fast atom bombardment mass spectrometry of a 6-o-methylglucose polysaccharide

Fast Atom Bombardment Mass Spectrometry of a 6-0- Methylglucose Polysaccharide

~ ~

Anne Dell? Department of Biochemistry, Imperial College of Science and Technology, London SW7 2AZ, UK

Clinton E. Ballou Department of Biochemistry, University of California, Berkeley, California 94720, USA

Fast atom bombardment mass spectra of a 6-O-methylglucose polysaccharide of molecular weight 2852 dalton are described. Fragmentation pathways in both positive and negative modes are defined. The presence of cationized sequence ions in the positive spectra is confirmed by salt dosing experiments. Positive spectra obtained using monothioglycerol as the supporting matrix are compared with data obtained using glycerol. The potential importance of three dimensional structure in controlling the fragmentation behaviour of large molecules under F AB conditions is discussed.

INTRODUCTION

The new technique of fast atom bombardment (FAB) mass spectrometry“’ has already had considerable impact in the area of biological polymer structure determination because of its ability to provide good quality data on relatively large, highly polar compounds. FAB mass spectrometry has achieved much of its suc- cess in the peptide area where, for example, it has been used to aid the characterization of a variety of novel peptides including an acetylated form of a -melanocyte stimulating hormone,3 a cardioactive ne~ropept ide ,~ dynorphins from the adrenal gland5 and many protein derived peptides.6-’1 These studies have enabled the delineation of fragmentation pathways for small peptides, and have indicated that FAB mass spectrometry when coupled with high-field magnet facilities,” can be used to define the molecular weights of large polypep- tides and small proteins.13

Many biologically important peptides and proteins exist in glycosylated forms, and FAB mass spectrometry is potentially a powerful tool for studying these molecules. It is clear that a detailed understanding of the FAB behaviour of both carbohydrates and peptides will be of considerable value in glycopeptide structural analysis. To date, however, reports on the FAB spectra of complex carbohydrates, both with respect to fragmentation mechanisms and to the maximum size of molecule amenable to analysis, are still limited.l4.I5

We are currently developing methods for glycopeptide structure determination and, as part of this work, we have been examining the FAB behaviour of a variety of carbohydrates. In this paper we report our FAB mass spectral studies of a 6-O-methylglucose polysaccharide (code AGMGP) derived from a Mycobacterial glycolipid. l6 We have recently revised the structure of this molecule using data obtained from a variety of chemical and physical techniques, with mass spectrometry playing a key role in defining its precise size.” The positive FAB spectrum of AGMGP has been briefly described in our paper reporting the structural revision of the i Author to whom correspondence should be addressed.

