Characterization of Heparin Oligosaccharide Mixtures as Ammonium

William LaMarr , Francyne Kubaski , Roberto Giugliani , Aratrik Guha , Eriko Yasuda , William Mackenzie , Seiji Yamaguchi , Yasuyuki Suzuki , Tada...
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Anal. Chem. 1998, 70, 2060-2066

Characterization of Heparin Oligosaccharide Mixtures as Ammonium Salts Using Electrospray Mass Spectrometry Wengang Chai,† Jinli Luo,‡ Chang Kee Lim,‡ and Alexander M. Lawson*,†

MRC Glycosciences Laboratory, Imperial College School of Medicine, Northwick Park Hospital, Harrow, Middlesex HA1 3UJ, U.K., and MRC Toxicology Unit, University of Leicester, Lancaster Road, Leicester LE1 9HN, U.K.

Among glycosaminoglycans, polysulfated heparin chains provide the greatest challenge to characterization due to high polarity, structural diversity, and sulfate lability. The present report demonstrates how electrospray mass spectrometry (ESMS) can be used to derive compositional information from pure and mixed fractions of heparin tetra- to decasaccharide fragments prepared by controlled digestion of heparin with heparinase I. It also describes an improved procedure for fractionation of heparin oligosaccharides up to octadecasaccharides. Ammonium salts prove to be superior to sodium salts, particularly for analysis of mixed components. In the mass spectrum of a hexasaccharide fraction, the identification of six major mass peaks that represent the known hexasaccharide structures confirms that ESMS analysis of heparin oligosaccharide fragments gives a close representation of their constituent composition. In addition to the previously identified components, several unreported oligosaccharides were detected in the spectra of octa- and decasaccharide fractions. The ESMS identification of the three major species in a decasaccharide fraction was confirmed after HPLC subfractionation and reanalysis. ESMS detection sensitivity of low picomole amounts of oligosaccharides can be readily achieved.

the intact chains are being made available for testing in biological assays.3-5 The most complex of the GAG chains are heparin and heparan sulfate, and their unambiguous characterization, even as fragments, is difficult. Alternating (1-4)-linked glucosamine (GlcN) and hexuronic acid (HexA) constitute their primary structure, with heterogeneity arising from variation in sulfation patterns, different degrees of GlcN N-acetylation, and isomerization of glucuronic acid (GlcA) to iduronic acid (IdoA). The most highly sulfated and common regions6 of heparin contain three sulfates per disaccharide unit, 6-O- and 2-N-disulfated-GlcN and 2-O-sulfated-HexA. Mass spectrometry (MS) has been an important method for high sensitivity characterization of GAG oligosaccharides.3,4,7-14 Due to the regular linear sequence of each class of GAG polysaccharides, structural details, such as chain length and composition, in terms of HexA and GlcN, and SO3 and acetyl (Ac) substitution, can be derived from molecular mass information of sufficient accuracy.7 Among MS techniques, negative-ion liquid secondary ion mass spectrometry (LSIMS) has been applied to heparin oligosaccharides with thioglycerol3,8,9 or triethanolamine as matrix.10 When analyzed as free acids or ammonium salts,

* Corresponding author. Tel: (44) 181-869 3250. Fax: (44) 181-869 3253. E-mail: [email protected]. † Imperial College School of Medicine. ‡ University of Leicester. (1) Bourin, M.-C.; Lindahl, U. Biochem. J. 1993, 289, 313-330. (2) Stringer, S. E.; Gallagher, J. T. Int. J. Biochem. Cell Biol. 1997, 29, 709714.

