Liquid Chromatography/Mass Spectrometry Sequencing Approach for Highly Sulfated Heparin-derived Oligosaccharides
2004; Elsevier BV; Volume: 279; Issue: 4 Linguagem: Inglês
10.1074/jbc.m304772200
ISSN1083-351X
AutoresCharuwan Thanawiroon, Kevin G. Rice, Toshihiko Toida, Robert J. Linhardt,
Tópico(s)Carbohydrate Chemistry and Synthesis
ResumoLiquid chromatography/mass spectrometry (LC/MS) is applied to the analysis of complex mixtures of oligosaccharides obtained through the controlled, heparinase-catalyzed depolymerization of heparin. Reversed-phase ion-pairing chromatography, utilizing a volatile mobile phase, results in the high resolution separation of highly sulfated, heparin-derived oligosaccharides. Simultaneous detection by UV absorbance and electrospray ionization-mass spectrometry (ESI-MS) provides important structural information on the oligosaccharide components of this mixture. Highly sensitive and easily interpretable spectra were obtained through post-column addition of tributylamine in acetonitrile. High resolution mass spectrometry afforded elemental composition of many known and previously unknown heparin-derived oligosaccharides. UV in combination with MS detection led to the identification of oligosaccharides arising from the original non-reducing end (NRE) of the heparin chain. The structural identification of these oligosaccharides provided sequence from a reading frame that begins at the non-reducing terminus of the heparin chain. Interestingly, 16 NRE oligosaccharides are observed, having both an even and an odd number of saccharide residues, most of which are not predicted based on biosynthesis or known pathways of heparin catabolism. Quantification of these NRE oligosaccharides afforded a number-averaged molecular weight consistent with that expected for the pharmaceutical heparin used in this analysis. Molecular ions could be assigned for oligosaccharides as large as a tetradecasaccharide, having a mass of 4625 Da and a net charge of –32. Furthermore, MS detection was demonstrated for oligosaccharides with up to 30 saccharide units having a mass of >10,000 Da and a net charge of –60. Liquid chromatography/mass spectrometry (LC/MS) is applied to the analysis of complex mixtures of oligosaccharides obtained through the controlled, heparinase-catalyzed depolymerization of heparin. Reversed-phase ion-pairing chromatography, utilizing a volatile mobile phase, results in the high resolution separation of highly sulfated, heparin-derived oligosaccharides. Simultaneous detection by UV absorbance and electrospray ionization-mass spectrometry (ESI-MS) provides important structural information on the oligosaccharide components of this mixture. Highly sensitive and easily interpretable spectra were obtained through post-column addition of tributylamine in acetonitrile. High resolution mass spectrometry afforded elemental composition of many known and previously unknown heparin-derived oligosaccharides. UV in combination with MS detection led to the identification of oligosaccharides arising from the original non-reducing end (NRE) of the heparin chain. The structural identification of these oligosaccharides provided sequence from a reading frame that begins at the non-reducing terminus of the heparin chain. Interestingly, 16 NRE oligosaccharides are observed, having both an even and an odd number of saccharide residues, most of which are not predicted based on biosynthesis or known pathways of heparin catabolism. Quantification of these NRE oligosaccharides afforded a number-averaged molecular weight consistent with that expected for the pharmaceutical heparin used in this analysis. Molecular ions could be assigned for oligosaccharides as large as a tetradecasaccharide, having a mass of 4625 Da and a net charge of –32. Furthermore, MS detection was demonstrated for oligosaccharides with up to 30 saccharide units having a mass of >10,000 Da and a net charge of –60. The structural elucidation of complex carbohydrates remains one of the most difficult challenges for chemists, often requiring the application of multiple analytical approaches (1.Davies J.G. Henrissat B. Biochem. Soc. Trans. 2002; 30: 291-297Crossref PubMed Google Scholar, 2.Cataldi T.R. Campa C. de Benedetto G.E. Fresenius' J. Anal. Chem. 2000; 368: 739-758Crossref PubMed Scopus (208) Google Scholar, 3.Koketsu M. Linhardt R.J. Anal. Biochem. 