Applications of Fast Atom Bombardment in Bioorganic Chemistry

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13 Applications of Fast Atom Bombardment in Bioorganic Chemistry Dudley H. Williams University Chemical Laboratory, Lensfield R o a d , Cambridge C B 21EW,England

The advent of FAB mass spectrometry has allowed the routine molecular weight determination of polar molecules, without derivatization,

up to ca 3,000

Daltons, and in exceptional cases, within 1 mass unit to the region of 8,000 Daltons. This advance, coupled with FAB fragmentation, and enzymic digestion techniques, has allowed the rapid solution of a number of problems in protein and peptide chemistry - problems which were hitherto rather difficult

to solve. Examples

are given. The discovery of FAB mass spectrometry i n 198l by Barber and co1 2 workers has, along with SIMS, and Californium plasma desorption mass spectrometry,^ revolutionized the study of polar molecules by mass spectrometry. This paper i s concerned with the application of FAB mass spectrometry to solve problems i n peptide and protein chemistry. The work to be presented has, when done i n the author's laboratory, been carried out on a Kratos MS-50 mass spectrometer, f i t t e d with a magnet possessing a mass range of 3000 Daltons at f u l l accelerating voltage (8KV). Xenon atoms of k-9 KeV t r a n s l a t i o n a l energy have been used as bombarding p a r t i c l e s into a matrix most commonly consisting of thioglycerol/diglycerol (1:1), or g l y c e r o l . The instrument i s f i t t e d with a post-acceleration detector, so that the t r a n s l a t i o n a l energies of incoming ions can 0097-6156/85/0291-0217$06.00/0 © 1985 American Chemical Society

DESORPTION MASS SPECTROMETRY

218

be increased by 9KeV, before impinging on an aluminized button. This device i s indicated schematically i n F i g . 1. The most s i g n i f i c a n t advantages of the FAB method are the a b i l i t y to study underivatized peptides, a reduction i n sample size, and the f a c i l i t y to study larger peptides. Peptides containing between k and 30 amino acid residues are conveniently studied with our present equipment. A sample size of approximately 0.1

nanomoles i s generally

s u f f i c i e n t f o r molecular weight determination i n either the p o s i t i v e or negative ion mode, but does not normally allow the sequence of amino acids to be determined.

Larger sample sizes, t y p i c a l l y between

1 and 5 nanomoles, afford some sequence information. Sequence ions are observed i n the p o s i t i v e and negative ion modes from both N- and C-termini of the peptide and this may enable the complete sequence of the peptide to be

determined.

The amount of sequence information available i n the positive and negative ion FAB mass spectra of peptides varies considerably. The abundance of sequence ions i s i n part dependent on sample size but this i s c l e a r l y not the only factor since large sample sizes ( 50 nanomoles) often f a i l to provide extensive sequence information f o r some peptides. Sequence information i s frequently not complete and the absence of either C or Ν terminal sequence ions does not allow the complete sequence determination. EI mass spectrometry of derivatized peptides complements FAB mass spectrometry but suffers from the disadvantage that larger sample sizes are usually required. For sample sizes less than

20

nanomoles and f o r high molecular weight peptides, alternative methods of generating sequence information are desirable. Carboxypeptidase digestion to generate a mixture of C-terminal fragments and chemical removal of N-terminal amino acid residues v i a the subtractive Edman degradation, coupled with FAB mass spectrometry o f f e r a promising alternative. As an example where sequence information i s available without the use of Edman or carboxypeptidase degradation, a study of calcineurin Β may be c i t e d . This work also allowed, most importantly, the determination of an N-terminal blocking group as myristic acid. Calcineurin Β i s a calcium-binding protein f i r s t found i n

13.

W I L L I A M S

219

FAB in Bioorganic Chemistry

bovine brain, and early work on t h i s protein demonstrated that the NH^-terminus

of calcineurin Β was blocked. When calcineurin Β was

cleaved with cyanogen bromide, and applied to an HPLC column, a peptide

(CB-l) lacking a free NH^-terminal amino acid was eluted as

a sharp peak at 57% a c e t o n i t r i l e . The amino acid composition of CB-l showed that i t was a decapeptide containing only two hydrophobic amino acids. This indicated that the blocking group was much more hydrophobic than the a c e t y l , formyl or pyrrolidone carboxylic acid groups most commonly found at the N-termini of proteins. Peptide CB-l had