(3) Chai, W.; Hounsell, E. F.; Bauer, C. J.; Lawson, A. M. Carbohydr. Res. 1995, 269, 139-156. (4) Chai, W.; Kogelberg, H.; Lawson, A. M. Anal. Biochem. 1996, 237, 88102. (5) Larnkjaer, A.; Nykjaer, A.; Olivecrona, G.; Thøgersen, H.; Østergaard, P. B. Biochem. J. 1995, 307, 205-214. (6) Casu, B. In Heparin: Chemical and Biological Properties, Clinical Applications; Lane, D. A., Lindahl, U., Eds.; Edward Arnold: London, 1989; pp 2549. (7) Chai, W.; Luo, J.; Lim, C. K.; Lawson, A. M. Proceedings of the 45th ASMS Conference on Mass Spectrometry and Allied Topics, Palm Springs, CA, June 1-5, 1997; Abstract WPH 251. (8) Reinhold, V. N.; Carr, S. A.; Green, B. N.; Petitou, M.; Choay, J.; Sinay, P. Carbohydr. Res. 1987, 161, 305-313. (9) Dell, A.; Rogers, M. E.; Thomas-Oates, J. E.; Huckerby, T. N.; Sanderson, P. N.; Nieduszynski, I. A. Carbohydr. Res. 1988, 179, 7-19. (10) Mallis, L. M.; Wang, H. M.; Loganathan, D.; Linhardt, R. J. Anal. Chem. 1989, 61, 1453-1458. (11) McNeal, C. J.; Macfarlane, R. D.; Jardine, I. Biochem. Biophys. Res. Commun. 1986, 139, 18-24. (12) Juhasz, P.; Biemann, K. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4333-4337. (13) Juhasz, P.; Biemann, K. Carbohydr. Res. 1995, 270, 131-147. (14) Chai, W.; Green, B. N.; Lawson, A. M. Proceedings of the 41st ASMS Conference Mass Spectrometry Allied Topics, San Francisco, CA, May 30June 4, 1993; p 85.

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It has become apparent that glycosaminoglycan (GAG) oligosaccharide sequences occurring in free form or as components of proteoglycans participate in a number of important biological processes and interact with a wide range of proteins.1,2 To gain a better insight into their functional significance, a detailed knowledge is required of the GAG structures involved. As part of a strategy for identifying the specific sequences that might be responsible for activity, an increasing number of characterized GAG oligosaccharides derived from partial depolymerization of

© 1998 American Chemical Society Published on Web 04/14/1998

extensive desulfation takes place, ions representing oligosaccharide species with only one sulfate being the most abundant in the spectra of chondroitin sulfate and heparin oligosaccharides.3,4 While sodium salts are much more stable and sulfate loss is greatly reduced, clustered sodiated ions dominate the quasimolecular ion region and complicate assignment of molecular masses, particularly of the heavily sulfated heparin oligosaccharides.3 Hence, neither salt is satisfactory for large oligosaccharides in samples with mixed components such as size-homogeneous fractions obtained from gel filtration chromatography. Other MS ionization techniques have been used for heparin oligosaccharides, such as plasma desorption mass spectrometry with the surfactant tridodecylmethylammonium chloride as ionpairing agent,11 although sensitivity and resolution were not adequate at that time to derive composition. Matrix-assisted laser desorption can also provide high-sensitivity detection of heparinderived oligosaccharides12,13 when used in conjunction with specially synthesized polybasic peptides or selected proteins as noncovalent complexing agents. Such peptide/protein complexes have considerably higher masses than the oligosaccharides alone and require the mass range of time-of-flight instruments. The present report illustrates the use of electrospray mass spectrometry (ESMS) to analyze heparin fragments up to decasaccharides, either in complex mixtures or as purified individual components, and extends earlier studies of heparin disaccharides.14 The ability to discern mixtures of oligosaccharide fragments is particularly important in biological and structural studies of GAGs, as the larger oligosaccharides derived from relatively abundant sources prove very difficult to purify. Similarly, for in vivo GAG oligosaccharide ligands that may be isolated only in very small amounts from enzymatic or chemical release from polysaccharides, high-sensitivity characterization has to contend with mixtures of oligosaccharides, as multiple-step purification is often precluded. In the present study, fractions of chain-lengthhomogeneous oligomers were prepared by partial heparinase I digestion, in which the polysaccharide is cleaved at the nonreducing side of IdoA to give 4,5-unsaturated hexuronic acid (∆UA)terminating oligosaccharides, and an improved procedure of fractionation by gel filtration chromatography is described. The assignment of ion species in the spectra of mixtures was supported by comparison with those ions expected from known documented structures. EXPERIMENTAL SECTION Materials. Heparin sodium salt from porcine intestinal mucosa, heparinase I (EC 4.2.2.7), heparin disaccharide standard ∆UA(2S)-GlcNS(6S) (I-S), and bovine serum albumin (BSA) were purchased from Sigma Chemical Co. (Dorset, U.K.). Bio-Gel P-6 (200-400 mesh) and ion-exchange resin AG50W-X8 were obtained from Bio-Rad Laboratories Ltd. (Hemel Hempstead, U.K.) and Sephadex G-10 from Pharmacia Biotech (Milton Keynes, U.K.). All other reagents and solvents used were of analytical grade. Preparation of Heparin Oligosaccharide Fragments. Heparin was partially depolymerized, essentially as described,3,15 by digestion with heparinase I, and the reaction stopped at ∼30% completion. Briefly, 200 mg of heparin was incubated with (15) Linhardt, R. J.; Wang, H. M.; Loganathan, D.; Lamb, D. J.; Mallis, L. M. Carbohydr. Res. 1992, 225, 137-145.