molec~le . ’~ We now present a full discussion of the FAB data, including spectra obtained in both positive and negative modes. The effects on the positive spectra of salt dosing and the replacement of the glycerol matrix by monothioglycerol are described. Structures are pro- posed for the major ions present in the negative spectra. Our understanding of the FAB behaviour of carbohydrates in both positive and negative modes has been considerably aided by these results on AGMGP.

~~~~~

EXPERIMENTAL

AGMGP was obtained by enzyme digestion of MGP, a polysaccharide containing four additional sugar residues. Experimental details for the isolation of MGP and AGMGP are given in Ref. 17.

Fast atom bombardment mass spectrometry was car- ried out using a VG ZAB 1F high field magnet mass spectrometer (mass range 3300 at 8 kV) fitted with a FAB source. Xenon was used as the bombarding gas and the FAB gun was operated at 8 kV.

Samples of,AGMGP were dissolved in 5 % acetic acid (5-10 pg 11.1- ) and a 1 p1 aliquot was added to a thin smear of glycerol or monothioglycerol on the stainless steel target. For the salt dosing experiments 1-5 mg of NaCI, KCI or LiCl were dissolved in 10 ml of water and 1 p,l of the resulting solution was added to the mixture of AGMGP/S0/~ acetic acid/glycerol on the target.

Spectra were obtained using a 500 s linear mass scan from m/z 3000 to m / z 10 and were recorded on UV sensitive chart paper. Spectra were mass marked by manual counting.

RESULTS AND DISCUSSION

The revised structure of AGMGP is shown in Fig. 1, and its positive FAB spectrum, obtained using glycerol as a matrix, is presented in Fig. 2. AGMGP is an acidic polysaccharide which is isolated as the alkali metal salt

CCC-0306-042X/83/0010-0050$03.50

50 BIOMEDICAL MASS SPECTROMETRY, VOL. 10, NO. 1, 1983 @ Wiley Heyden Ltd, 1983

FAST ATOM BOMBARDMENT OF A 6-0-METHYLGLUCOSE POLYSACCHARIDE

r I

L I CO,H

Figure 1. Revised structure of AGMGP, a 6-0-methylglucose polysaccharide containing 16 hexose residues and a glyceric acid moiety at the reducing terminus.

after saponification and enzymic digestion of the parent glycolipid. Thus the signals at m / z 2875 and 2891 are attributed to the protonated molecular ions of the sodium and potassium salts, respectively. When a sufficient quantity of AGMGP is loaded into the glycerol matrix (=lo kg), complete suppression of glycerol ionization occurs and the spectrum shown in Fig. 2 is obtained. The most striking feature of this spectrum is the presence of fragment ion clusters, containing signals 2,16 and 18 u apart, which are repeated at sugar unit intervals (176 u for monomethylglucose residues and 16211 for glucose residues) from just above m / z 700 up to the molecular ion. Below m / z 700 a different fragmentation pattern is evident and this part of the spectrum is rationalized as follows: m / z 177, 353 and 529 are assigned to oxonium ion species of the following structure (a ) :

R = H (m /z 177) R = 6-0-MeGlc (m/z 353) R = (6-0-MeGl~)~ ( m / z 529) RO

I

Facile loss of up to two molecules of water from each of these sequence ions gives rise to the signals at mlz 141,159,299,317,335 and 493. Analogous cleavage at the fourth sugar residue yields the ion at mlz 705. At this point in the spectrum the fragmentation pattern becomes more complex with the appearance of major signals 22 (mlz 727) and 38 (mlz 743) mass units higher than the signal assigned to the oxonium sequence ion at m / z 705. A further degree of complexity is introduced by the presence of flanking signals two mass units away from each of the major ions, presumably due either to hydrogen transfers dur- ing fragmentation or to redox-type reactions in the matrix. The signals at m / z 727 and 743 can be assigned to sodium containing sequence ions resulting from cleavage on the nonreducing and reducing sides, respectively, of the glycosidic oxygen with charge retention on the non-reducing end of the molecule (6 and c).

foMe c O M e

ONa (b 1

Alternatively, m / z 743 could be potassium cationized. However, the salt dosing experiments described later indicate that a major contributor to m / z 743 is a sodium containing species.

