fast atom bombardment mass spectrometry of protein and carbohydrate biopolymers

trends in analytical chemistry, vol. 8, no. lo,1989 375

Fast atom bombardment mass spectrometry of protein and carbohydrate biopolymers Anne Dell and Mark E. Rogers London, U.K.

Within the past decade significant advances in the fEld of biological mass spectrometry have resulted in unique ap- proaches to the structural analysis of biopolymers. This arti- cle pinpoints key instrumental developments and outlines strategies based upon high field fast atom bombardment mass spectrometry which are allowing hitherto elusive biopolymer structural data to be obtained

Introduction Although mass spectrometrists have been wres-

tling with biopolymers for more than thirty years, the nineteen eighties will be seen by future gener- ations as the first decade of real success for biological mass spectrometry (MS). This success is largely due to two key instrumental developments in the late nineteen seventies - high field magnet mass spectrometers’ and fast atom bombardment (FAB) ionisation - which came together in 1980 to begin a revolution in the MS analysis of biopolymers. For the first time it was possible to obtain mass spectra on intact polypeptides, oligosaccharides, oligonu- cleotides and other small biopolymers without the need for prior derivatisation. Molecules that were huge by MS criteria (above about 300 dalton) and were multiply charged in their natural state could, nevertheless, be induced to desorb from a liquid ma- trix as a singly charged molecular on species (e.g. [M+H]+, (M+Na]+, [M-H]-) which could be fo- cussed and detected at good sensitivity. Spectra were soon obtained on picomolar quantities of several small proteins up to 10 000 daltons; more recently the mass limits for detection of small proteins by FAB-MS have been extended to 24 000 daltons as a result of improvements in ionisation and detection of high mass ions3. However, genuine MS problem solving in the biopolymer area is still largely con- fined to the low mass domain up to about 6000 daltons. Some strategies for ensuring that biopolymers of any size and complexity can be successfully analysed in this mass range are highlighted below.

Sequencing small biopolymers Peptides

Natural fragmentation is present in the FAB spectra of all peptides of 3000 daltons or less provided

they are sufficiently pure. The fragment ions arise via chemical ionisation pathways leading to the pro- duction of N- and C-terminal fragments (see Fig. 1)4-6. These can be conveniently differentiated, to assist interpretation, by first treating the sample with a 1:l mixture of acetic anhydride-[2H6]acetic anhydride in methanol to label the amino terminus. N-terminal fragment ions will then appear as 1:l doublets 3 U apart while C-terminal ions appear as singlet ammonium ions with accompanying alkyl sat- ellites 15 U to lower mass.

Natural fragmentation may not be sufficient to de- termine a complete sequence but microchemical and enzymic manipulations of the peptide followed by FAB analysis of the products usually affords the missing information. This approach was used in a study of a cardioactive peptide (called CPB) isolated from the giant neurone of the gastropod Aplysia7. This was the first neuropeptide of completely un- known sequence to be characterised by FAB-MS. The FAB spectrum of CPB, which was obtained from 1 pug of impure material, was extremely complex, and no firm assignment, other than the molecular weight of 1140 defined by the [M+H]+ signal at 1141, could be made. The 1:l acetyl-deuteroacetyl

________ (-OH + H+

___ __.... t-OH

Fig. I. Peptide fragments at the amide bond and between the amide nitrogen and the a-carbon producing N-terminal sequence ions if the charge is retained on the N-terminal fragment (upper) and C-terminal sequence ions if the charge k retained on the C-terminal fragmen t (lower) .

01659936/89/$03.00. (Q Elsevier Science Publishers B.V.

376 trena5 in analytical chemistry, vol. 8, no. IO, 1989

RO, RO &I-k 0 0

R'O 0

__-_-- RO X RO OR

Fig. 2. Permethylated (R = methyl), andperacetylated (R = acetyl) oligosaccharides cleave at the glycosidic linkage to yield non- reducing end fragment ions. Cleavage is preferred if X is an aceta- mido group but X can also be OMe or OAc. (R’ = OMe or OAc or another sugar residue).

derivative was then prepared and the resulting FAB spectrum showed an intense molecular ion doublet at m/z 1183, 1186 together with weak N-terminal doublets and C-terminal singlets which defined the partial sequence Met-Asn-Tyr-Leu-Ala-Phe-. The amino acid analysis of CPB had indicated that a sin- gle basic residue, arginine, was present. Trypsin is an enzyme which cleaves peptides on the C-terminal side of basic residues and is a powerful tool to use in conjunction with FAB-MS. FAB-MS of the tryptic digest of CPB gave an abundant molecular ion at m/z 1011, revealing that the digestion had removed a C- terminal amidated methionine residue from CPB. Combining this result with the partial sequence de- duced from the natural fragmentation, the complete sequence of CPB was assigned as follows: Met-Asn- Tyr-Leu-Ala-Phe-Pro-Arg-MetNHz.