heparinase I (100 units) in 5 mM sodium phosphate buffer (pH 7.1, 6 mL) containing 0.2 M NaCl and BSA (1 mg) at 30 °C for 30 h. The digestion mixture, after desalting on a Sephadex G-10 column (1.6 cm × 36 cm, with elution by H2O at a flow rate of 20 mL/h) and lyophilization, was fractionated on a gel filtration column of Bio-Gel P-6 (1.6 cm × 90 cm) with elution by 0.2 M NH4Cl (pH 3.5) at a flow rate of 15 mL/h. Eluate was monitored on-line by refractive index and UV at 232 nm. The pooled fractions were freeze-dried and desalted on a Sephadex G-10 column (1.6 cm × 36 cm) before analysis and further chromatography. Heparin oligosaccharide fragments containing nonreducing terminal ∆UA after heparinase I digestion were quantified by their UV absorbance at 232 nm with disaccharide I-S used as standard for calibration. Microscale conversion of the sodium salts of heparin oligosaccharides into ammonium salts was carried out on a minicolumn of cation exchange (AG50W-X8, H form). Typically, 50-100 µL of gel was used for samples containing a few nanomoles. Oligosaccharides were eluted with 3 times bed volume of water and 1-2 µL of ammonium acetate solution (0.2 M) added to the eluate before lyophilization. HPLC Separation. Bio-Gel P-4 fractions were chromatographed by strong anion exchange (SAX) HPLC on an analytical column, S5-SAX (4.6 mm × 250 mm, Phase Separations Ltd., Clwyd, Wales), using a titanium-lined Gilson liquid chromatograph system fitted with a variable-wavelength UV detector monitored at 232 nm. Elution was carried out with a linear gradient of NaCl (solvent A, 0.2 M NaCl, and solvent B, 1.5 M NaCl; pH 3.5). Heparin decasaccharides were fractionated by a gradient of 70% B to 90% B in 40 min at a flow rate of 1 mL/min. The major HPLC subfractions were collected, desalted on a Sephadex G10 column, and freeze-dried. Electrospray Mass Spectrometry. ESMS was carried out on a VG Quattro quadrupole instrument (Micromass Ltd., Altrincham, U.K.) fitted with an atmospheric pressure ionization electrospray source. A Varian 9012 HPLC pump (Walton-onThames, Surrey, U.K.) was used for solvent delivery. Sample solutions (5-200 pmol/µL) were injected through a 10-µL Rheodyne loop into the mobile phase (ACN/0.5 mM NH4HCO3, 1:1 v/v) at a flow rate of 10 or 20 µL/min into the electrospray source. For the HPLC-purified decasaccharides, ACN/H2O (1:1 v/v) was used as sample solvent and mobile phase. The source temperature was maintained at 90 °C, and flow rates of drying and nebulizing gas (nitrogen) were optimized at 350 and 10 L/h, respectively. The cone voltage was maintained at 60 V, and the capillary and HV electrode potentials were at 2.66 and 0.22 kV, respectively. Full-scan data were acquired over a mass range of 150-900 Da. The raw data were processed and transformed into values of molecular masses on a mass scale using a VG MassLynx data system (Micromass Ltd.). RESULTS AND DISCUSSION Influence of Salt on ESMS of Heparin Tetrasaccharides. Heavily sulfated sugar heparin oligosaccharides are normally prepared as sodium salts since the free acid is very labile and self-catalyzed decomposition readily occurs. Previously, it has been shown that, under LSIMS conditions, sodium salts are much more stable than ammonium salts, the latter fragmenting readily Analytical Chemistry, Vol. 70, No. 10, May 15, 1998

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Table 1. Heparin Oligosaccharide Components Identified from ESMS and the Deduced Compositions fractions

found

composition

calcd

tetrasaccharides

1155.1 1075.1 995.0 1732.5 1652.5 1614.3 1572.0 1534.4 1492.4 2309.9 2230.1 2191.6 2150.4 2111.8 2031.9 2888.5 2807.3 2768.8 2727.1 2689.6 2646.6 2609.1