2000; 283: 136-145Crossref PubMed Scopus (45) Google Scholar, 4.Packer N.H. Harrison M.J. Electrophoresis. 1998; 19: 1872-1882Crossref PubMed Scopus (45) Google Scholar, 5.Mulloy B. Mol. Biotechnol. 1996; 6: 241-265Crossref PubMed Scopus (16) Google Scholar). Glycosaminoglycans (GAGs), 1The abbreviations used are: GAG, glycosaminoglycan; LC, liquid chromatography; MS, mass spectrometry; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; ESI-MS, electrospray ionization-MS; HPLC, high performance liquid chromatography; RPIP, Reversed-phase ion-pairing; TrBA, tributylammonium acetate; dp, degree of polymerization; GlcA, glucuronic acid; IdoA, iduronic acid; GlcN, glucosamine; HexN, hexosamine; P, pyranose; S, sulfo; Ac, acetyl; NRE, non-reducing end.1The abbreviations used are: GAG, glycosaminoglycan; LC, liquid chromatography; MS, mass spectrometry; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; ESI-MS, electrospray ionization-MS; HPLC, high performance liquid chromatography; RPIP, Reversed-phase ion-pairing; TrBA, tributylammonium acetate; dp, degree of polymerization; GlcA, glucuronic acid; IdoA, iduronic acid; GlcN, glucosamine; HexN, hexosamine; P, pyranose; S, sulfo; Ac, acetyl; NRE, non-reducing end. and heparin in particular, have proven to be extremely difficult to analyze because of high negative charge, polydispersity, and sequence heterogeneity (6.Esko J.D. 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A. 2000; 97: 10365-10370Crossref PubMed Scopus (112) Google Scholar) have been applied in the past to solve many of these complex structures. A successful combination of detection, separation, and spectral techniques might provide a critical advantage in understanding GAG structure.NMR spectroscopy is an invaluable tool for the structural elucidation of GAGs, affording saccharide composition, ring conformation, glycosidic linkage, and sulfation patterns (17.Pervin A. Gallo C. Jandik K.A. Han X.J. Linhardt R.J. Glycobiology. 1995; 5: 83-95Crossref PubMed Scopus (195) Google Scholar, 18.Yang H.O. Gunay N.S. Toida T. Kuberan B. Yu G.L. Kim Y.S. Linhardt R.J. Glycobiology. 1995; 10: 1033-1040Crossref Scopus (61) Google Scholar, 27.Mikhailov D. Linhardt R.J. Mayo K.H. Biochem. J. 1997; 328: 51-61Crossref PubMed Scopus (85) Google Scholar). Unfortunately, this technique is limited by the requirement of relatively large amounts (10 nmol to 1 μmol) of pure oligosaccharide sample. Mass spectrometry (MS) is another powerful technique for structural elucidation of GAGs. Soft ionization methods, including electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), have been amazingly successful in the analysis of neutral oligosaccharides and their peptide and lipid conjugates (28.Peter-Katalinic J. Mass Spectrom. Rev. 1994; 13: 77-98Crossref Scopus (78) Google Scholar, 29.Fura A. Leary J.A. Anal. Chem. 1993; 65: 2805-2811Crossref PubMed Scopus (115) Google Scholar, 30.Reinhold V.N. Reinhold B.B. Costello C.E. Anal. Chem. 1995; 67: 1772-1784Crossref PubMed Scopus (316) Google Scholar, 31.Harvey D.J. J. Chromatogr. A. 1996; 720: 429-446Crossref PubMed Scopus (145) Google Scholar). The analysis of polyanionic GAG-oligosaccharides, such as those derived from heparin and heparan sulfate, is still in its infancy (28.Peter-Katalinic J. Mass Spectrom. Rev. 1994; 13: 77-98Crossref Scopus (78) Google Scholar, 32.Khoo K.H. Morris H.R. McDowell R.A. Dell A. Maccarana M. Lindahl U. Carbohydr. Res. 1993; 244: 205-223Crossref PubMed Scopus (32) Google Scholar).MS studies have focused on the analysis of oligosaccharides prepared through the controlled enzymatic depolymerization of polysaccharides. MALDI time-of-flight (TOF) MS (19.Juhasz P. Biemann K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4333-4337Crossref PubMed Scopus (147) Google Scholar, 26.Shriver Z. Raman R. Venkataraman G. Drummond K. Turnbull J. Toida T. Linhardt R.J. Biemann K. Sasisekharan R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10365-10370Crossref PubMed Scopus (112) Google Scholar) and ESI-MS (20.Chai W.G. Luo J.L. Lim C.K. Lawson A.M. Anal. Chem. 1998; 70: 2060-2066Crossref PubMed Scopus (102) Google Scholar, 23.Siegel M.M. Tabei K. Kagan M.Z. Vlahov I.R. Hileman R.E. Linhardt R.J. J. Mass Spectrom. 