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PLASMA DESORPTION MASS SPECTROMETRY : THE STABILITY OF MOLECULAR IONS
L. Silly, R. Cotter
To cite this version:
L. Silly, R. Cotter. PLASMA DESORPTION MASS SPECTROMETRY : THE STABIL- ITY OF MOLECULAR IONS. Journal de Physique Colloques, 1989, 50 (C2), pp.C2-37-C2-40.
�10.1051/jphyscol:1989207�. �jpa-00229403�
JOURNAL DE PHYSIQUE
Colloque C2, supplbment au n02, Tome 50, fbvrier 1989
PLASMA DESORPTION MASS SPECTROMETRY : THE STABILITY OF MOLECULAR IONS
L. SILLY and R . J . COTTER
Middle Atlantic Mass Spectrometry Facility, Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of
Medicine, Baltimore, MD 21205, U.S.A.
Resume
L e s l a r g e u r s d e p i c s e t l e s i n t e n s i t e s r e l a t i v e s d e s i o n s m o l C c u l a i r e s m o n o e t m u l t i - c h a r g e s a p p a r a i s s a n t d a n s l e s s p e c t r e s d e m a s s e p a r p l a s m a d e s o r p t i o n s o n t u t i l i s e e s c o m m e u n e s o n d e p o u r l a s t a b i l l t e d e s i o n s m o l e c u l a i r e s . O e u x p e p t i d e s q u i o n t d e s p o i d s m o l e c u l a i r e s e t d e s s t r u c t u r e s t e r t i a i r e s i d e n t i q u e s m a i s d e s s t r u c t u r e s p r i m a i r e s e t d e s p o i n t s i s o - e l e c t r i q u e s d i f f k r e n t s o n t e t 8 p r e p a r e s e n s o l u t i o n s d e p H d i v e r s a v a n t d l & t r e a d s o r b e s s u r n i t r o c e l l u l o s e . L e s l a r g e u r s d e p i c s d e s i o n s m o l e c u l a i r e s r e s u l t a n t s o n t k t 6 o p t i m i s e e s p r e s d e l e u r s p o i n t s i s o - e l e c t r i q u e s r e s p e c t i f s . D e s e t u d e s s i m i l a i r e s o n t e t e e n t r e p r i s e s e n a j o u t a n t d u g l u t a t h i o n , u n e m a t r i c e q u i a d e s e f f e t s s u r l e s s p e c t r e s P D M S e q u i v a l e n t s .3 l a n i t r o c e l l u l o s e .
A b s t r a c t
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The peak w i d t h s and r e l a t i v e i n t e n s i t i e s o f t h e s i n g l y - andm u l t i p l y - c h a r g e d m o l e c u l a r i o n s a p p e a r i n g i n plasma d e s o r p t i o n mass s p e c t r a a r e used as a probe f o r t h e s t a b i l i t y o f m o l e c u l a r i o n s . Two p e p t i d e s , lysozyme and
l a c t a l b u m i n , w h i c h have s i m i l a r m o l e c u l a r w e i g h t s and t e r t i a r y s t r u c t u r e s , b u t d i f f e r e n t p r i m a r y s t r u c t u r e s and i s o e l e c t r i c p o i n t s , were p r e p a r e d i n s o l u t i o n s o f d i f f e r e n t pH, b e f o r e a d s o r p t i o n t o n i t r o c e l l u l o s e f o i l s . The peak w i d t h s o f t h e r e s u l t a n t m o l e c u l a r i o n s i g n a l s were m i n i m i z e d n e a r t h e i r r e s p e c t i v e i s o e l e c t r i c p o i n t s , w h i c h i s c o n s i s t e n t w i t h r e p o r t s on t h e s t a b i l i t y o f t h e i r t e r t i a r y
s t r u c t u r e s a n d / o r e n z y m a t i c a c t i v i t i e s n e a r t h e i s o e l e c t r i c p o i n t . S i m i l a r s t u d i e s were c a r r i e d o u t w i t h t h e a d d i t i o n o f g l u t a t h i o n e , a m a t r i x w h i c h has e f f e c t s on PDMS s p e c t r a s i m i l a r t o t h a t o f t h e n i t r o c e l l u l o s e s u r f a c e .