6SO3 5SO3 4SO3 9SO3 8SO3 7SO3, 1Ac 7SO3 6SO3, 1Ac 6SO3 12SO3 11SO3 10SO3, 1Ac 10SO3 9SO3, 1Ac 8SO3, 1Ac 15SO3 14SO3 13SO3, 1Ac 13SO3 12SO3, 1Ac 12SO3 11SO3, 1Ac

1155.0 1074.9 994.8 1732.4 1652.4 1614.3 1572.3 1534.3 1492.2 2309.9 2229.8 2291.8 2149.8 2111.7 2031.6 2887.4 2807.3 2769.3 2727.3 2689.2 2647.2 2609.1

hexasaccharides

octasaccharides

decasaccharides

Figure 1. ES mass spectra of heparin tetrasaccharide fraction as sodium (a) and ammonium salts (b). (Lower panels, raw data; upper panels, transformed spectrum.)

by consecutive losses of sulfate during ionization.4 Experimental conditions for ESMS were established with heparin tetrasaccharides and then employed for larger oligosaccharides. In the negative-ion ES spectrum of the sodium salts of tetrasaccharides, clustered sodiated quasimolecular ions dominated the spectrum, and sulfate loss was negligible (Figure 1a). The major components identified were isomeric tetrasaccharides with five (1075.0 Da) and six (1154.9 Da) sulfates. The pentasulfated tetrasaccharide species3 were collectively more abundant than the hexasulfated species. In contrast to LSIMS, where [M - nH + (n - 1)Na]- are the dominant ions, ES produced ions carrying less sodium, such that the most abundant peak within each cluster was the monosodiated species (e.g., 1097.0 and 1176.9 Da, Figure 1a). This was presumed to result from inclusion of the low concentration of NH4HCO3 in the mobile carrier solution and partial exchange of sodium for ammonium cation during the spray ionization process. As shown below, ammonium adducts do not normally occur in abundance in the spectrum. When the oligosaccharides were analyzed as ammonium salts, obtained by microscale conversion from the sodium salts, the spectrum of the tetrasaccharide fraction was much simpler (Figure 1b). Ions equivalent to [M - nH]n- (where M represents the free acid) were the main peaks, although small amounts of sodium salts were still present. The extensive desulfation of ammonium salts observed in LSI mass spectra did not occur. The major components (1075.1 and 1155.1 Da) were readily identified, and, in addition, a tetrasulfated species (995.0 Da) was also apparent. 2062 Analytical Chemistry, Vol. 70, No. 10, May 15, 1998

Higher detection sensitivity of ammonium salts than that of sodium salts was achieved by the reduced distribution of ion current among the cation adducts. Improvements in detection sensitivity and mass accuracy have been observed previously in ESMS of ammonium salts of oligonucleotides by the reduction of cation adduction.16 Fractionation of Heparin Oligosaccharide Fragments as Ammonium Salts for ESMS. A fractionation procedure was optimized to resolve di- to octadecasaccharides (Figure 2) of comparable amounts and generate them in ammonium form for ESMS without the need of cation exchange. This convenient method was used to isolate heparin oligosaccharides at the >100mg level and extends the yield and range of large-sized heparin fragments beyond what was previously possible.17 It involved controlled digestion of heparin with heparinase I (30% completion), and direct gel filtration of the digest mixture on Bio-Gel P-6 (Figure 2), and elution with NH4Cl. Desalting was carried out on a short column of Sephadex G-10. Detection by UV and RI, which also give an evaluation of quantity and purity, indicated good resolution of the di- to decasaccharide fractions (F1-F5, Figure 2). Higher fractions containing the dodeca- to octadecasaccharides (F6-F9, Figure 2) were less well resolved but clearly apparent. ESMS Identification of Heparin Hexa-, Octa-, and Decasaccharide Fractions. In the ES spectrum of the pooled gel filtration hexasaccharide fraction (F3, Figure 2), although the pattern of the multiply charged ions from the several components was complex (Figure 3, lower panel), six major mass peaks (1732.5, 1652.5, 1614.3, 1572.0, 1534.4, and 1492.4 Da) were readily identified in the transformed spectrum, indicating hexasaccharides of six different compositions (Figure 3, upper panel, and Table 1). Ammonium adducts of the major components were also (16) Limbach, P. A.; Crain, P. F.; McCloskey, J. A. J. Am. Soc. Mass Spectrom. 1995, 6, 27-39. (17) Pervin, A.; Gallo, C.; Jandik, K. A.; Han, X.-J.; Linhardt, R. J. Glycobiology 1995, 5, 83-95.