1997; 32: 760-772Crossref PubMed Scopus (31) Google Scholar, 33.Col R.D. Silverstro L. Naggi A. Torri G. Baiocchi C. Moltrasio D. Cedro A. Viano I. J. Chromatogr. 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In contrast, ESI-MS utilizes a flowing stream containing the analyte, provides for very gentle ionization, and can be easily combined with on-line liquid-phase separation techniques, such as high performance liquid chromatography (HPLC) and capillary electrophoresis.True molecular weight distributions of polysaccharides are difficult to measure by MS because of their polydispersity (P = MW/MN > 1). MALDI-TOF analysis of synthetic polymers, which are in many respects similar to polysaccharides (e.g. polydisperse, long-chain oligomers with distinct repeating units), cannot provide accurate molecular weight distributions of polymers with P > 1.2 (35.Monstaudo G. Montaudo M.S. Puglisi C. Samperi F. Rapid Commun. Mass Spectrom. 1995; 9: 453-462Crossref Scopus (289) Google Scholar, 36.Jackson C. Larsen B. McEwen C. Anal. Chem. 1996; 68: 1303-1308Crossref Scopus (127) Google Scholar). Prior fractionation is required to simplify these polydisperse mixtures prior to MALDI-TOF (37.Deery M.J. 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A. 2000; 884: 23-30Crossref PubMed Scopus (14) Google Scholar) is difficult to interface with MS due to the high ionic strength mobile phases required to elute multiply charged oligosaccharides. Reversed-phase ion-pairing (RPIP)-HPLC, relying on tetraalkyl ammonium salts, provides excellent chromatographic resolution; however, these ion-pairing reagents are non-volatile and are required at high concentrations, making them incompatible with ESI-MS. Volatile ion-pairing reagents (42.Guan F. Isii A. Seno H. Watanabe-Suzuki K. Kumazuwa T. Suzuki O. J. Chromatogr. B. 1999; 731: 155-165Crossref PubMed Scopus (33) Google Scholar, 43.Storm T. Reemtsma T. Jekel M. J. Chromatogr. A. 1999; 854: 175-185Crossref PubMed Scopus (120) Google Scholar), post-column removal of ion-pairing reagent with an in-line membrane (44.Conboy J.J. Henion J.D. Martin M.W. Zweigenbaum J.A. Anal. 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The present study applies RPIP-HPLC/ESI-MS to the analysis of highly charged heparin-derived oligosaccharides to obtain both molecular weight and sequence information on the present heparin polymer.EXPERIMENTAL PROCEDURESMaterials—Bovine lung heparin, sodium salt (≥140 USP units/mg, 500,000 units) was from Sigma. Heparinase (heparin lyase I, EC 4.2.2.7) was from IBEX (Montreal, Canada). Acetonitrile, HPLC-grade, and all other chemicals, of the purest grade available, were obtained from Aldrich. Ultrapure water was obtained using a Milli-Q system (Millipore, Billerica, MA).Preparation of Heparin Oligosaccharide Mixtures—The heparin oligosaccharide mixture was prepared from bovine lung heparin by controlled enzymatic depolymerization with heparinase as described previously (17.Pervin A. Gallo C. Jandik K.A. Han X.J. Linhardt R.J. Glycobiology. 1995; 5: 83-95Crossref PubMed Scopus (195) Google Scholar). Briefly, 1 mg of heparin was digested with 0.2 milliunits of heparinase (EC 4.2.2.7) in 200 μl of 50 mm sodium phosphate buffer (pH 7.0) at 30 °C. The reaction mixture was incubated until the digestion was 30% complete, and then the mixture was boiled at 100 °C for 2 min to inactivate the enzyme. The mixture was freeze-dried and redissolved in 100 μl of double distilled water.Chromatographic Conditions—HPLC separations were performed on a5-μm Discovery C18 column (4.6 × 250 mm) from Supelco (Bellefonte, PA). Eluent A was water/acetonitrile (80:20), and eluent B was water/acetonitrile (35:65). Tributylamine (15 mm) and ammonium acetate (50 mm) were added to both eluents. The mobile phase pH was adjusted to 7.0 with acetic acid. The sample (20 μl, 10 mg/ml) was injected, and a linear gradient (from 0 to 100% eluent B in 120 min) at a flow rate of 0.5 ml/min was used for elution.Off-line UV-LC measurements were performed using a Shimadzu HPLC system consisting of two Shimadzu LC-10Ai pumps and a Shimadzu UV-visible spectrophotometric detector (Model SPD-10A). The elution profiles were monitored by absorbance at 232 nm at 0.02 absorbance units at full scale. On-line HPLC