1271 as determined by FAB mass spectrometry

in the p o s i t i v e ion mode [ (M + N a ) = 129 *·, (M + H ) = 1272] and i n +

the negative

+

1

ion mode [(M - H)~ = 1270]. The number of carboxylic

acid groups was determined from the p o s i t i v e ion FAB mass spectrum of of 60 was observed, which

the e s t e r i f i e d peptide. An increase i n

corresponds to the formation of two methyl esters plus methanolysis of the C-terminal homoserine lactone. The M of the amino acid comr ponent of CB-l was 1060, i n d i c a t i n g that the M was

of the blocking group

211. When calcineurin Β was digested with Staphylococcus aureus

proteinase, a peptide, SP1, lacking a free NH^-terminal amino acid was also eluted from HPLC at 57% a c e t o n i t r i l e . Amino acid analysis showed i t to be a tripeptide Gly,Asx,Glx. From the known s p e c i f i c i t y of S. aureus proteinase the C-terminal mass spectrometry established the

reside of SP1 must be Glu. FAB

of SP1 as 528, and e s t e r i f i c a -

tion of this peptide led to an increase i n

to 556, which corres

ponds to the formation of two methyl esters. Since SP1 has two free carboxyl groups, the sequence of SP1 must be X-(Gly,Asn)-Glu and the of the blocking group must be 211. The assignments of sequence ions observed i n the FAB mass spectrum of CB-l and i t s methyl ester are given i n Table I, and suggested that the sequence of CB-l was: Gly-Asn-Glu-Ala-Ser-Tyr-Pro-Leu-Glu-Hsl Assuming that the blocking group X i s linked to the NH^terminal glycine by the usual amide bond, then hydrolysis should = 228. This corresponds to the

y i e l d a carboxylic acid

of a

C ^ saturated f a t t y acid, the mass spectra are consistent with the supposition that X i s C

H

CO-. This p o s s i b i l i t y was confirmed by


220

Table I A Assignment of the sequence ions i n the FAB mass sectra of decapeptide Type of f ragmen t iona χ + + + + +

Na NAcy CAmin CAmin CAlk

-

NA NAcy CAmin CAlk

M -Values of fragment ions

M

determining Gly Asn Glu A l a Ser Tyr Pro Leu Glu Hsl peak b 621 708 968 108l 1210 129^ 856 953 1066 1294 1027 913 784 713 1294 1005 891 762 1272 897 768 129^ b 597 684 944 1057 1186 1270 582 832 929 1042 1171 1270 1003 889 760 689 1270 988 874 744 1270

Most of the sequence ions observed i n the p o s i t i v e ion ( + ) mode are cationised by NA . The base peaks i n the p o s i t i v e ion FAB mass spectra of peptides that lack basic functional groups as i n CB-l often correspond to the M of peptides cationised by the formation of adducts with Na and/or K , traces of which are usually present in the matrix. +

+

+

Table I B Assignment of the sequence ions i n the FAB mass spectra of the decapeptide ester Type of fragment ion X a

+ + + +

Na NAcy CAmin CAlk

Gly

1087

M -Values of the fragment ions r Asn Glu- Ala,Ser Pro Leu Glu- Homo-OMe -Ser-OMe -OMe b 982 1095 1238 1080 870 973 957 8l4

M

deter mining peak

1354 1354 1354 1354

fragment+ ions are named as follows : CAmin

CAlk

NAcy NA Bond cleavages are accompanied by a hydrogen transfer to the charged fragment except i n the NAcy case. Positive and negative signs indicate cationic and anionic fragment ions, respectively. ^Due to c y c l i c nature of the proline residue fragmentation of NA type does not produce fragment ions.

13.

WILLIAMS

221


i s o l a t i n g the blocking group as i t s methyl ester. On gas chromatography, this ester co-migrated with methyl myristate. We were also able to use FAB mass spectrometry to determine the amino acid sequence around the active s i t e serine i n the acyl transference domain of rabbit mammary f a t t y acid synthase.6

