Analysis of Keratan Sulfate Oligosaccharides by Electrospray

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Anal. Chem. 2005, 77, 902-910

Analysis of Keratan Sulfate Oligosaccharides by Electrospray Ionization Tandem Mass Spectrometry Yuntao Zhang,*,† Yutaka Kariya,‡ Abigail H. Conrad,† Elena S. Tasheva,† and Gary W. Conrad†

Division of Biology, Kansas State University, Manhattan, Kansas 66506, and Central Research Laboratories, Seikagaku Corporation, Higashiyamato-shi, Tokyo 207-0021, Japan

Keratan sulfate (KS) is a glycosaminoglycan consisting of repeating disaccharide units composed of alternating residues of D-galactose and N-acetyl-D-glucosamine linked β-(1-4) and β-(1-3), respectively. In this study, electrospray ionization tandem mass spectrometry (ESI-MS/MS) was employed to identify keratan sulfate oligosaccharides. Two nonsulfated disaccharide isomers and two monosulfated disaccharide isomers were distinguished through MS/MS. In MS1 spectra of multiply sulfated KS oligosaccharides, the charge state of the most abundant molecular ion equals the number of sulfates. Subsequent MS2 and MS3 spectra of mono-, di-, tri-, and tetrasulfated KS oligosaccharides and sialylated tetrasaccharides reveal diagnostic ions that can be used as fingerprint maps to identify unknown KS oligosaccharides. Based on the pattern of fragment ions, the compositions of an oligosaccharide mixture from shark cartilage KS and of two enzyme digests of bovine corneal KS were determined directly, without prior isolation of individual oligosaccharides by HPLC or other methods. Proteoglycans consist of core proteins to which glycosaminoglycans (GAGs), polyanionic polysaccharides composed of repeating disaccharide units, are covalently linked. They are found prominently on cell surfaces and within the extracellular matrix of all tissues. Based on the composition of their disaccharide units and the presence of sulfate or carboxyl groups, GAGs can be divided into four classes: (1) hyaluronic acid (HA), (2) heparin and heparan sulfate (HS), (3) chondroitin sulfate (CS) and dermatan sulfate (DS), and (4) keratan sulfate (KS).1 In contrast to other GAGs, KS does not contain uronic acids, and its repeating disaccharide unit is composed of alternating residues of D-galactose (Gal) and N-acetylD-glucosamine (GlcNAc) linked β-(1-4) and β-(1-3), respectively. In KS from most tissues, the hydroxyl groups at the C-6 positions of both Gal and GlcNAc residues are sulfated.2 Different degrees * Corresponding author. Tel: 785-532-6553. Fax: 785-532-6653. E-mail: [email protected]. † Kansas State University. ‡ Seikagaku Corp. (1) Yoon, J. H.; Brooks, R.; Halper, J. Anal. Biochem. 2002, 306, 298-304. (2) Conrad, G. W. In Carbohydratees in Chemistry and Biology; Ernst, B., Hart, G. W., Sinay¨ , P., Eds.; Wiley-VCH: New York, 2000; pp 717-727.

