fast atom bombardment mass spectrometric strategies for characterizing carbohydrate-containing...

BIOMEDICAL AND ENVIRONMENTAL MASS SPECTROMETRY, VOL. 16, 19-24 (1988)

Fast Atom Bombardment Mass Spectrometric Strategies for Characterizing Carbohydrate-containing Biopolymers

Anne Dell,? Neil1 H. Carman, Philip R. Tiller and Jane E. Thomas-Oates Department of Biochemistry, Imperial College of Science and Technology, London SW7 2AZ, UK

Fast atom bombardment mass spectrometric methodologies for carbohydrate structure determination are inti- mately linked to classical procedures such as derivatization and hydrolysis. This paper reviews the current status of carbohydrate fast atom bombardment mass spectrometry and reports on two new protocols which we are using to optimize sensitivity and fragmentation.

INTRODUCTION

Major advances have been made in carbohydrate structure analysis during the 1980s, largely as a result of advances in nuclear magnetic resonance (NMR) and mass spectrometric methodologies. Fast atom bombardment (FAB) mass spectrometry has played a vital role in this structural revolution' and is beginning to provide answers to complex problems which have previously been totally intractable. In this paper we briefly review the progress to date, and describe novel strategies that we are devising to help probe the structures of minute quantities of carbohydrates which have putative roles in molecular recognition.

There exists an enormous diversity of carbohydrate- containing biopolymers, ranging from bacterial and plant polysaccharides having molecular weights of mil- lions of daltons (but, despite their size, often constituted of simple repeat units), through to the relatively small (200-1 0 000-dalton) complex oligosaccharides present in glycoconjugates. The current workable mass range of carbohydrate FAB mass spectrometry is about 4000 daltons for underivatized samples. Reduction in hydro- gen bonding capability e.g. by derivatization, extends this mass range to about 6000 daltons routinely, and to 20000 daltons or more for the special 'mapping' technique used for lactosaminoglycans.' Since the majority of carbohydrate-containing biopolymers (with the exception of glycolipids) are well in excess of the routine mass range, FAB mass spectrometry can only be applied to the products of enzymic or chemical digestion. Fortunately, a variety of procedures is now available for generating oligosaccharides and small glycoconjugates from all major classes of biopolymers. This makes FAB mass spectrometry a universally applicable technique in carbohydrate analysis. Further- more, once oligomers of appropriate size are available, they are all amenable to similar FAB mass spectrometric strategies, irrespective of their origin or structural diversity.

t Author to whom correspondence should be addressed.

FAB mass spectrometry of native samples, if successful, defines molecular weight and provides valuable information on sugar composition (isomers are not, however, differentiated). Importantly, FAB mass spectrometry also reveals the presence of functional groups such as methyl, acyl, phosphate, sulphate, etc., which are frequently difficult to define by classical carbohydrate methodologies. The presence of novel cyclic carbohydrates can also be indicated by the masses of the molecular ions.3 However, definite proof of the exis- tence of cyclic structures (as well as generation of information on virtually all structural features which can be defined by FAB mass spectrometry) requires the prior preparation of derivatives. Many structural problems, particularly those involving glycoprotein~,".~ are best solved by strategies incorporating derivatization prior to all FAB mass spectrometric experiments.

To date, the peracetyl and permethyl derivatives have proven to be the most useful for FAB mass spectrometry. Since these are the same derivatives as those chosen for most classical methodologies, FAB mass spectrometry can be conveniently incorporated into structural programmes already well established in carbohydrate research. This is particularly important because FAB mass spectrometry provides data which are, by and large, complementary to those afforded by gas chromatography/mass spectrometry and enzymatic analysis.

Peracetyl and permethyl derivatives ionize to give abundant molecular ions plus reproducible and predict- able fragment ions which permit unambiguous sequencing and the facile assignment of branching patterns. High sensitivity is achieved with both derivatives, in some cases two orders of magnitude better than that obtained with the native materials. At the present time, most FAB mass spectrometric analyses of peracetylated or permethylated samples are successful starting with 0.5-5 pg of sample, a sensitivity sufficient for solving many important structural problems. Higher sensitivity will, however, be essential for the characterization of glycoconjugates and polysaccharides that can only be isolated in sub-microgram quantities.

One of the most exciting areas of current carbo-

0887-6 134/88/24OO19-06 $05.00 0 1988 by John Wiley & Sons, Ltd.