Cleavage at the fifth and subsequent glycosidic link- ages up to the tenth sugar residue yields the signals at m / z 903, 919 (5 residues), 1079, 1095 (6 residues), 1255, 1271 (7 residues), 1431, 1447 (8 residues), 1607, 1623 (9 residues) and 1783, 1799 (10 residues). The corresponding noncationized oxonium ions are of very low abundance above m / z 705. The next major signal, following the 1783, 1799 cluster, occurs at m / z 1959 which corresponds in mass to a monomethylglucosyl addition to m / z 1783. The expected associated signal 161.1 higher at m / z 1975 (m/z 1799 plus 176u) however is absent. Instead, a signal is present 161.1 below m / z 1959 at m / z 1943. These data suggest that m / z 1959 is not a sequence ion of the type described above. At higher mass the characteristic sequence ion cluster reappears at m/r 2121, 2137, i.e. after a mass interval of methylglucosyl plus glucosyl from m / z 1783, 1799. A branch point of the molecule is suggested by these data with m / z 1943 and 1959 arising from loss of a complete glucose molecule from the 12-hexose unit, the sequence ions of which are present at m / t 2121, 2137. This conclusion is consistent with the revised structure of AGMGP (see Fig. 1). The remainder of the spectrum can be interpreted in a similar manner, i.e. m / z 2283,2299, sequence ion cleavage at residue 13; m/z 2429, 2445, loss of the branching glucose from the 15 hexose unit; m / z 2607, 2623, sequence ion cleavage at residue 15; m / z 2713, loss of a branching glucosyl residue from the molecular ion; m / z 2787, loss of glyceric acid from the molecular ion.

To help confirm the above assignments, and in par- ticular to prove that sodium cationization was taking place, a number of salt dosing experiments were per- formed on AGMGP in glycerol. Addition of potassium chloride or lithium chloride to the matrix yielded potassium and lithium cationized molecular ion species, respectively. In each instance the most abundant molecular ion signal corresponded to the potassium or lithium cationized sodium salt of AGMGP although significant amounts of potassium or lithium cationized potassium or lithium salts were also present. These results suggest that the sodium ion of the glycerate salt does not equilibrate readily with other ions in the matrix. All signals in the AGMGP spectrum that had been assigned to sodium cationized species (see above) shifted appropriately after potassium or lithium dosing. Shifts were only partial, however, as illustrated in Fig. 3 which shows the effects of salt dosing on three fragment ion clusters. Note the appearance of new signals 16 u higher for potassium dosing (Fig. 3(b)) and 16 u lower for lithium dosing (Fig. 3(c)). Only partial shifts are expected in view of the molecular ion data, which indicates that a high proportion of the molecules retain a strongly bound sodium ion.

It is interesting that a minimum length of carbohy- drate chain (4 residues) appears to be necessary before significant cationization of the sequence ions occurs in the glycerol matrix. Moreover, the degree of cationization of the lower mass sequence ions is influenced by the type of matrix used. These observations are exemplified by the results shown in Figs 4 and 5. Figure

BIOMEDICAL MASS SPECTROMETRY, VOL. 10, NO. 1, 1983 51

A. DELL AND C. E. BALLOU

353 159

177 141

299

317

335

529

551 567 493

b & - 1079 903 1919

*03 815

1431 1167

1623

1447

1695

1799 , 1959

2137

2875

I I 2187 2713

Figure 2. The positive FAB spectrum of AGMGP obtained from 10 Fg of sample loaded into a glycerol matrix.

52 BIOMEDICAL MASS SPECTROMETRY, VOL. 10, NO. 1, 1983


1959 2137 1799

1799 LI 2137

1959

( b i

1783 2121

1767 1799 L L 2105 L 1959 2137 cr

2153 1975 L

( C i

1815

Figure3. Partial positive FAB spectra of AGMGP (a) 5 pg of sample loaded into a glycerol matrix (b) 5 +g of sample loaded into a glycerol matrixfollowed by 1 @I of asolution of potassium chloride inwater (100 pg m1-l (c)5 pg of sample loaded intoa glycerol matrixfollowed by 1 &I of a solution of lithium chloride in water (100 p g ml-’1.

