Field Desorption Mass Spectra of Gastrine Peptides and...
Transcript of Field Desorption Mass Spectra of Gastrine Peptides and...
This work has been digitalized and published in 2013 by Verlag Zeitschrift für Naturforschung in cooperation with the Max Planck Society for the Advancement of Science under a Creative Commons Attribution4.0 International License.
Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschungin Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung derWissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht:Creative Commons Namensnennung 4.0 Lizenz.
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 .
[1] K. Biemann, in G. R. Waller (ed.): Biochemical Applications of Mass Spectrometry, p. 405, Wiley -Interscience, New York 1972.
[2] P. J. Arpino and F. W. McLafferty, in F. C. Nachod, J. J. Zuekerman, and E. W. Randall (ed.): Determination of Organic Structures by Physical Methods, Vol. 6, pp. 1-89, Academic Press, New York 1976.
[3] H. R. Morris, in R. H. Pain and B. J. Smith (ed.): New Techniques in Biophysics and Cell Biology, p. 149, Wiley-Interscience, New York 1973.
[4] B. C. Das and E. Lederer, in A. Niederwieser and E. Pataki (eds.): New Techniques in Amino Acids, Peptide, and Protein Analysis, p. 175, Ann Arbor Sei. Publ., Ann Arbor 1971.
[5] H. Nau, H. J. Förster, J. A. Kelley, and K. Bie-mann, Biomed. Mass Spectrom. 2, 326 (1975).
[6] H. Nau, Angew. Chem. 88, 74 (1976); and refer-ences therein.
[7] B. G. Dawkins, P. J. Arpino, and F. W. McLaf-ferty, Biomed. Mass Spectrom. 5, 1 (1978).
[8] H. D. Beckey and H. R. Schulten, Angew. Chem. 87, 425 (1975).
[9] H. R. Schulten and H. D. Beckey, Org. Mass Spectrom. 6, 885 (1972).
[10] H. U. Winkler and H. D. Beckey, Biochem. Biophys. Res. Commun. 46, 391 (1972).
[11] S. Asante-Poku, G. W. Wood, and D. E. Schmidt (Jr.), Biomed. Mass Spectrom. 2, 121 (1975).
[12] I. Lüderwald, M. Przybylski, H. Ringsdorf, D.
Silberhorn, K. Zech, and W. Voelter, Adv. Mass Spectrom. 7 B, 1513 (1978).
[13] J. Beacham, P. H. Bentley, R. A. Gregory, G. W. Kenner, J. K. Macleod, and R. C. Sheppard, Nature (London) 209, 586 (1969).
[14] S. D. Nelson, Y. Vaishuav, M. Przybylski, 25th Int. Conference on "Mass Spectrometry and Allied Topics", Abstr. p. 509, Washington, U.S. A., 1977.
[15] W. Voelter, E. Kraas, and S. Fuchs, J. Pak. Chem. Soc., in press.
[16] S. D. Nelson, J. Hinson, and J. R. Mitchell, Biochem. Biophys. Res. Commun. 69, 900 (1976).
[17] S. D. Nelson, J. R. Mitchell, J. A. Timbrell, W. R. Snodgrass, and G. B. Corcoran, Science 193, 901 (1976).
[18] I. Lüderwald, M. Przybylski, H. Ringsdorf, D. Silberhorn, H. Kalbacher, and W. Voelter, Z. Naturforsch. 33b, 805, 809 (1978).
[19] F. W. Röllgen and H. R. Schulten, Org. Mass Spectrom. 10, 660 (1975).
[20] H. J. Veith, Angew. Chem. 88, 762 (1976). [21] M. Senn, R. Venkataraghavan, and F. W. McLaf-
ferty, J. Am. Chem. Soc. 88, 5593 (1966). [22] H. R. Schulten, in D. Glick (ed.): Methods of
Biochemical Analysis, Vol. 24, 313, D. Glick, Wiley-Interscience, New York 1977.
[23] J. van der Greef, N. M. M. Nibbering, H. R. Schulten, and W. D. Lehmann, Z. Naturforsch. 33b, 770 (1978).
[24] H. U. Winkler, R. J. Beuhler, and L. Friedman, Biomed. Mass Spectrom. 3, 201 (1976).