l p e p t i d e s o f

v a r i o u s s izes a r e p r e s e n t l y s u b j e c t o f f u r t h e r w o r k

i n o u r l a b o r a t o r y .

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