4 shows a partial mass spectrum of AGMGP obtained after the addition of sodium chloride to the matrix. Although sufficient salt was added to cationize com- pletely the parent molecule (Fig. 4(b), m/z 2897 corresponds to the sodium cationized sodium salt of AGMGP), no cationization of the first three sequence ions was obtained (Fig. 4(a)). In contrast to this result, the use of monothioglycerol, instead of glycerol, as the matrix resulted in significant cationization of all sequence ions, including those at low mass. A typical spectrum (above m / z 290; below this mass, signals from the matrix dominate the spectrum) is reproduced in Fig. 5 . Sodium cationization is clearly evident at the second sequence ion ( m J z 375, 391) and przdominates at higher mass (e.g. m / z 551,567,727,743, etc.). These results suggest that the absence of sodium cationized sequence ions in the low mass region of the spectrum of AGMGP in glycerol may reflect conformational restraints imposed on the molecule by the glycerol matrix. It is possible that AGMGP as the glycerate salt

2091

Figure 4. Partial positive FAB spectra of AGMGP obtained from 10 w g of sample loaded into a glycerol matrix followed by 1 p,I of a solution of sodium chloride in water (100 pg ml-’).

exists as a ‘crown ether’ type of complex in glycerol, with the sodium ion strongly bound to a number of sugar residues located towards the reducing end of the molecule. The chelated sodium ion is then available for cationization of the higher mass sequence ions, directing fragmentation such that cleavage takes place on either side of the glycosidic oxygen.

A detailed examination of the AGMGP positive FAB spectra (e.g. Fig. 2) reveals other fragmentation pathways in addition to those outlined above. For example, a series of ions separated in mass by 176 u is evident at m / z 815, 991, 1167, 1343, 1519, 1695 and 1871. Each of these signals is 7 2 u above the higher mass component of the sodium cationized sequence ion clusters previously described. These ions are also found in the spectrum obtained using monothioglycerol as matrix and are therefore not glycerol adduct ions as suggested in our preliminary report on the FAB spectrum of AGMGPI7. A plausible structure for this ion series is given below (d) :

xo F)..jMe + N{ X = (6-0-MeGlc).

OH I (4

If this explanation is correct the ion series should terminate at residue 11 (subsequent sugar residues are not methylated at position 6) and should be replaced


A. DELL AND C. E. BALLOU

353

1079 1095 919

887 903

1431 1447 1255 I271

1623

1519 1695 ’.

Y

2875 I

Figure 5. Positive FAB spectrum of AGMGP obtained from 10 pg of sample loaded into a monothioglycerol matrix.

by a series 58 u above the corresponding sodium cationized glycosidically cleaved sequence ion, i.e. structure e :

The signal at m / z 2195 which is 58 u above m / z 2137 supports this hypothesis. Higher mass signals are insufficiently intense to confirm or contradict the pro- posed fragmentation pathway.

Under negative FAB conditions AGMGP gives excellent data and a typical spectrum (above m/z 400, lower mass sample ions are obscured by background peaks) is reproduced in Figure 6. The molecule yields abundant [M - HI- ions at m/z 285 1 and a weak signal is also present for the doubly charged ion [M- 2Hr- at m/z 1425 (confirmed by the presence of its C

isotope peak at m / z 1425.5). The remainder of the spectrum is dominated by two series of ions at m/z 411, 587, 763, 939, 1115, 1291, 1467, 1643, 1819, 1995, 2157, 2319, 2481 and 2643 (for convenience referred to in the following discussion as Series A) and at m/z 467,643,819,995,1171,1347, 1523,1699 and 1875 (Series B). In addition, loss of branching glucosyl residues from the molecular ion yields the signals at m/z 2689 and 2527. Note that series A ions continue at sugar unit intervals from m / z 41 1 up to the molecular ion, while Series B ions occur 56u above each of the Series A ions but only as far as m / z 1875. Plausible structures for Series A (f) and Series B ( g ) ions are shown below:



