Fourier Transform Mass Spectrometry - American Chemical Society

Chapter 8

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Analytical Applications of Laser Desorption-Fourier Transform Mass Spectrometry for Nonvolatile Molecules R. S. Brown and C. L. Wilkins Department of Chemistry, University of California, Riverside, CA 92521

Laser desorption Fourier transform mass spectrometry (LD-FTMS) results from a series of peptides and polymers are presented. Successful production of molecular ions of peptides with masses up to ~2000 amu is demonstrated. The amount of structurally useful fragmentation diminishes rapidly with increasing mass. Preliminary results of laser photodissociation experiments in an attempt to increase the available structural information are also presented. The synthetic biopolymer poly(phenylalanine) is used as a model for higher molecular weight peptides and produces ions approaching m/z 4000. Current instrument resolution limits are demonstrated utilizing a poly(ethyleneglycol) polymer, with unit mass resolution obtainable to almost 4000 amu.

L a s e r d e s o r p t i o n F o u r i e r t r a n s f o r m mass s p e c t r o m e t r y (LD-FTMS) has emerged an an a l t e r n a t i v e s o f t i o n i z a t i o n method f o r samples w h i c h a r e d i f f i c u l t t o a n a l y z e by more c o n v e n t i o n a l mass s p e c t r o m e t r i c methods. S i n c e i t s i n i t i a l d e m o n s t r a t i o n (J_) f o r o r g a n i c samples, t h e r e has been s t e a d y p r o g r e s s i n a p p l i c a t i o n s o f LD-FTMS t o a wide v a r i e t y o f samples. These have i n c l u d e d such d i v e r s e s u b s t a n c e s as s i m p l e p e p t i d e s ( 2 , 3 ) , sugars ( 4 ) , polymers ( 2 , 5 , 6 ) , p o r p h y r i n s (7,8) and o l i g o s a c c h a r i d e s (9,10) o f v a r i o u s m o l e c u l a r w e i g h t s . A l t h o u g h s t i l l a c o m p a r a t i v e l y new t e c h n i q u e , f o r many samples LD-FTMS compares f a v o r a b l y w i t h more e s t a b l i s h e d i o n i z a t i o n methods such as f a s t atom bombardment (FAB) and f i e l d d e s o r p t i o n ( F D ) , t h e former h a v i n g become the method o f c h o i c e f o r most n o n - v o l a t i l e p o l a r samples. LD-FTMS o f f e r s s e v e r a l advantages over FAB, i n c l u d i n g the l a c k o f need f o r a l i q u i d m a t r i x , w h i c h i s a source of h i g h c h e m i c a l background i n the FAB method. A d d i t i o n a l l y , the absence o f a l i q u i d m a t r i x removes the n e c e s s i t y t h a t the sample be s o l u b l e . Indeed, i n s o l u b l e samples can be a p p l i e d d i r e c t l y i n s o l i d form t o a sample probe and a n a l y z e d by LD-FTMS w i t h good r e s u l t s ( 6 ) . Many o r g a n o m e t a l l i c s , m e t a l l o p o r p h y r i n s and polymers do n o t y i e l d s p e c t r a when examined by 0097-6156/87/0359-0127$06.00/0 © 1987 American Chemical Society