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INTRODUCTIOND u r i n g t h e l a s t s e v e r a l y e a r s , a number o f new mass s p e c t r a l i o n i z a t i o n t e c h n i q u e s have been developed, w h i c h employ h i g h l y e n e r g e t i c p h o t o n and p a r t i c l e beams. These i n c l u d e : l a s e r d e s o r p t i o n / I / , s e c o n d a r y i o n mass s p e c t r o m e t r y 121, f a s t atom bombardment / 3 / and plasma d e s o r p t i o n mass s p e c t r o m e t r y 141. These t e c h n i q u e s a r e o f c o n s i d e r a b l e i n t e r e s t t o b i o c h e m i s t s , because ( d e s p i t e t h e h i g h p r i m a r y e n e r g i e s ) t h e y have enabled t h e d e s o r p t i o n o f i n t a c t m o l e c u l a r i o n s o f complex b i o m o l e c u l es and t h e i r subsequent mass a n a l y s i s by mass s p e c t r o m e t r y . I n a d d i t i o n , t h e absence o f s u b s t a n t i a l and i n f o r m a t i v e gas phase f r a g m e n t a t i o n o f m o l e c u l e s above 5000 d a l t o n s suggests t h a t such i o n s a r e formed w i t h unusual s t a b i l i t y and w i t h v e r y 1 i t t l e i n t e r n a l energy.
I n t h e p a r t i c l e t e c h n i q u e s , p r i m a r y i o n s have been used i n b o t h t h e k i l o v o l t and m e g a v o l t r e g i o n . The l a t t e r were f i r s t employed f o r b i o m o l e c u l a r a n a l y s i s b y M a c f a r l a n e and Torgerson 141, who r e c o r d e d t h e i r mass s p e c t r a u s i n g a t i m e - o f - f l i g h t a n a l y z e r . W h i l e t h e m a j o r c o h e r e n t i o n s i g n a l f o r l a r g e p e p t i d e s i s observed as t h e m o l e c u l a r i o n peak,
c o n s i - d e r a b l e f r a g m e n t a t i o n does o c c u r i n t h e a c c e l e r a t i n g and d r i f t r e g i o n s o f t h e
t i m e - o f - f l i g h t a n a l y z e r , b u t c o n t r i b u t e s p r i m a r i l y t o t h e i n c o h e r e n t b a s e l i n e and t h e peak w i d t h /5-8/, and i s l a r g e l y due t o t h e l o s s o f s m a l l p e r i p h e r a l groups /5,9,10/.
Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:1989207
C2-38 JOURNAL
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PHYSIQUEI n t h e PDMS t e c h n i q u e , samples a r e d e p o s i t e d on a t h i n aluminum o r a l u m i n i z e d m y l e r f o i l by e l e c t r o s p r a y i n g a c i d i c s o l u t i o n s o f t h e sample /11/. Heavy fragments from t h e spontaneous f i s s i o n o f Cf-252 p e n e t r a t e t h e f o i l and desorb secondary i o n s , whose masses a r e measured b y t h e i r f l i g h t t i m e s a f t e r a c c e l e r a t i o n . R e c e n t l y , two a l t e r n a t i v e
approaches t o t h e s i m p l e e l e c t r o s p r a y i n g t e c h n i q u e have been developed. These i n c l u d e t h e a d d i t i o n o f t h e t r i p e p t i d e g l u t a t h i o n e t o t h e a c i d i c s o l u t i o n used t o e l e c t r o s p r a y t h e sample /12/ and t h e use o f n i t r o c e l l u l o s e s u r f a c e s f o r a d s o r p t i o n o f t h e sample /13/.
What i s remarkable i s t h a t t h e y have i d e n t i c a l e f f e c t s on t h e mass s p e c t r a : an i n c r e a s e i n t h e m o l e c u l a r i o n s i g n a l r e l a t i v e t o t h e i n c o h e r e n t b a s e l i n e , an i n c r e a s e i n t h e abundance o f m u l t i p l y - c h a r g e d i o n s , and a s i g n i f i c a n t decrease i n t h e s p e c t r a l peak w i d t h s
/12,14,15/. I n b o t h cases, i t has been suggested t h a t such f e a t u r e s r e f l e c t a l o w e r i n g o f t h e b i n d i n g energy between sample and s u b s t r a t e /12,15/. A d d i t i o n a l l y , A l a i
,
e t . a1.