587

I

467 643

763

819

939

995 1115

1171 1291

1467 1523 1425.5 I

1643 1699

1777 1819

1875 1995

a&Mm.&n

2319 L

2157

w.Lu..Luuy--

2527 k 2481 ..

A - & .LI

2689 L 2643

b -.

Figure 6. Negative FAB spectrum of AGMGP obtained from 10 p.g of sample loaded into a glycerol matrix.

The series A ion at m l z 411 is thus produced by cleavage across the third sugar unit from the nonreducing end of the molecule, generating a stable enolate anion. Subsequent signals in this series are rationalized as arising in a similar manner. It should be noted that the negative spectrum does not reveal the positions of the branching glucosyl residues since these residues are

apparently cleaved off with equal facility to the back- bone cleavage thereby producing a spectrum equivalent to that expected for a nonbranched molecule. Series B ions can only be generated if a 6-0-methyl group is present on the sugar residue undergoing cleavage. Hence the termination of this series at residue 11 (see Fig. 1).


A. DELL AND C . E. BALLOU

In other studies of glucan polymers18 we have obser- ved Series A-type ions as the major fragment ions in the negative FAB spectra. Hence formation of Series

dimensional structure in controlling the fragmentation behaviour of large molecules under FAB conditions.

A ions appears to be a general fragmentation mechan- ism for underivatized oligosaccharides. Acknowledgements

In conclusion, our studies on AGMGP have allowed us to define fragmentation pathways for this complex molecule in both positive and negative FAB modes' Further, the salt dosing and monothioglycerol ex@- ments have suggested the potential importance of three

A.D. is grateful for grant support from the Medical and Science and Engineering Research Councils and thanks Professors H. R. Morris and A. L. Burlingame for their interest in this work. C.E.B. acknowl- edges support from United States Public Health Service Grant AI-12522 and National Science Foundation Grant PCM80-23388.

REFERENCES

1. M. Barber, R. S. Bordoli, R. D. Sedgwick and A. N. Tyler, Chem. Commun. 325 (1981).

2. M. Barber, R. S. Bordoli, R. D. Sedgwick and A. N. Tyler, Nature (London) 293,270 (1981).

3. A. Dell, T. Etienne, M. Panico, H. R. Morris, G. P. Vinson, B. J. Whitehouse, M. Barber, R. S. Bordoli, R. D. Sedgwick and A. N. Tyler, Neuropeptides 2, 233 (1982).

4. H. R. Morris, M. Panico, A. Karplus, P. E. Lloyd and B. Riniker Nature (London) in press.

5. S. Lemaire, L. Chouinard, D. Denis, M . Panico and H. R. Morris, Biochem. Biophys. Res. Commun. 108, 51 (1982).

6. H. R. Morris, A. Deli, A. T. Etienne, M. Judkins, R. A. McDowell, M. Panico and G. W. Taylor, Pure Appl. Chem. 54, 267 (1982).

7. H. R. Morris, A. Dell, M. Judkins, R. A. McDowell, M. Panico and G. W. Taylor, Peptides: Synthesis-Structure-Function Proceedings 7th Am. Peptide Symp., edited by D. H. Rich and E. Gross, pp. 745-755 Pierce Chemical Co. (1981).

8. H. R. Morris, M. Panico, M. Barber, R. S. Bordoli. R. D. Sedgwick and A. N. Tyler, Biochem. Biophys. Res. Commun. 101, 623 (1981).

9. D. H. Williams, C. V. Bradley, S. Santikarn, and G. Bojesen, Biochem. J. 201, 105 (1982).

10. D. H. Williams, G. Bojesen, A. D. Auffret, and L.C.E. Taylor, FEBS Lett. 128,37 (1981).

11. D. H. Williams, C. Bradley, G. Bojesen, S. Santikarn and L. C. E. Taylor J. Am. Chem. SOC. 103,5700 (1981).

12. H. R. Morris, A. Dell and R. A.McDowell, Biomed. Mass Spectrom. 8,463 (1981).

13. A. Dell and H. R. Morris, Biochem. Biophys. Res. Commun. 106. 1456 (1982).

14. A. L. Burlingame, A. Dell and D. H. Russell, Anal. Chem. 54, 363R (1982).

15. H. Egge, J. Dabrowski, P. Hanfland, A. Dell and U. Dabrowski, Advances in Experimental Medicine and Biology, in press.

16. C. E. Ballou, Accrs Chem. Res. 1,366 (1968). 17. L. C. Forsberg, A. Dell, D. J. Walton and C. E. Ballou, J. Biol.

Chem. 257,3555 (1982). 18. A. Dell, unpublished work.

Received 25 June 1982


fast atom bombardment mass spectrometry of a 6-o-methylglucose polysaccharide

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