20 A. DELL ET AL.

hydrate research is the characterization of glycoproteins, particularly those on cell surfaces where the carbohydrates are believed to be involved in cell-cell recognition. FAB mass spectrometry has already played an important role in characterizing such substances, e.g. the assignment of detailed structures of large polylactos- aminoglycans present in cell surface glycoproteins has only been possible since the application of FAB mass spe~trometry.~.~ The FAB spectra obtained from permethylated samples of glycoprotein-derived glycans are especially simple to interpret because the molecules fragment at all the N-acetylhexosamine (HexNAc) residues, yielding patterns of signals that reveal the type of glycan. For example, the identification of the di- sialylated biantennary structure (1) is a trivial problem, requiring only a few hours of analysis time because of the simple, but diagnostic, set of fragment ions produced from its permethyl derivative.

376 825 NeuAqGal-GlcNAqMan \ 2492

376 825 ,M~~-GICNA~GICNAC-AS~

Neu AqGal-GlcNAaMan

(1 )

Fragment ions, derived from the branches of permethylated N-linked glycans, reveal the patterns of sub- stitution and define whether N-acetyl lactosamine repeats are present. The overall size of the molecule is given by the mass of the fragment ion arising from cleavage of the chitobiose core, or from the molecular ion, if present. As the molecules become larger, e.g. tetra-antennary, or bi- and tri-antennary structures with several lactosamine repeats in the branches, the obser- vation of chitobiose cleavage ions or molecular ions becomes more and more difficult. This is unfortunate because valuable structural information relating to het- erogeneity, the precise lengths of branches, and the presence of minor constituents, i.e. data which may be vital for understanding the fine tuning of molecular recognition, can no longer be obtained by FAB mass spectrometry. We are addressing this problem, and the related one of improving sensitivity, by investigating derivatives containing a permanent positive charge. Results from one such derivative are presented below.

Some oligosaccharides, e.g. those derived from bacterial polysaccharides, do not have the regularly spaced HexNAc residues present in glycoproteins. Such molecules may not produce sufficient glycosidic cleavage ions to allow complete sequence assignment. We have introduced a methanolysis/FAB mass spectrometric strategy for solving such problems, and details of the method and its application to oligosaccharides from a meningitis bacterium are described below.

EXPERIMENTAL

Materials

The majority of reagents, including trimethyl p - aminophenyl ammonium chloride (TMAPA), and syn-

thetic oligosaccharides were obtained from Sigma. The Shigella flexneri octasaccharide was a gift from Dr N. Carlin, National Bacteriological Laboratory, Sweden. Oligosaccharides from Neisseria meningitidis were provided by Dr H. Jennings, NRC, Canada.

Methods

The majority of the experimental procedures are the same as previously except for the following.

Derivatization of reducing termini. TMAPA (10 mg, 45 pmol) was dissolved in water (350 pl) containing acetic acid (41 pl). This aminating solution (5 pl) was added to the carbohydrate (10 nmol) and the mixture heated at 90°C for 5 min. A reducing reagent was prepared by dissolving TMAPA (4 mg, 18 pmol) and sodium cyano- borohydride (10 mg, 160 pl) in water (350 pl) containing acetic acid (41 pl). This solution (20 pl) was added to the reaction mixture and incubated at 90 "C for a further 60 min, after which the sample was freeze-dried. The product was either purified by high-performance liquid chromatography (HPLC) prior to FAB mass spectrometric analysis, or it was peracetylated and analysed directly by FAB mass spectrometry without further purification.

HPLC of TMAPA-treated oligosaccharides. HPLC analyses were carried out using a Waters Associates HPLC system, having a Model 440 absorbance detector oper- ated at 254 nm. TMAPA-treated samples were chro- matographed on a Phase Sep Spherisorb S5 amino column (25 cm x 4.6 mm). Elution was carried out using acetonitrile/water at a flow rate of 1 ml min-'. The system was run isocratically with water for 5 min, followed by a linear gradient from water to 100% acetonitrile over 60 min. The TMAPA-tagged maltoheptaose sample eluted at 35 min.