/12/have noted t h e use o f g l u t a t h i o n e i n t h e r e f o l d i n g o f lysozyme /16/, and suggested t h a t f o l d e d p e p t i d e s a r e more l o o s e l y bound t o t h e s u r f a c e and a r e desorbed w i t h l o w e r i n t e r n a l energy. R o e p s t o r f f , e t . a1
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/17/ have a l s o suggested t h a t t h e use o f n i t r o c e l l u l o s e s u r f a c e s o r t h e g l u t a t h i o n e m a t r i x r e s u l t s i n t h e d e s o r p t i o n o f " c o o l e r 1 ' m o l e c u l a r ions, w h i l e C h a i t /18/ has shown t h a t m o l e c u l a r i o n s o f p o r c i n e i n s u l i n desorbed fromn i t r o c e l l u l o s e undergo l e s s m e t a s t a b l e f r a g m e n t a t i o n t h a n t h o s e from e l e c t r o s p r a y e d f o i l s . I n t h i s s t u d y , two p e p t i d e s : bovine m i l k c ( - l a c t a l b u m i n and c h i c k e n egg w h i t e lysozyme, w i t h s i m i l a r m o l e c u l a r w e i g h t s (14,176 and 14,307 amu r e s p e c t i v e l y ) , s i m i l a r t e r t i a r y s t r u c t u r e s , b u t d i f f e r e n t i s o e l e c t r i c p o i n t s ( P I = 5 and 11 r e s p e c t i v e l y ) , a r e compred.
I n b o t h cases, t h e i s o e l e c t r i c p o i n t can be c o r r e l a t e d w i t h b i o l o g i c a l a c t i v i t y and s t a b l e conformations. Davies and coworkers /19/ have s t u d i e d t h e enzymatic and l y t i c a c t i v i t y o f lysozyme as a f u n c t i o n o f t h e i o n i c s t r e n g t h o f s o l u t i o n s o f t h e p e p t i d e a t d i f f e r e n t pH.
A t l o w i o n i c s t r e n g t h , maximum enzymatic a c t i v i t y o c c u r s a t a pH o f 9.2, w h i l e l y t i c a c t i v i t y i s v e r y h i g h above pH 10. Saint-Blancard, et. a l . /20/ have c o n f i r m e d t h e l y t i c a c t i v i t y a t h i g h pH, and suggested t h a t t h e i s o e l e c t r i c p o i n t r e p r e s e n t s a v e r y s t a b l e c o n f o r m a t i o n a l s t r u c t u r e . For l a c t a l b u m i n , Hanssens e t . a l . /21/ have used f l u o r e s c e n c e measurements t o determine t h a t maximum s t a b i l i z a t i o n o f c o n f o r m a t i o n o c c u r s i n t h e range o f pH 5-4, near t h e i s o e l e c t r i c p o i n t . Thus, we examined b o t h t h e abundances o f m u l t i p l y charged i o n s and t h e peak w i d t h s i n t h e mass s p e c t r a o f lysozyme and l a c t a l b u m i n , w h i c h were prepared i n s o l u t i o n s o f d i f f e r e n t pH p r i o r t o a d s o r p t i o n on n i t r o c e l l u l o s e .
A d d i t i o n a l l y , s i n c e t h e p e p t i d e g l u t a t h i o n e has a l s o been r e p o r t e d as a r e f o l d i n g r e a g e n t /16/, we measured abundances and peak w i d t h s as w e l l f o l l o w i n g t h e a d d i t i o n o f i n c r e a s i n g amounts o f g l u t a t h i o n e t o t h e sample s o l u t i o n .
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EXPERIFIENTAL METHODSMass s p e c t r a l measurements were made u s i n g a BIO-ION Nordic (Uppsala, Sweden) BIN 10K plasma d e s o r p t i o n mass spectrometer, equipped w i t h a 10 uCi source o f Cf-252. Data were a c q u i r e d on a PDP 11/73 microcomputer, w i t h a t i m e r e s o l u t i o n o f 1 ns/channel. Chicken egg w h i t e lysozyme and b o v i n e mi.lk 4 - l a c t a l b u m i n were purchased from Sigma Co. I n t h e f i r s t experiment samples were d i s s o l v e d i n aqueous s o l u t i o n s o f a c e t i c a c i d and ammonium h y d r o x i d e a t d i f f e r e n t c o n c e n t r a t i o n s t o a c h i e v e a range o f pH f r o m 1 t o 11, d e p o s i t e d on n i t r o c e l l u l o s e - c o a t e d f o i l s , and a n l y z e d a f t e r 6-8 hours. I n t h e second experiment, 100 ug o f p e p t i d e was d i s s o l v e d i n s o l u t i o n s o f g l a c i a l a c e t i c a c i d c o n t a i n i n g v a r i o u s c o n c e n t r a t i o n s o f g l u t a t h i o n e , and t h e n e l e c t r o s p r a y e d o n t o a1 uminized m y l a r f o i l s .