Oligosaccharides and glycoconjugates The molecular ions present in the FAB spectra of

carbohydrate-containing biopolymers provide valuable compositional information, including the nature and number of attached moieties such as acyl, sul- phate, phosphate, etc. Native biopolymers exhibit some glycosidic fragmentation which is of variable usefulness for sequence determination. To ensure reliable fragmentation, samples may be converted to their peracetyl or permethyl derivatives*. These fragment in a well-defined manner (see Fig. 2) to af- ford abundant non-reducing and sequence ions which are valuable for sequence assignment of all glycans, but are especially useful for the analysis of complex glycoproteins (see later).

FAB-MS and the biotechnology industry The use of high field FAB-MS in conjunction with

classical protein and carbohydrate chemistry has proven to be a powerful and cost effective method for structural analysis of genetically engineered pro-

teins and glycoproteins of interest to the biotechnology industry’. Methods have been developed and refined for determining the identity of blocked N- terminal residues, and for detecting the presence or absence of C-terminal truncation or ‘ragged ends’ - two areas that pose extreme problems for conven- tional classical techniques. In addition, FAB-MS can answer questions concerning post ribosomal modification such as glycosylation, phosphorylation and S-S bridge formation. All of these studies are based on FAB mapping procedures which exploit a unique feature of peptide FAB-MS, namely that mixtures of peptides examined at the nanomolar or sub-nanomolar level do not fragment despite exhibiting fragment ions when examined as pure materials. The absence of fragment ions means that spectra are rela- tively simple to interpret even when a complex mixture is being examined. In the FAB mapping procedure a protein or glycoprotein is converted to a mixture of peptides by treatment with a specific protease such as trypsin. The products of the digestion are analysed by FAB-MS without pre-fractionation and the spectrum exhibits [M+H]+ signals for most or all of the tryptic peptides derived from the sample. These can be mapped onto the anticipated structure, thereby defining the integrity or otherwise of the primary sequence. Errors of translation, deletion, in- sertion, or point mutation (excepting inversions in sequence), and post-translational modification of processing, can be detected and assigned by this powerful technology. C-terminal ragged ends are re- vealed by the presence of molecular ions for truncated C-terminal peptides. N-terminal modification results in a shift to higher mass of the anticipated signal for the N-terminal tryptic peptide. Simple bio- chemical, enzymatic or chemical procedures such as cyanogen bromide digestion can be used to verify assignments in the spectrum (cyanogen bromide cleaves peptides on the C-terminal side of methionine). Mass shifts after sequential Edman degradation (observing the molecular ions of truncated peptides in the new FAB maps) provide N-terminal sequence information; putative assignments of signals to N-terminally blocked peptides are corroborated if the signals do not shift after treatment with the Ed- man reagents.

Sequential Edman degradation together with FAB-MS of unfractionated or partially purified mixtures is a very powerful combination of techniques. This strategy has been exploited in a number of studies, including establishing the point mutations which give rise to sequence variations in pathological vari- ants of Antithrombin, a plasma glycoprotein which is vital in inhibiting clot formation and thrombosis”. Peptide FAB maps were created from several dig-

trends in apaIytica1 chemistry, vol. 8, no. IO, 1989 377

o-o ?

o-o O-O 9 \z 0.0-&0-~0-&+0-070-o70-070-~0-dt. ”

>.-•?kAsn (a) ’

0 NeuAc 0Gal 0 GlcNAc 0 Man A Fuc

“.-&-As” 1 b) 0-0-o>.