Figure 2. Bio-Gel P-6 profile of heparin oligosaccharide fragments prepared by controlled digestion with heparinase I.

Figure 3. ES mass spectrum of heparin hexasaccharide fraction as ammonium salts. (Lower panel, raw data; upper panel, transformed spectrum.)

observed (e.g., 1670.1 Da). Complexity of the primary spectrum can lead to artifactual peaks from transformation; however, these peaks can often be recognized and excluded by their peak shapes being too wide, too narrow, or nonsymmetrical. At least nine hexasaccharides have been reported to date from lyase digestion of heparin. Their compositions are all represented by the identified ES mass peaks (Figure 3 and Table 1): a fully sulfated hexasaccharide with nine sulfate groups, 1732.5 Da;15 two octasulfated hexasaccharides, 1652.5 Da;18,19 one heptasulfated, 1572.0 Da;20 its three monoacetylated hexasaccharide analogues, 1614.3 Da;19-21 and two hexasaccharides each containing six sulfates and one acetyl, 1534.4 Da.20,22 However, the hexasaccha(18) Horne, A.; Gettins, P. Carbohydr. Res. 1992, 225, 43-57. (19) Larnkjaer, A.; Hansen, S. H.; Østergaard, P. B. Carbohydr. Res. 1995, 266, 37-52. (20) Tsuda, H.; Yamada, S.; Yamane, Y.; Yoshida, K.; Hopwood, J. J.; Sugahara, K. J. Biol. Chem. 1996, 271, 10495-10502.

ride with six sulfates (1492.4 Da, Figure 3) has not been reported. The identification of six major mass peaks that cover the range of known structures would support the view that the ES spectrum gives a close representation of the components in the fraction. Limited numbers of heparin octasaccharides and larger structures have been isolated and well characterized. Among octasaccharides, the fully sulfated sequence (12 sulfates) was the first described by Linhardt and colleagues,15 and a monoundersulfated octasaccharide (11 sulfates) was identified by LSIMS and NMR.3 Ions corresponding to the compositions of these two octasaccharides were present in the ES spectrum of octasaccharide fraction (F4, Figure 2) at 2309.9 and 2230.1 Da, respectively (Figure 4 (21) Linhardt, R. J.; Rice, K. G.; Merchant, Z. M.; Kim, Y. S.; Lohse, D. L. J. Biol. Chem. 1986, 261, 14448-14454. (22) Linhardt, R. J.; Wang, H. M.; Loganathan, D.; Bae, J. H. J. Biol. Chem. 1992, 267, 2380-2387.

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Figure 4. ES mass spectrum of heparin octasaccharide fraction as ammonium salts. (Lower panel, raw data; upper panel, transformed spectrum.)

Figure 5. ES mass spectrum of heparin decasaccharide fraction as ammonium salts. (Lower panel, raw data; upper panel, transformed spectrum.)

and Table 1). Further structures were also indicated by the masses 2150.4 (10 sulfates), 2031.9, 2111.8, and 2191.6 Da, representing monoacetylated octasaccharides (8, 9, and 10 sulfates, respectively, Figure 4 and Table 1). These previously unidentified structures await isolation and detailed characterization. Only two decasaccharide sequences have been reported and structurally elucidated: a fully sulfated (15 sulfates, 2888.5 Da) and a monoundersulfated decasaccharide (14 sulfates, 2807.3 Da).5,17 From the spectrum of the decasaccharide fraction (F5, Figure 2), the presence of at least seven major components was detected (Figure 5 and Table 1). Among previously unidentified components were the decasaccharides containing 12 (2646.6 Da) and 13 sulfates (2727.1 Da) and the monoacetylated analogues, with 11 (2609.1 Da), 12 (2689.6 Da), and 13 sulfates (2768.8 Da). 2064 Analytical Chemistry, Vol. 70, No. 10, May 15, 1998

The spectra of hexa-, octa-, and decasaccharide fractions indicated that sulfation did not exceed the expected fully sulfated substitution of three sulfates per disaccharide unit. Additional 3-O-sulfation of GlcN in a pentasaccharide sequence is important in antithrombin III-binding to heparan sulfate and heparin chains.23 This 3-O-sulfated GlcN is normally located at the reducing side of a nonsulfated GlcA residue and, hence, could be present in these oligosaccharide fractions that do not have components exceeding the overall three sulfates per disaccharide unit. Among the many undersulfated oligosaccharides, only the monoundersulfated species detected by ESMS did not contain an acetyl group. The absence of N-acetyl suggests the initial biosynthetic Ndeacetylation/N-sulfation is complete24 and that sulfate is missing (23) Lindahl, U.; Thunberg, L.; Ba¨ckstro¨m, G.; Riesenfeld, J.; Nording, K.; Bjo¨rk, I. J. Biol. Chem. 1984, 259, 12368-12376.