Methanolysis. The N. meningitidis oligosaccharide (coded L5-F3) was permethylated using the Hakomori procedure. The product (10 pg) was transferred to a Reacti- vial (Pierce Chemical Co.) and 20 p1 methanolic HCl was added. A 1 pl aliquot was removed for FAB mass spectrometric analysis and the remaining solution was incubated at 40°C for 40 min, followed by 60°C for a further hour. Aliquots (1 pl) were removed after 2 min, 20 min, 40 min, 45 min, 70 rnin and 100 min and analysed immediately by loading directly into the FAB matrix. The exact time-course can be altered during the reaction if the FAB mass spectra indicate that the reaction is proceeding either very slowly or very rapidly.

FAB mass spectrometry. FAB mass spectrometry was carried out as previously described7 on a VG Analytical high-field ZAB mass spectrometer fitted with an M-Scan FAB gun.

FAST ATOM BOMBARDMENT MASS SPECTROMETRIC STRATEGIES 21

RESULTS AND DISCUSSION

Improvement of sensitivity

We showed, a number of years ago, that reducing end derivatives such as o x i m e ~ ~ . ' ~ could improve sensitivity and assist sequence assignment. Subsequently, other groups have described the FAB mass spectrometric behaviour of oligosaccharides 'tagged' with chromo- phores or fluorophores which were introduced to facilit- ate HPLC Im provements in sensitivity and quality of sequence data were similarly reported for these derivatives. On their own, reducing end derivatives are not as useful as peracetyl or permethyl derivatives, owing to lower sensitivity and less useful fragmentation. Indeed, the majority of structural problems that have been solved using strategies incorporating HPLC and subsequent FAB mass spectrometry (see, for example, Refs 5 and 13) have exploited the superior detection capabilities of radioactive isotopes (readily introduced via reduction) and the superior FAB behaviour of permethyl derivatives. Despite this, reducing end derivatives can play a valuable role in carbohydrate FAB mass spectrometry, but they are best used in conjunction with other derivatives such as the pera~ety1.l~ In this respect, it occurred to us that a potential route to higher sensitivity, particularly at high mass, could be to incorporate a moiety containing a permanent positive charge into the reducing end and then to peracetylate or permethylate. A suitable 'tag' would be one which is compatible with subsequent derivatization steps, which gives enhanced molecular ion abundance, and which does not completely direct fragmentation away from the non-reducing ends, since the HexNAc cleavages are essential for facile sequencing and mapping of glycoproteins. The reducing end reagent, TMAPA (2), incorporated by reduction of the Schiffs base, appears to meet these specifications.

n L

The behaviour of TMAPA was investigated using maltoheptaose. Treatment of maltoheptahose with TMAPA under reducing conditions yielded 3 which, after HPLC

L

3

purification, gave the anticipated molecular ion (M+) at m/z 1287. A signal : noise ratio of approximately 10 : 1 was obtained using 0.5 pg of sample, while similar quality data on native maltoheptaose required about 5 pg of material. No fragment ions were present in the spectrum of 3 but, after peracetylation of either the HPLC purified sample, or of the crude reaction

3 3 i i 619

1- ,

813 841 I . ,I ., , L .

1129 ? , 1101 I

1389 1417 1611

19" 1993

2253 'M'*2295

2211 I 1 Figure 1. Positive FAB spectrum of a 0.5 pg sample of maltoheptaose after tagging with TMAPA and peracetylation. Matrix signals are indicated by X.

product, excellent quality spectra were obtained showing significant fragmentation. A representative spectrum is shown in Fig. 1. The major molecular ion at m/z 2295 corresponds to the per-0-acetylated molecule having an additional acetyl group on the secondary nitrogen. Mono- and di-underacetylated molecules afford the signals at m/z 2253 and 2211. The fragmentation pattern is complex but is readily rationalized. Three major series of ions are present: (i) b-cleavage ions' at m/z 1965, 1677, 1389, 1101, 813, 525; (ii) ring cleavage ions' containing a formyl group on the glycosidic oxygen which occur 28 u higher than each of the ions in (i); and (iii) A-type cleavage ions' at m/z 1771, 619 and 331. Series (i) and (ii) retain the TMAPA moiety, whilst (iii) are non-reducing and are also relatively weak. For comparison, the spectrum of peracetylated aniline-tagged maltoheptaose is shown in Fig. 2. This molecule lacks the permanent positive charge. It is clear that the permanent positive charge has improved molecular ion abundance relative to fragment ion abundance and has, as expected, switched the fragmentation mode from A-type cleavage to b-cleavage. Importantly, however, some non-reducing ions are still present in the TMAPA spectrum.