The

synthase was labelled i n the acyl transferase domain(s) by the formation of O-ester intermediates a f t e r incubation with ["^C]a c e t y l - or malonyl-CoA ( F i g . 2A). The modified protein was

then

digested with elastase (Fig. 2B), and radioactive material isolated v i a successive p u r i f i c a t i o n steps on Sephadex G-50

and reverse phase

HPLC. The isolated peptides were then sequenced by FAB MS. The data summarized i n Table II established the sequences of both the acetyl and malonyl hexapeptides to be N-acyl-Ser-leu-Gly-Glu-Val-Ala. The N-terminal location of the acyl group was confirmed by showing that the molecular ions were i d e n t i c a l a f t e r treatment with acetic anhydride, which would acetylate a free amino group. Since the linkages between the acetyl and malonyl groups and intact f a t t y acid synthase are sensitive to hydroxylamine ( l M, pH 9-5,

2.Oh,

38°C; unpublished work), the i n i t i a l acylation cannot be at an amino group. An O-MJ

migration must have occurred a f t e r proteolytic

digestion. Our own work i s now evolving, i n part, to use FAB mass spectrometry to determine which portions of some genes are expressed in the frog Xenopus l a e v i s . The genes i n question are those coding for some of the peptides which are secreted through the frog skin when the frog i s stimulated (by handling, or by infection with epinephrine or nor-epinephrine). These peptides are s i g n i f i c a n t because of the remarkable structural, and physiological a c t i v i t y , s i m i l a r i t i e s which exist between them and a number of mammalian neuropeptides. If known (and sequenced) frog skin peptides are used as a guide to synthesize appropriate c-DNA probes, the genes which code f o r these peptides can be isolated and sequenced. However, i t is then a problem to know which other parts of the gene are expressed i n peptide production. FAB mass spectrometry has proved to be a very powerful tool i n solving this problem. FAB MS has been used to show that a large number of hitherto unidentified peptides are +

excreted by Xenopus l a e v i s , although f o r most of these only MH

ions

222


• 8kV

ION BEAM

+ 6kV + 4kV

\////

SLIT -3-5kV

-9kV

F i g u r e 1.

Schematic

i l l u s t r a t i o n of the post-acceleration

detector.

CORE OF FATTY/ ACID SYNTHASE

-^SCoA I *CH C0 H 2

2

"0-C-CH —C0 H 2

Elastase F i g u r e 2. elastase

Schematic disgestion.

2

Digestion ( 6 h , 37 C)

i l l u s t r a t i o n o f p r o t e i n r a d i o - l a b e l l i n g and

13.

WILLIAMS

223


Table II a

Amino acid sequence information derived from negative ion FAB mass spectra of the malonyl- and acetyl-hexapepties 0

659

486/470

Malonyl — Ser —

373/35^

316/300^

•Ala-OH

Leu

359

488

587

659

Val-

•Ala-OH

H+

N-Malony1-hexapept ide b

-C0„

486/470 ^ 373/357^

3l6/30(^

A c e t y l — Ser

H

+

N-Ac ety1-hexap ep t i de

Sequence ions observed i n the negative ion FAB mass spectra of the above peptides are as follows:

-CA

- NH -

-CAlk

-CHR-

-C0-

-NH-

-NA where the -CA and -NA cleavages occur with hydrogen transfer to the charged fragment. bSome decarboxylation of the malonyl group to produce an acetyl group was observed i n the FAB mass spectra of the N-malonylhexapeptide.


224

are observed i n peptide mixtures. However, when these molecular ions are considered i n the l i g h t of known gene sequences, and i n the l i g h t of possible processing sites i n the peptides which they would produce i f transcribed and translated, f u l l peptide sequences corresponding to the molecular ions can be proposed. These peptide sequences can then be validated (or otherwise) by one or two cycles of Edman degradation, with re-determination of the molecular weight by FAB (as outlined previously i n this a r t i c l e ) after each cycle.7 Although the above work i s not completed, the p r i n c i p l e by which FAB mass spectrometry can be used i n conjunction with known gene sequences, and i n p a r t i c u l a r to check t h e i r accuracy, can be Q i l l u s t r a t e d by reference to the recent work of Gibson and Biemann. It i s obvious that i t i s prudent to check the correctness