measurements were carried out on an Agilent 1100 series pumping system (Agilent Technologies, Inc.) consisting of a G1312A binary gradient pump, a G1322A vacuum degasser, a G1367A well plater sampler, and a GB65B multiple wave-length UV/VIS detector.Mass Spectrometric Conditions—ESI mass spectra were obtained using an Agilent 1100 series Classic G2445A LC/MSD trap (Agilent Technologies, Inc.). Optimization was performed by direct injection (6 μl/min, 1 mg/ml) of trisulfated heparin disaccharide (Fig. 1, n = 0) with mobile phase A at a flow rate of 0.5 ml/min. The electrospray interface was set in negative ionization mode with the skimmer potential –19.7 V, capillary exit –48.4 V, and a source of temperature of 350 °C to obtain maximum abundance of the standard disaccharide ions in full scan spectra (200–2200 Da, 10 full scans/s). Nitrogen was used as a drying (12 liters/min) and nebulizing gas (60 p.s.i.). Total ion chromatograms and mass spectra were processed using Agilent Chemstation A.07. Software versions were as follows: 4.0 LC/MSD trap control 5.0 and Data Analysis 2.0 (Agilent Technologies, Inc.). Monoisotopic masses are reported throughout the text. For MS simplification experiments, the post-column addition reagents were (optimally 5 mm tributylammonium acetate (TrBA) in CH3CN) directly infused into the ESI source using an Agilent syringe pump at flow rate of 6 μl/min. Both the sample and the post-column addition reagent were sprayed at the same time to ensure in-source mixing.RESULTS AND DISCUSSIONHeparin (and heparan sulfate) are structurally diverse, high molecular weight (MWavg > 10,000), polydisperse (P ≈ 1.1–1.4) mixtures (9.Linhardt R.J. Toida T. Witczak Z.B. Nieforth K.A. Carbohydrates as Drugs. Marcel Dekker, Inc., New York1997: 277-341Google Scholar, 47.Edens R.E. Al-Hakim A. Weiler J.M. Rethwisch D.G. Fareed J. Linhardt R.J. J. Pharm. Sci. 1992; 81: 823-827Abstract Full Text PDF PubMed Scopus (158) Google Scholar). Heparinase is used in reducing the molecular weight of this mixture to prepare low molecular weight heparins (8.Linhardt R.J. Gunay N.S. Sem. Thromb. Hemost. 1999; 25: 5-15Crossref PubMed Scopus (38) Google Scholar) and to facilitate the isolation and purification of specific heparin oligosaccharide components, through tedious and repetitive chromatography (10.Hileman R.E. Fromm J.R. Weiler J.M. Linhardt R.J. BioEssays. 1998; 20: 156-167Crossref PubMed Scopus (514) Google Scholar). Bovine lung heparin was selected as the starting material in this study as it has a reduced structural complexity, when compared with porcine intestinal heparin, because of its relatively high proportion of trisulfated disaccharide sequences of the structure →4) 2-O-sulfo-α-l-idopyranosuronic acid (1→4) 2-deoxy-2-sulfamido-6-O-sulfo-α-d-glucopyranose (1→ [→4) α-l-IdoAp2S (1→4) α-d-GlcNpS6S (1→] (10.Hileman R.E. Fromm J.R. Weiler J.M. Linhardt R.J. BioEssays. 1998; 20: 156-167Crossref PubMed Scopus (514) Google Scholar). Controlled, partial (30%), heparinase-catalyzed depolymerization yielded a mixture comprised primarily of oligosaccharides ranging from disaccharide (degree of polymerization (dp) 2) to oligosaccharides larger than tetradecasaccharide (dp14) as confirmed by size exclusion chromatography and gradient PAGE (17.Pervin A. Gallo C. Jandik K.A. Han X.J. Linhardt R.J. Glycobiology. 1995; 5: 83-95Crossref PubMed Scopus (195) Google Scholar, 47.Edens R.E. Al-Hakim A. Weiler J.M. Rethwisch D.G. Fareed J. Linhardt R.J. J. Pharm. Sci. 1992; 81: 823-827Abstract Full Text PDF PubMed Scopus (158) Google Scholar) (data not shown). This oligosaccharide mixture is known to contain oligosaccharides having primarily an even number of saccharide residues. Furthermore, one oligosaccharide sequence predominates (Fig. 1), and disaccharides (dp2) through tetradecasaccharides (dp14) have been purified from this mixture and characterized by multidimensional NMR spectroscopy (17.Pervin A. Gallo C. Jandik K.A. Han X.J. Linhardt R.J. Glycobiology. 