TMAPA-tagged maltoheptaose was permethylated using the sodium hydroxide procedure of Ciucanu and Kerek." An abundant molecular ion for the fully methylated molecule was observed at m/z 1623, from a sample loading of 0.1 pg. No fragment ions were present

22 A. DELL ET AL.

+ Hex,

I

Hex; 559 619

Hexi

1195 I

Hex; 1483

1423 I

Hex: 1771

1729

Figure 2. Positive FA6 spectrum of a 0.5 pg sample of maltoheptaose after tagging with aniline and peracetylation. The [M + H]+ signal at m/z 21 96 corresponds to the per-U-acetylated molecule. Additional acetylation on the secondary nitrogen is indicated by the signal at rn/z 2238. The signals at m/z 2250 and 2292 contain a single TFA group replacing an acetyl group. The major fragment ions result from A-type cleavages.

but this was not surprising since the molecule lacked HexNAc residues. To investigate the potential application of TMAPA-tagging for molecules containing HexNAc residues, we used an octasaccharide derived from Shigella jlexneri, having the sequence Rha- GlcNAc-Rha-Rha-Rha-GlcNAc-Rha-Rha (coded 4). The FAB spectrum of TMAPA-tagged permethylated 4 is shown in Fig. 3. The molecular ion signals (m/z 1715 and 1729) are very abundant, whilst the fragment ions are relatively weak. Fragment ions resulting from cleavage at each of the HexNAc residues, m/z 434 (Rha- HexNAc') and m/z 1201 (Rha-HexNAc- Rha,-HexNAc') are present, proving that the important A-type ions are still produced despite the fixed charge. Thus the TMAPA-tagged oligosaccharide exhibits the required behaviour of compatibility with

permethylation, high molecular ion abundance, and observable A-type HexNAc cleavages. We are now extending these studies to oligosaccharides released from glycoproteins by chemical or enzymic treatment.

Methanolysis as an aid to sequence analysis

The use of derivatization to direct fragmentation in FAB mass-spectrometry of carbohydrates has been very successful. However, in some molecules, especially highly branched ones, the favoured cleavages may not provide sufficient data for complete sequencing. To overcome this problem we have devised a strategy in which FAB mass spectrometric analyses of chemical

FAST ATOM BOMBARDMENT MASS SPECTROMETRIC STRATEGIES

1729

23

Rha ti exNAc*

I I 434

R ha Hex NAc Rho3HexNAc* 1201

Figure 3. Positive FAB spectrum of Rha-GlcNAc-Rha,-GlcNAc-Rha, tagged with TMAPA and permethylated. Two molecular ions are observed at m/z 1715 and 1729. The latter corresponds to molecules that have been methylated on the secondary nitrogen. HexNAc cleavage ions are present at m/z 434 and 1201.

hydrolyses are carried out whilst the reaction is proceeding. The sample is first permethylated and checked by FAB mass spectrometry to establish whether methyl- ation is reasonably complete. If so, the permethylated sample is subjected to a time-course methanolysis during which aliquots are removed at frequent intervals and FAB spectra acquired. The masses of the molecular ions of the hydrolytic fragments define the number of free hydroxyl groups present and these data are used to assign sequences and branching patterns. For example, hydrolytic removal of one or more residues without the generation of a free hydroxyl group indicates that those residues are at the reducing end. Similarly, the pro- duction of two free hydroxyl groups indicates the hydrolysis of two different branches.

Our structural studies on Neisseria meningitidis core lipopolysaccharides'6 have provided us with a range of oligosaccharides which we have used to optimize the methanolysis protocol. One of these samples (coded L5-F3) gave the following data. Negative FAB mass spectrometry of the native material afforded molecular ions at m / z 1310 and 1352, corresponding to composi- tions of Hex,HexNAc,Hep,KDO, and its mono- acetylated counterpart, respectively. Signals 18 u below m/z 1310 and 1352 indicate that some of the molecules

are lactonized. Positive FAB mass spectrometry gave corroborative data. Positive FAB mass spectrometry of perdeuteroacetylated L5-F3 located the acetyl group on a non-reducing HexNAc residue and, together with data from the permethyl derivative, defined the partial structure (5).