Buchanan; Fourier Transform Mass Spectrometry ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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FAB-MS, due t o t h e i r i n s o l u b i l i t y i n the l i q u i d m a t r i x . Thus, LD-FTMS s h o u l d be c o n s i d e r e d complementary t o FAB-MS. Due t o the r e l a t i v e l y s m a l l number of l a b o r a t o r i e s equipped w i t h LD-FTMS, the method i s not as w e l l known or as h i g h l y d e v e l oped as FAB i o n i z a t i o n which i s now a v a i l a b l e f o r most commercial magnetic s e c t o r and quadrupole i n s t r u m e n t s . The p u l s e d n a t u r e of the l a s e r s t y p i c a l l y employed are i d e a l l y matched w i t h mass s p e c t r o m e t e r s c a p a b l e of s i m u l t a n e o u s d e t e c t i o n or r a p i d a n a l y s i s . Thus FTMS or time of f l i g h t (TOF) i n s t r u m e n t s are best s u i t e d f o r LD a p p l i c a t i o n s . However, the l a t t e r possesses l i m i t e d mass r e s o l u t i o n . T h i s c h a p t e r w i l l attempt t o p r e s e n t the c u r r e n t c a p a b i l i t i e s and l i m i t a t i o n s of LD-FTMS and t o address i t s f u t u r e potential. Experimental The g e n e r a l e x p e r i m e n t a l arrangement f o r LD-FTMS has been p r e s e n t e d i n d e t a i l elsewhere 02) and o n l y a b r i e f d e s c r i p t i o n w i l l be p r o v i d e d h e r e . For sample d e s o r p t i o n / i o n i z a t i o n , a p u l s e d c a r bon d i o x i d e l a s e r i s f o c u s s e d t o a p p r o x i m a t e l y a 1 mm diameter spot v i a a ZnSe l e n s onto a s t e e l c i r c u l a r probe t i p (10 mm diame t e r ) a t t a c h e d t o a d i r e c t i n s e r t i o n probe h o l d i n g the sample. F o l l o w i n g the l a s e r p u l s e , an a p p r o p r i a t e d e l a y ( u s u a l l y 3-5 seconds) i s i n t r o d u c e d t o a l l o w the h i g h l o c a l p r e s s u r e ( ΐ ο - Μ ο t o r r u n c o r r e c t e d i o n i z a t i o n gauge r e a d i n g ) of desorbed n e u t r a l s to be pumped away, l e a v i n g the i o n s w h i c h are t r a p p e d t o be d e t e c t e d w i t h improved r e s o l u t i o n . Such d e l a y s s h o u l d be s u b s t a n t i a l l y reduced i n the newer d i f f e r e n t i a l l y pumped d u a l c e l l d e s i g n . S u b s e q u e n t l y , the i o n s a r e e x c i t e d t o a l l o w t h e i r image c u r r e n t s to be r e c o r d e d u s i n g a h i g h speed a n a l o g t o d i g i t a l (A/D) con v e r t e r . The t r a n s i e n t s i g n a l thus o b t a i n e d i s then a p o d i z e d and F o u r i e r t r a n s f o r m e d t o produce the mass spectrum f o r the i o n s r e s u l t i n g from a s i n g l e l a s e r p u l s e . A l t h o u g h s i g n a l a v e r a g i n g may be employed, the c u r r e n t s p e c t r a a l l r e s u l t from s i n g l e l a s e r p u l s e s u s i n g ~ l - 5 μg of sample. No d e f i n i t i v e comparisons of the e f f e c t s of l a s e r s o p e r a t i n g a t d i f f e r e n t wavelengths f o r l a s e r d e s o r p t i o n have been r e p o r t e d . For p h o t o d i s s o c i a t i o n e x p e r i m e n t s , a s p e c i a l probe was c o n s t r u c t e d t o r e p l a c e the 12.7 mm d i a m e t e r d i r e c t i n s e r t i o n probe n o r m a l l y employed. I t c o n s i s t s of a h o l l o w s t a i n l e s s s t e e l tube w h i c h has a 38 mm f o c a l l e n g t h q u a r t z l e n s vacuum s e a l e d a t one end and an extended h o l l o w probe t i p a t the o t h e r . The beam of a Lambda P h y s i k exciraer l a s e r o p e r a t i n g a t 308 nm was passed through the probe and l e n s i n t o the FTMS c e l l t h r o u g h a s m a l l h o l e i n the c e n t e r of the t r a p p l a t e as shown i n F i g u r e 1. - 6

Sample P r e p a r a t i o n 50-100 μg of each p e p t i d e was d e p o s i t e d onto the s t a i n l e s s s t e e l probe t i p from a m e t h a n o l / a c e t i c a c i d s o l u t i o n (1 drop a c e t i c a c i d i n 1 ml of methanol) r e s u l t i n g i n sample consumption of ~1 μg per l a s e r p u l s e based on a 1 mm l a s e r spot and u n i f o r m sample coverage. S i m i l a r amounts of the o t h e r samples were a p p l i e d from a methanol s o l u t i o n . S e n s i t i v i t y i s v e r y dependent upon sample c o m p o s i t i o n and the r e s u l t s p r e s e n t e d a r e t y p i c a l f o r these 2