of

the amino acid sequence derived from the base sequence of the gene not only at the NH^ and COOH termini, which i s the common practice, but throughout the entire protein. This would help to uncover any s i g n i f i c a n t errors as well as address the p o s s i b i l i t y of postt r a n s l a t i o n a l modifications. For checking the correctness of a proposed structure the prot e i n i s hydrolyzed at s p e c i f i c sites - i . e . , with trypsin - to produce a pool of smaller peptides, which i s then p a r t i a l l y separated by HPLC into f i v e or six f r a c t i o n s . Each f r a c t i o n i s then subjected to FAB-MS, which allows the determination of the molecular weights of most or a l l of the peptides present i n each f r a c t i o n . These values are then compared with the molecular weights of the t r y p t i c peptides predicted from the DNA deduced amino acid sequence. The fractions can then also be subjected to Edman dégradâtion(s) and the molecular weights of the shortened peptides determined by FAB-MS to i d e n t i f y the NH^-terminal amino acid(s) of each peptide from the change i n i t s molecular weight. For example, a t r y p t i c digest of the protein Gln-tRNA synthetase was separated into six crude fractions by HPLC to remove the enzyme, reagents, s a l t s , and other contaminants and also to reduce the complexity of the o r i g i n a l digest, which, i n turn, reduces the complexity of the resulting FAB mass spectra. There were two predicted t r y p t i c peptides of M^s 736 1379,

and

respectively, which i s 10 daltons less than two unmatched ex-

perimentally found peptides (M s 7^6

and 1389). The former cover the

13.

WILLIAMS


amino acid positions 7-12 and 1-12,

225

i . e . , they are overlapping

peptides from the NH^ terminus of Gln-tRNA synthetase. Only the p a i r serine/proline d i f f e r s by 10 daltons. The preliminary sequence i n deed contained serine i n position J, which i s common to both, and the condon used f o r serine was TCG, which could be converted to prol i n e condon CCG i f base 19 were C rather than T. Re-inspection of the DNA sequence analysis data revealed that not only base 19 i s C but also base l 8 . Because both CGT and CGC code f o r arginine, the corrected sequence now contains an arginineproline bond, which trypsin s p l i t s very slowly. This i s probably the reason why both t r y p t i c peptides (positions 1-12 and 7-12) were observed. The following are the preliminary and f i n a l DNA and amino Q acid sequences of t h i s region. AGT-GAG-GCA-GAA-GCC-CGT-TCG-ACT-AAC-TTT-ATC-CGT Ser-Glu-Ala-Glu-Ala-Arg-Ser-Thr-Asn-Phe-Ile-Arg AGT-GAG-GCA-GAA-GCC-CGC-CCG-ACT-AAC-TTT-ATC-CGT Ser-Glu-Ala-Glu-Ala-Arg-Pro-Thr-Asn-Phe-Ile-Arg

Conclusion

It i s evident from the foregoing examples that FAB mass spectrometry i s a powerful technique i n several aspects of protein and peptide chemistry. This i s p a r t i c u l a r l y true i n determining the nature of blocking groups i n proteins, i n checking the sequences of bases i n genes (by examining the resulting protein), and i n determining which portions of genes are expressed (when the peptide products are readily available as r e l a t i v e l y pure components, or simple mixtures).

Acknowledgments The author thanks the SERV (UK) f o r support of that portion of the work carried out at the University of Cambridge. Literature Cited 1. M. Barber, R. S. Bondoli, R. D. Sedgwick, and A. N. Tyler, J . Chem. Soc. Chem. Commun., 1981, 325. 2. See, f o r example, H. Grad, N. Winograd, and R. G. Cooks, J . Am. Chem. Soc., 1977, 99, 7725.

DESORPTION

226

MASS

SPECTROMETRY

3. R. D. MacFarlane and D. F. Torgerson, Science, 1976, 191, 920. 4. C. V. Bradley, D. H. Williams, and M. R. Hanley, Biochem. Biophys. Res. Commun., 1982, 104, 1223; see also Y. Shimonishi, Y. M. Hong, T. Kitagishi,

T. Matsuo, H. Matsuda, and I. Katakuse, Eur. J.

Biochem., 198O, 112, 251. 5. A. Aitken, P. Cohen, S. Santikarri, D. H. Williams, A. G. Calder, A. Smith, and C. B. Klee, FEBS Letters, 1982, 150, 3l4. 6. A. D. McCarthy, A. Aitken, D. G. Hardie, S. Santikarn, and D. H. Williams, FEBS Letters, 1983, l60, 296. 7. B. W. Gibson, L. Poulter, and D. H. Williams, unpublished work. 8. B. W. Gibson and K. Biemann, Proc. Natl. Acad. Sci U.S.A., 1984, 81, 1956. RECEIVED

April 24, 1985

Applications of Fast Atom Bombardment in Bioorganic Chemistry

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