Hex-Hex

\ (Hep.KD0)

Ac,,, -HexNAc-(Hep,Hex) / 5

The complete sequence was obtained using the methanolysis protocol. Approximately 10 pg L5-F3 were permethylated and 1 pg analysed by FAB mass spectrometry. The resulting spectrum showed that the sample was completely methylated and that the KDO was fully lactonized ([M + H I C at m / z 1630). The remainder of the permethylated sample was then subjected to a time-course methanol/HCl treatment (see Experimental) using FAB mass spectrometry to monitor aliquots while the reaction was proceeding. Significant regions of the FAB spectra obtained at three key time-

24 A. DELL ET AL.

1630

(a) 1630

964 1182

Figure 4. Methanolysis of L5-F3: (a) Data obtained immediately after addition of methanol/HCI, (b) Data obtained after 10 min methanolysis at 40 "C. (c) Data obtained after methanolysis at 40 "C for 60 min and 60 "C for 20 min.

points are reproduced in Fig. 4. Spectrum (a) contains a new signal at m/z 1400 corresponding to the composition Hex,HexNAc,Hep, , consistent with the removal of a KDO residue from the reducing end. Spectrum (b) contains a new signal at m/z 1182 attributable to Hex,HexNAc,Hep, with one free hydroxyl group arising from removal of a non-reducing Hex from Hex,HexNAc,Hep, . Spectrum (c) contains a signal at m/z 964, first observed as a minor component in spectrum (b), that is consistent with the composition Hex,HexNAc,Hep, with two free hydroxyl groups. This indicates the loss of an additional non-reducing terminal hexosyl residue. These data define the structure of L5-F3 as 6.

Hex-Hex

\

CONCLUSION

FAB mass spectrometry now has an excellent track- record for solving a broad spectrum of carbohydrate structural problems. Many more exciting problems await solution because current technology is not suffi- ciently sensitive. We are confident that conjoint chemical/FAB mass spectrometric strategies of the type reported in this paper will help to take carbohydrate FAB mass spectrometry from the microgram to the nanogram range of sensitivity.

Acknowledgements

Ac,,, -HexNAc-Hep' I I Hex 6

We thank the Medical Research Council for financial support. PRT is a recipient of an SERC studentship.

REFERENCES

1. A. Dell, Adv. Carbohydr. Chem. Biochem. 45. 19 (1 987). 2. J. E. Oates, A. Dell, M. Fukuda and M. N. Fukuda, Carbohydr.

Res. 141, 149 (1985). 3. A. Dell, J. Oates, C. Lugowski. E. Romanowska, L. Kenne and

B. Lindberg, Carbohydr. Res. 133, 95 (1 984). 4. M. Fukuda, A. Dell and M. N. Fukuda, J. Biol Chem. 259,

4782 (1 984). 5. H. Sasaki, B. Bothner, A. Dell and M. Fukuda, J. Eiol. Chem.

262,12059 (1 987). 6. M. Fukuda, B. Bothner, P. Ramsamooj, A. Dell, P. R. Tiller, A.

Varki and J. C. Klock, J. Biol Chem. 260, 12957 (1 985). 7. A. Dell and J. E. Oates, in Analysis of Carbohydrates b y GLC

and MS, ed. by C. J. Biermann and G. McGinnis, CRC Press, Boca Raton, Florida, 1988.

8. W. T. Wang, N. C. LeDonne, Jr, B. Ackerman and C. C. Sweeley, Anal. Biochem. 141, 366 (1 984).

9. A. Dell, J. E. Oates, H. R. Morris and H. Egge, lnt. J. Mass. Spectrom. /on. Phys. 46, 41 5 (1 983).

10. E. A. Nothnagel, M. McNeil, P. Albersheim and A. Dell, Plant Physiol. 71, 91 6 (1 983).

11. G. R. Her, S. Santikarn. V. N. Reinhold and J. C. Williams, J. Carbohydr. Chem. 6, 129 (1 987).

12. B. L. Gillece-Castro, S. J. Fisher, A. L. Tarentino, D. L. Peter- son and A. L. Burlingame, Arch. Biochem. Biophys. 256, 194 (1987).

13. M. Fukuda, S. R. Carlsson, J. C. Klock and A. Dell, J. Biol. Chem. 261,12796 (1 986).

14. B. Lindberg, K. Leontein, U. Lindquist, S. B. Svenson, G. Wrangsell, A. Dell and M. Rogers, Carbohydr. Res., 174, 31 3, (1988).

15. 1. Ciucanu and F. Kerek, Carbohydr. Res. 131, 209 (1 984). 16. Research being carried out in collaboration with Dr H. J. Jen-

nings, NRC, Canada.

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