Figure 6. SAX-HPLC separation of heparin decasaccharide fraction F5 from Bio-Gel P-6 chromatography.

from a HexA residue. This is supported by the isolation and identification of a comprehensive range of monoundersulfated hexa- to tetradecasaccharides that all contain a GlcA residue next to the reducing terminal GlcNS(6S).17 Further undersulfated oligosaccharides all contain acetylated species due to incomplete N-deacetylation of GlcNAc. Fractionation of Decasaccharides by HPLC and Identification by ESMS. The decasaccharide fraction (F5, Figure 2) was further fractionated by SAX HPLC, and three major peaks, F5-5, F5-6, and F5-7 (Figure 6), were collected and their ESMS spectra obtained to confirm the assignment of major ion species detected in the mixture. The mass spectra of HPLC subfractions showed much simpler patterns of multiply charged ions (Figure 7a-c, lower panels) than in the spectrum of mixed components. The composition of the decasaccharide in fraction F5-5 was deduced as a monoundersulfated decasaccharide (2807.7 Da, Figure 7a) and that of F5-6 as the fully sulfated analogue (2887.8 Da, Figure 7b). The major mass peak in the spectrum of fraction F5-7 at 2769.6 Da indicated it to be a monoacetylated decasaccharide containing 13 sulfates that has not been described previously. Despite not being the most heavily sulfated species this decasaccharide was the last eluting component from the SAX column. It has been observed previously that the elution sequence in strong anion-exchange chromatography does not always follow sulfate content in size-homogeneous structures (W. Chai, E. F. Hounsell, D. Bailey, and A. M. Lawson, unpublished results) and may be ascribed to the acetyl group playing a role in the chromatography. This underlines the need for mass spectrometric analysis of heparin oligosaccharides to derive compositional information. The transformed spectra clearly demonstrated that the major ions detected were from the intact molecules and degradation products were minimal. In the spectrum of F5-6 (Figure 7b, upper panel), the fully sulfated species appeared as the main mass peak at 2887.8 Da, and the minor component at 2807.9 Da was considered to arise mainly from a component in the adjacent fraction due to incomplete separation (see HPLC profile in Figure 6). The purified fraction F5-5 was used to assess the sensitivity that can be achieved for decasaccharides. By injection of 5 µL of sample solution at a concentration of 5 pmol/µL, the molecular (24) Lindahl, U.; Kjelle´n, L. Thromb. Haemost. 1991, 66, 44-48.

Figure 7. ES mass spectra of major HPLC subfractions of heparin decasaccharides as ammonium salts (a, F5-5; b, F5-6; c, F5-7). (Lower panels, raw data; upper panels, transformed spectrum.)

mass peak was detected with a signal-to-chemical noise ratio greater than 4:1. However, when the sample solution is dilute, sodium and potassium adducts become prominent and complicate the spectrum. This salt interference was assumed to be due to the highly anionic nature of heparin oligosaccharides, readily acquiring strong cations from low-level salt contamination in the instrument inlet system. More careful preparation of sample solution and the instrument will further improve the detection sensitivity. In summary, ESMS of heparin oligosaccharide fragments as ammonium salts provides a valuable method for their analysis, even when in complex mixtures of size-homogeneous fractions. The close representation in the spectrum of each component in a mixture allows rapid screening of oligosaccharide fragments present in fractions typically prepared by gel filtration chromaAnalytical Chemistry, Vol. 70, No. 10, May 15, 1998

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tography. Oligosaccharide chain length and composition can be deduced, in terms of HexA, GlcN, SO3, and Ac, from the accuracy of mass measurement. Characterization of heparin oligosaccharides by this ESMS method in combination with binding and inhibition assays provides an opportunity to derive structural requirements, such as size, charge density, and sequence, in relation to biological activity.

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ACKNOWLEDGMENT This work was supported, in part, by Program Grant E400/ 622 from the UK Medical Research Council. Received for review November 20, 1997. Accepted March 5, 1998. AC9712761