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c l a s s e s o f compounds. P o l y ( p h e n y l a l a n i n e ) was i n s o l u b l e and was d e p o s i t e d from a methanol s u s p e n s i o n produced by s o n i c a t i o n . A p p r o x i m a t e l y 500 μg o f p o l y ( p h e n y l a l a n i n e ) was r e q u i r e d t o i n s u r e adequate coverage o f t h e probe t i p , due t o t h e i r r e g u l a r s u r f a c e produced as a r e s u l t o f e v a p o r a t i o n o f t h e s o l v e n t . R e s u l t s and D i s c u s s i o n P e p t i d e s . One o f t h e a p p l i c a t i o n s t h a t has b e n e f i t e d most from the development o f s o f t i o n i z a t i o n methods f o r mass s p e c t r o m e t r y has been the a n a l y s i s o f p o l a r b i o l o g i c a l m a t e r i a l s . P e p t i d e s , i n p a r t i c u l a r , have been e x t e n s i v e l y s t u d i e d by a v a r i e t y o f s o f t i o n i z a t i o n methods, p r i m a r i l y FAB, w i t h e x c e l l e n t r e s u l t s b e i n g o b t a i n e d f o r p e p t i d e s i n t h e 1000-10000 d a l t o n range. LD-FTMS a l s o has proved t o be v e r y s u c c e s s f u l f o r a n a l y s i s o f s i m p l e pep t i d e s and an e x a m i n a t i o n o f these r e s u l t s s h o u l d h e l p d e l i n e a t e the a n a l y t i c a l p o t e n t i a l o f LD-FTMS i n t h i s i m p o r t a n t a r e a . The LD-FTMS spectrum o f t h e d e c a p e p t i d e A n g i o t e n s i n I i s shown i n F i g u r e 2 and i s a good example o f t y p i c a l r e s u l t s f o r p e p t i d e s of t h i s s i z e . An abundant p r o t o n a t e d m o l e c u l a r i o n (M+H) i s o b s e r v e d , as w e l l as t h e N-terrainus sequence i o n s (Zg)" " t o (Zj) w i t h d e c r e a s i n g r e l a t i v e i n t e n s i t y f o r the s m a l l e r fragments; t h e r e a r e no abundant fragment i o n s w i t h m/z lower than 900. A l t h o u g h a low abundance C-terrainus sequence i o n (Ag)" " i s o b s e r v e d , t h e N-terminus i o n s u s u a l l y predominate, i n a d d i t i o n t o fragments r e s u l t i n g from l o s s e s o f n e u t r a l s such as H2O, NH3 and 002· T h i s i s i n c o n t r a s t w i t h FAB r e s u l t s i n w h i c h C-terminus i o n s a r e u s u a l l y observed ( 1 1 ) . As m o l e c u l a r w e i g h t i s i n c r e a s e d , l e s s f r a g m e n t a t i o n i n d i c a t i v e o f t h e p e p t i d e ' s sequence i s o b s e r v e d , as c a n be seen i n t h e spectrum o f t h e u n d e c a p e p t i d e , Substance P, i n F i g u r e 3. Here, the p r o t o n a t e d m o l e c u l a r i o n dominates t h e spectrum w i t h the sodium c a t i o n i z e d s p e c i e s (M+Na) a l s o observed. Fragment i o n s a r e l i m i t e d t o t h e f i r s t sequence i o n from t h e N-terminus as w e l l those a r i s i n g from l o s s e s o f H2O and NH3. ( Z 9 ) and (A9)"*" i o n s a t m/z = 1094 and m/z = 1087 a r e a l s o observed i n low r e l a t i v e abun dance, as i s an i o n which may a r i s e from a minor i m p u r i t y a t m/z 1104. T h i s r e d u c t i o n i n u s e f u l f r a g m e n t a t i o n a t h i g h e r masses i s a l s o observed i n FAB-MS. Use o f h i g h e r l a s e r power d e n s i t y i n c r e a s e s s t r u c t u r a l l y r e l e v a n t f r a g m e n t a t i o n a t t h e expense o f the m o l e c u l a r i o n and r e s u l t s i n lower s i g n a l t o n o i s e r a t i o s (i_*e_. decreased s e n s i t i v i t y ) . The e f f e c t s o f l a s e r power on p o r p h y r i n f r a g m e n t a t i o n a l s o have been r e p o r t e d CO. The LD spectrum o f t h e t r i d e c a p e p t i d e n e u r o t e n s i n ( F i g u r e 4 ) , shows s e v e r a l d i f f e r e n c e s . Here f r a g m e n t a t i o n r e s u l t s almost e x c l u s i v e l y from s m a l l n e u t r a l l o s s e s . A p r o t o n a t e d m o l e c u l a r i o n i s s t i l l one o f t h e major i o n s observed (m/z = 1673) and some c a t i o n i z e d ( K ) fragment s p e c i e s a r e apparent as w e l l as a s m a l l amount o f c a t i o n i z e d m o l e c u l a r i o n (1000 amu) i s q u e s t i o n a b l e due t o the preponderance o f m o l e c u l a r c o m p o s i t i o n s a t these masses w h i c h a r e w i t h i n even a 1 ppm e r r o r . +

+

2

2

Conclusions The a n a l y t i c a l a p p l i c a t i o n s o f LD-FTMS have j u s t begun t o be developed. As more work i s done, LD-FTMS w i l l c o n t i n u e t o be a p p l i e d t o a n a l y s i s o f d i f f i c u l t samples. I t i s a p r o m i s i n g t e c h n i q u e f o r s t a b l e samples such as p o l y m e r s , o r g a n o m e t a l l i c s and


FOURIER TRANSFORM MASS SPECTROMETRY

138

(A)

443

r

4

N(CH CH ) 2

3

(M-CI)

2

(M-HCI*Naj] 465

481

ο 150

200

250

r

, M i

"

ι

1

Γ 400

300 350 MASS IN A.M.U.