1995; 5: 83-95Crossref PubMed Scopus (195) Google Scholar).Extensive experiments were undertaken to optimize the HPLC separation of both small (dp2–dp6) and large (dp10–dp14) heparin-derived oligosaccharides (48.Thanawiroon C. Linhardt R.J. J. Chromatogr. A. 2003; 1014: 215-223Crossref PubMed Scopus (64) Google Scholar). RPIP-HPLC used an MS-friendly mobile phase including: volatile ion-pairing reagents, tributylamine; volatile inorganic salt, ammonium acetate; and an organic modifier, acetonitrile. The choice of volatile ion-pairing reagent represents a compromise between low alkyl chain length affording high volatility, required for compatibility with on-line MS and longer alkyl chain length affording higher on-column retention of heparin-derived oligosaccharides associated with enhanced resolution (48.Thanawiroon C. Linhardt R.J. J. Chromatogr. A. 2003; 1014: 215-223Crossref PubMed Scopus (64) Google Scholar). TrBA provided optimal retention with MS compatibility. Organic cosolvent, solvent pH, temperature, gradient profiles of volatile inorganic salt, and various ion-pairing reagents were first optimized, in off-line RPIP-HPLC using UV detection, to obtain efficient oligosaccharide separation (48.Thanawiroon C. Linhardt R.J. J. Chromatogr. A. 2003; 1014: 215-223Crossref PubMed Scopus (64) Google Scholar). Ammonium formate and ammonium acetate gradients gave low peak intensities and complicated spectra due to ion suppression. RPIP-HPLC utilizing an acetonitrile gradient at a fixed concentration of ammonium acetate (50 mm), pH 7.0, and TrBA (15 mm) afforded both high chromatographic resolution and excellent MS analyses (Fig. 2).Fig. 2RPIP-HPLC separation of heparin oligosaccharides obtained from controlled (30%) heparinase depolymerization of bovine lung heparin. A total ion chromatogram using negative ESI-MS detection (upper trace) with peaks numbered and a UV chromatogram at 232 nm (lower trace) with dp of peaks corresponding to the oligosaccharides in Fig. 1 are shown. The inset shows the expanded view of both the total ion chromatogram and the UV chromatograms of higher oligosaccharides assigned to dp16–dp28 by peak counting. Assignments for labeled peaks, based on ESI-MS spectra, are given in Table I.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In the mass spectral analysis of highly sulfated oligosaccharides, the partial ion-pairing of sulfo groups with various cations (i.e. sodium, potassium, ammonium, or ion-pairing reagent) can pose significant problems in ESI-MS analysis, affording a large number of pseudomolecular ions having different m/z values, resulting in highly complex spectra and decreased sensitivity (20.Chai W.G. Luo J.L. Lim C.K. Lawson A.M. Anal. 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Mass Spectrom. 1997; 8: 155-160Crossref Scopus (36) Google Scholar). In DNA analysis, post-column addition of organic solvent, ion-pairing reagent, and organic acids and organic bases, as a sheath liquid directly within the ion source, decreases the surface tension and increases the volatility of the electrosprayed solution, simplifying the mass spectra (49.Cai J. Henion J.D. J. Chromatogr. A. 1995; 703: 667-692Crossref Scopus (307) Google Scholar, 50.Huber C.G. Krajete A. J. Chromatogr. A. 2000; 870: 413-424Crossref PubMed Scopus (55) Google Scholar, 51.Cheng X. Gale D.C. Udseth H.R. Smith R.D. Anal. Chem. 1995; 67: 586-593Crossref Scopus (83) Google Scholar, 52.Stephenson Jr., J.L. McLuckey S.A. Anal. Chem. 1998; 70: 3533-3544Crossref PubMed Scopus (119) Google Scholar, 53.Griffey R.H. Sasmor H. Grieg M.J. J. Am. Soc. Mass Spectrom. 1997; 8: 155-160Crossref Scopus (36) Google Scholar).In the analysis of heparin-derived oligosaccharides, we also expected to observe a reduction in charge states from added acids (formic acid, acetic acid) and bases (ammonium hydroxide, piperidine, and imidazole) by influencing the proton transfer reactions, simplifying spectral complexity, and enhancing peak intensity. The addition of CH3CN as sheath liquid resulted in improvement in ion intensity and the substantial spectral simplification, possibly by weakening of the solventsolute and ion-counter-ion interactions and the subsequent release of free analyte ions to the evaporation process. The most intense ion peaks of each oligosaccharide caused by cation adduction are always associated with the TrBA adduct. The post-column addition of a low concentration (5 mm) of TrBA ion-pairing reagent in acetonitrile provided the most simplified spectra, suppressing the Na+/NH4+ adduct
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