Fig. 3. (a) One of the major structures present in adult Band 3 glycoprotein; the fragment ions observed in the FAB spectrum of the permethylated derivative are formed by cleavages at the positions indicated with arrows. (b) The major structure present in HEMP- AS Band 3; the arrows indicate the major cleavage sites.

ests and potential variant amino acid sequences were located by a computer comparison of the molecular ions with those predicted from the sequence. The data produced allowed the location of an important antithrombin variant within the sequence spanning residues 371 to 399. Eleven steps of Edman followed by MS conclusively proved that the variant structure contained a histidine at position 393 where the normal structure has an arginine residue. This result gave a clear insight into why the variation is pathological, since the arginine-393 to serine-394 bond is the one split by thrombin binding in the normal phys- iological situation. Substitution of arginine by histidine would prevent the normal binding by thrombin.

One of the most important post translational events for many proteins is the formation of disul- phide bridges between appropriate cysteine residues in the primary protein sequence. Problems asso- ciated with low specific activity in recombinant protein engineering can sometimes be related to the au- thenticity of the S-S bridges in the product since scrambled or incorrect S-S bridge configurations lead invariably to a functionally defective product. Based on the FAB mapping procedure described above, an entirely new strategy for the important task of S-S bridge assignment in proteins has been devised”. This final stage of protein primary structure determination has traditionally been very diffi- cult, involving long and exhaustive procedures that raise the possibility of scrambling during analysis. By contrast, the FAB procedure is straightforward and rapid. The protein is enzymically cleaved using con- ditions that minimise S-S bridge cleavage, i.e. neu- tral or acidic pH, and a FAB map is obtained. This map is compared with a second map obtained after treatment of the digest mixture with a mild reducing agent. Peptides which contain an intra-molecular S-S bridge will shift by two mass units as a result of conversion of the S-S bridge into a di-thiol; a high mass signal derived from two peptides joined by an

S-S bridge will disappear and be replaced by two signals at lower mass. If necessary, assignments can be confirmed by Edman degradation followed by a re- peat of the FAB map. These methods can be used to study the denaturation and refolding of proteins by time course analysis of S-S bridges.

Glycoproteins and cell surface antigens FAB-MS strategies for analysing glycans from gly-

coproteins can be conveniently divided into those which accomplish an initial screen for presence and type of glycosylation and those which, in conjunction with classical carbohydrate procedures, furnish de- tailed structural information, All have been de- signed to be applicable to a wide range of molecules irrespective of size and complexity. The protocols used ensure that in the final analysis the FAB data is being acquired in the high sensitivity low mass region of the spectrum (i.e. below about 5000 U). Sensitive screening for 0-glycosylation is rapidly and sensiti- vely effected by FAB-MS analysis of peracetylated or permethylated oligosaccharides released from the glycoprotein by alkaline elimination. The products of glycoprotein acetolysis produce characteristic ions in the FAB spectra which reveal the presence of high mannose and/or complex N-glycans. Sites of N- glycosylation in the protein sequence can, if re- quired, be ascertained by comparing tryptic FAB maps before and after the glycoprotein has been treated with N-glycanase, an enzyme which removes N-linked glycans12>13. Unambiguous sequencing of 0- and N-glycans is best achieved using permethy-

378 trends in analytical chemistry, vol. 8, no. lo,1989

lated derivatives which reproducibly fragment at each amino-sugar residue (see Fig. 2) thereby af- fording information on branching, sites of sialylation, fucosylation, sulphation as well as branch length. The fragmentation pattern displayed by a permethylated sample is very reproducible. Hence, a comparison of data obtained from two related glycoproteins, e.g. natural and recombinant or normal and abnormal, will reveal whether the non-reducing structures are identical or, if not, the exact nature of the differences. For example, FAB-MS of permethylated N-glycans from normal human Band 3, the anion transporter of erythrocytes, gave a characteristic map of fragments derived from cleavage at each N-acetylglucosamine residue in the long polylactosa- minoglycan chains (see Fig. 3). In contrast, an analo- gous map from the Band 3 of individuals suffering from a type of genetic anaemia called HEMPAS showed none of the fragment ions characteristic of polylactosaminoglycans and instead showed ions corresponding to a truncated molecule (see Fig. 3), indicating that a block in the biosynthesis had pre- vented chain elongation14.

tivity. The development of a new generation of mass spectrometers tailor made for biological applications and featuring post-acceleration array detection of ions up to 10 000 daltons in mass should help to achieve the goal of femtomolar sensitivity. These methods promise to revolutionise protein and carbohydrate structure analysis over the next decade and at the same time accelerate our understanding of many biological processes.