450

500

(Β)

(M-Clf-(HCOOH) ,397

-(C H ) -(C H )N(C H ) 326 ?

2

5

2

443

4

4

465 48I ο . 200

150

250 SS IN A

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m .

4 0 0

450

500

( a ) L a s e r d e s o r p t i o n p o s i t i v e i o n spectrum o f Rhodamine Β u t i l i z i n g o n l y t h e CO2 l a s e r w i t h e j e c t i o n o f a l l i o n s below m/z = 443. (b) P o s i t i v e i o n spectrum as i n ( a ) but w i t h UV i r r a d i a t i o n (308 nm) a f t e r t h e pump down d e l a y .


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p o r p h y r i n s , as w e l l as many i n s o l u b l e samples. C o n t i n u e d work i n the a r e a i s e x p e c t e d t o improve b o t h t h e mass range and s e n s i t i v i t y w h i l e b e t t e r i n s t r u m e n t a t i o n and u n d e r s t a n d i n g o f t h e i o n dynamics o f FTMS s h o u l d r e s u l t i n t h e r e a l i z a t i o n o f the h i g h r e s o l u t i o n promise o f FTMS a t h i g h mass. G i v e n i t s p r e s e n t s t a t e of development, LD-FTMS c a n p r o v i d e u n i q u e c a p a b i l i t i e s f o r s o l u t i o n o f a v a r i e t y o f a n a l y t i c a l problems. Acknowledgments Support from t h e N a t i o n a l I n s t i t u t e s GM-30604 i s g r a t e f u l l y acknowledged.

o f H e a l t h under g r a n t

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

15.

McCrery, D.A.; Ledford, E . G . , Jr.; Gross, M.L. Anal. Chem. 1982, 54, 1435-1437. Wilkins, C . L . ; Weil, D.A.; Yang, C . L . C . ; Ijames, C.F. Anal. Chem. 1985, 57, 520-524. Wilkins, C . L . ; Yang, C.L.C. Int. J. Mass Spectrom. Ion Processes 1986, 72, 195-208. McCrery, D.A.; Gross, M.L. Anal. Chim. Acta, 1985, 178, 103-116. Brown, R.S.; Weil, D.A.; Wilkins, C.L. Macromolecules 1986, 19, 1255-1260. Brown, C.E.; Kovacic, P.; Wilke, C . E . ; Cody, R.B.; Kinsinger, J.A. J . Polymer Sci. Poly. Lett. E d . , 1985, 23, 453-463. Brown, R.S.; Wilkins, C.L. Anal. Chem. 1986, 58, 3196-3199. Brown, R.S.; Wilkins, C.L. J. Am. Chem. Soc. 1986, 108, 2447-2448. Coates, M.L.; Wilkins, C.L. Biomed. Mass Spectrom. 1985, 12, 424-428. McCrery, D.A.; Gross, M.L. Anal. Chim. Acta, 1985, 178, 91-103. König, W.A.; Aydin, M.; Schulze, V.; Rapp, V . ; Hohn, M.; Pesch, R.; Kalikhevitch, V.N. Int. J. Mass Spectrom. Ion Phys. 1983, 46, 403-406. Cody, R.B., J r . ; Amster, I.J.; McLafferty, F.W. Proc. Natl. Acad. Sci. USA 1985, 82, 6367-6370. Alai, M.; Demirev, P.; Fenselau, C . ; Cotter, R. Anal. Chem. 1986, 58, 1303-1307. Jonsson, G.P.; Hedin, A.B.; Hakansson, P . L . ; Sundquist, B.U.; Save, G.B.S.; Nielsen, P . F . ; Roepstorff, P.; Johansson, K . E . ; Kamensky, I . ; Lindberg, M.S.L. Anal. Chem. 1986, 58, 1084-1087. Bowers, W.D.; Delbert, S.S.; McIver, R.T. Anal. Chem. 1986, 58, 969-972.

RECEIVED September 14, 1987


Fourier Transform Mass Spectrometry - American Chemical Society

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