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RESULTS AiID DISCUSSIOrlF i g u r e 1 shows t h e peak w i d t h s o f t h e s i n g l y - , doubly- and t r i p l y - c h a r g e d m o l e c u l a r i o n s o f l a c t a l b u m i n d e p o s i t e d on n i t r o c e l l u l o s e as a f u n c t i o n o f pH. Near t h e i s o e l e c t r i c p o i n t ( P I = 5 ) t h e peak w i d t h s reach a minimum, w h i l e t h e m o l e c u l a r i o n i n t e n s i t i e s and t h e r a t i o o f h i g h e r charged s p e c i e s ( d a t a n o t shown) reach a maximum. F i g u r e 2 shows t h e r e s u l t s from a s i m i l a r experiment w i t h lysozyme ( P I = 11) where t h e minimum peak w i d t h s a r e achieved a t v e r y h i g h pH.
When lysozyme and l a c t a l b u m i n a r e " t i t r a t e d " w i t h g l u t a t h i o n e ( d a t a n o t shown) m o l e c u l a r i o n i n t e n s i t i e s and charge s t a t e s i n c r e a s e , w h i l e peak w i d t h s decrease u n t i l t h e m o l a r r a t i o ( g l u t a t h i o n e / p e p t i d e ) i s about 20 t o 30, a f t e r which t h e s e parameters remain f a i r l y c o n s t a n t /22/. I n b o t h cases, a v e r y c l e a r " e n d p o i n t " i s observed. The decreased p e a k w i d t h s for lysozyme (70-8011s) and l a c t a l b u n i n (100-120IIs) a r e comparable t o t h e minima i n 51e n i t r o - ~ l l u l c s e ex,xzi.zzts.
Figure 1. Peak widths of t h e singly-, doubly- and triply-charged no;eculai- ions of lactalbumin as a function of pH.
Figure 2. Peak widths of the singly-, doubly- and triply-charged rooleculai- ions of lysozyr~ie as a function of pH.
JOURNAL DE PHYSIQUE
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CONCLUSIONSI n t e r a c t i o n s between p e p t i d e s and n i t r o c e l l u l o s e a r e hydrophobic /23,24/, w i t h t h e hydrophobic s u r f a c e s l o c a t e d p r i m a r i l y i n s i d e t h e n a t i v e p r o t e i n /25/. The b i n d i n g o f p e p t i d e s t o n i t r o c e l l u l o s e i n c r e a s e s w i t h t i m e , w h i c h i s r e f l e c t e d i n i n c r e a s i n g peak w i d t h s i n t h e mass s p e c t r a when t h e sample i s analyzed s e v e r a l hours a f t e r a d s o r p t i o n . I n t h i s case, t h e 6-8 h o u r " i n c u b a t i o n " p e r i o d serves t o " a m p l i f y " t h e e f f e c t s o f t h e pH o f t h e s o l u t i o n b e f o r e t h e p e p t i d e was adsorbed. Since t h e e f f e c t s o f pH on t h e
c o n f o r m a t i o n a l s t r u c t u r e o f t h e s e two p r o t e i n s has been w e l l documented /19-21/, i t i s c l e a r t h a t t h e peak w i d t h s r e f l e c t t h e t e r t i a r y s t r u c t u r e . I n t h e case o f t h e
n i t r o c e l l u l o s e s u r f a c e , p a r t i a l u n f o l d i n g a t extreme pH may a c c e l e r a t e exposure o f t h e hydrophobic r e s i d u e s t o t h e s u r f a c e and i n c r e a s e b i n d i n g .
On
t h e o t h e r hand, t h e g l u t a t h i o n e m a t r i x , we suspect, serves t o p r e s e r v e t h e n a t i v e s t r u c t u r e s t h r o u g h t h e e l e c t r o s p r a y process, and t h u s achieves t h e same e f f e c t .5
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ACKNOWLEDGEMENTST h i s work was supported by a g r a n t (DMB 85-15390) f r o m t h e N a t i o n a l Science Foundation and c a r r i e d o u t a t t h e M i d d l e A t l a n t i c Mass Spectrometry L a b o r a t o r y , an NSF Shared
I n s t r u m e n t a t i o n F a c i l i t y .
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