Acknowledgements The protein chemistry applications described in

this paper were carried out under the direction of Professor Howard R. Morris at Imperial College. We are grateful for financial support from the Medi- cal Research Council.

References 1

2

The characteristic non-reducing fragment ions observed in the spectra of permethylated glycans are present irrespective of the overall size of the molecule. This important feature of carbohydrate FAB- MS can be exploited in a variety of applications including the screening of cell surfaces for novel carbohydrate antigens. Studies of the N-glycans on the surface of normal granulocytes, chronic myelogenous leukaemia (CML) cells and acute myelogenous leukaemia cells provide an excellent illustration of the power of FAB-MS for mapping non-reducing structures on glycoproteins15@. The N-glycans were stripped from the cell surface by pronase digestion and carbohydrate-containing fractions were permethylated and analysed by FAB-MS. Each type of cell showed a unique FAB map revealing differences in sialylation fucosylation and elongation of branches. Importantly, a key signal at m/z 1622 in the CML map defined a uni

16 ue sialylated, difucosylated epit-

ope on this cell line .

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H. R. Morris, A. Dell and R. A. McDowell, Biomed. Mass Spectrom., 8 (1981) 463. M. Barber, R. S. Bordoli and R. D. Sedgwick, In H. R. Mor- ris (Editor), Soft Ionisation Biological Mass Spectrometry, Heyden, London, 1981, pp. 137-152. M. Barber and B. N. Green, Rapid Commun. Mass Spec- born., 1 (1987) 80. H. R. Morris, M. Panico, M. Barber, R. S. Bordoli, R. D. Sedgwick and A. N. Tyler, Biochem. Biophys. Res. Com- mun., lOl(1981) 623. K. Biemann and S. A. Martin, Mass Spectrom. Rev., 6 (1987) 1. D. H. Williams, C. V. Bradley, S. Santikarn and G. Bojesen, Biochem. J., 201(1982) 105. H. R. Morris, M. Panico, A. Karplus, P. E. Lloyd and B. Ri- niker , Nature, 300 (1982) 643. A. Dell, Adv. Carbohydr. Chem. Biochem., 45 (1987) 19. H. R. Morris and F. M. Greer, Trends Biotechnol., 6 (1988) 140.

13

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H. Erdjument, D. A. Lane, M. Panico, V. Di Marzo and H. R. Morris, J. Biol. Chem., 263 (1988) 5589. H. R. Morris and P. Pucci, Biochem. Biophys. Res. Com- mun., 126 (1985) 1122. A. Dell and J. E. Thomas-Oates, In C. J. Biermann and G. D. McGinnis (Editors), Analysis of Carbohydrates by GLC and MS, CRC Press, Boca Raton, FL, 1988. S. A. Carr and G. D. Roberts, Anal. Biochem., 157 (1986) 396. M. N. Fukuda, A. Dell and P. Scartezzini, .I. Biol. Chem., 262 (1987) 7195. M. Fukuda, E. Spooncer, J. E. Oates, A. Dell and J. C. Klock, J. Biol. Chem., 259 (1984) 10925.

The future High mass FAB-MS techniques are now available

which are capable of solving a wide range of biopolymer problems. The methods are already quite sensitive, applicable to sample sizes of the order of 100 ng to a few ,ug, require little or no prior knowledge of structure, and can be successfully applied to mixtures. The next generation of problems, e.g. charac- terisation of antigens on parasites, defining the structures of receptors, biopolymer messengers, etc. are already demanding a much higher level of sensi-

16 M. Fukuda, B. Bothner, P. Ramsamooj, A. Dell, P. R. Til- ler, A. Varki and J. C. Klock, J. Biol. Chem., 260 (1985) 12957.

17 K. L. Rinehart, Trends Anal. Chem., 2 (1983) 10.

Anne Dell received her BSc degree in chemistry from the Univer- sity of Western Australia in 1972 and her PhD from the University of Cambridge in 1975. Since 1975 she has been in the Department of Biochemistry, Imperial College, London where she is now a Reader in carbohydrate biochemistry. Mark E. Rogers received his BSc and PhD degrees in biochemistry from the Polytechnic of Central London in 1981 and 1985 re- spectively. He is currently a Lecturer in the Department of Clini- cal Endocrinology, Imperial College London.

fast atom bombardment mass spectrometry of protein and carbohydrate biopolymers

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