902 Analytical Chemistry, Vol. 77, No. 3, February 1, 2005

of sulfation have been observed in the cornea,3 various types of cartilage,4,5 and the brain.6 Keratan sulfate plays important roles in corneal transparency, nerve growth cone guidance, and cell adhesion.2 Thus, sensitive detection of all forms of KS would be very useful for understanding its normal role in vivo and for diagnosis of KS-related diseases. High-pH anion-exchange chromatography and nuclear magnetic resonance techniques have been applied to characterize the structure of KS oligosaccharides from human cornea.7-12 Fast-atom bombardment tandem mass spectrometry (FAB-MS/MS) has been used to analyze KS oligosaccharides from various biological tissues.13-17 With the development of ionization techniques, electrospray ionization tandem mass spectrometry (ESI-MS/MS) techniques have become more useful than those of FAB-MS/MS for the analysis of oligosaccharides.18-20 It has (3) Funderburgh, J. L.; Carterson, B.; Conrad, G. W. J. Biol. Chem. 1987, 262, 11634-11640. (4) Barry, F. P.; Rosenberg, L. C.; Gaw, J. U.; Koob, T. J.; Neame, P. J. J. Biol. Chem. 1995, 270, 20516-20524. (5) Lauder, R. M.; Huckerby, T. N.; Nieduszynski, I. A.; Plaas, A. K. Biochem. J. 1998, 330, 753-757. (6) Krusius, T.; Finne, J.; Margolis, R. K.; Margolis, R. U. J. Biol. Chem. 1986, 261, 8237-8242. (7) Lauder, R. M.; Huckerby, T. N.; Brown, G. M.; Bayliss, M. T.; Nieduszynski, I. A. Biochem. J. 2001, 358, 523-528. (8) Huckerby, T. N.; Lauder, R. M.; Brown, G. M.; Nieduszynski, I. A.; Anderson, K.; Boocock, J.; Sandall, P. L.; Weeks, S. D. Eur. J. Biochem. 2001, 268, 1181-1189. (9) Lauder, R. M.; Huckerby, T. N.; Nieduszynski, I. A. Glycobiology 2000, 10, 393-401. (10) Lewis, D.; Davies, Y.; Nieduszynski, I. A.; Lawrence, F.; Quantock, A. J.; Bonshek, R.; Fullwood, N. J. Glycobiology 2000, 10, 305-312. (11) Huckerby, T. N.; Nieduszynski, I. A.; Bayliss, M. T.; Brown, G. M. Eur. J. Biochem. 1999, 266, 1174-1183. (12) Whitham, K. M.; Hadley, J. L.; Morris, H. G.; Andrew, S. M.; Nieduszynski, I. A.; Brown, G. M. Glycobiology 1999, 9, 285-291. (13) Scudder, P.; Tang, P. W.; Hounsell, E. F.; Lawson, A. M.; Mehmet, H.; Feizi, T. Eur. J. Biochem. 1986, 157, 365-373. (14) Krusius, T.; Reinhold: V. N.; Margolis, R. K.; Margolis, R. U. Biochem. J. 1987, 245, 229-234. (15) Taguchi, T.; Iwasaki, M.; Muto, Y.; Kitajima, K.; Inoue, S.; Khoo, K. H.; Morris, H. R.; Dell, A.; Inoue, Y. Eur. J. Biochem. 1996, 238, 357-367. (16) Karlsson, N. G.; Karlsson, H.; Hansson, G. C. J. Mass Spectrom. 1996, 31, 560-572. (17) Kubota, M.; Yoshida, K.; Tawada, A.; Ohashi, M. Eur. J. Mass Spectrom. 2000, 6, 193-203. (18) Matsuo, T.; Seyama, Y. J. Mass Spectrom. 2000, 35, 114-130. (19) Careri, M.; Bianchi, F.; Corradini, C. J. Chromatogr., A 2002, 970, 3-64. (20) Dell, A.; Morris, H. R. Science 2001, 291, 2351-2356. 10.1021/ac040074j CCC: $30.25

© 2005 American Chemical Society Published on Web 01/04/2005

Figure 1. Mass spectra of keratan sulfate oligosaccharides: (A) Gal-β-1,4-GlcNAc; (B) Gal-β-1,4-GlcNAc(6S); (C) Gal(6S)-β-1,4-GlcNAc(6S); (D) Gal-β-1,4-GlcNAc(6S)-β-1,3-Gal(6S)-β-1,4-GlcNAc(6S); (E) Gal(6S)-β-1,4-GlcNAc(6S)-β-1,3-Gal(6S)-β-1,4-GlcNAc(6S); (F) Sia-R-2,3Gal-β-1,4-GlcNAc(6S)-β-1,3-Gal(6S)-β-1,4-GlcNAc(6S).

also been demonstrated that the ESI-MS/MS techniques can be used for compositional analysis of CS, DS, and HS polymers that have been digested by enzymes.21-26 More recently, KS mono- and disulfated disaccharides have been analyzed using liquid chromatography/turbo ion spray tandem mass spectrometry.27 However, systematic identification of the other kinds of KS oligosaccharides by ESI-MS/MS has not been reported so far. In this study, we used ESI-MS/MS techniques to distinguish two nonsulfated disaccharide isomers and two monosulfated disaccharide isomers and to characterize disulfated disaccharides, tri- and tetrasulfated tetrasaccharides, and sialylated tetrasaccharides. Then, the sequences of a hexasaccharide from shark cartilage KS and the compositions of enzyme digests of bovine corneal KS were determined.

EXPERIMENTAL SECTION Materials. Gal-β-1,4-GlcNAc(6S), Gal(6S)-β-1,4-GlcNAc(6S), Gal-β-1,4-GlcNAc(6S)-β-1,3-Gal(6S)-β-1,4-GlcNAc(6S), Gal(6S)-β1,4-GlcNAc(6S)-β-1,3-Gal(6S)-β-1,4-GlcNAc(6S), Sia-R-2,3-Gal-β-1,4GlcNAc(6S)-β-1,3-Gal(6S)-β-1,4-GlcNAc(6S), and an unidentified KS oliosaccharide mixture were kindly provided by Seikagaku (21) Zaia, J.; Costello, C. E. Anal. Chem. 2001, 73, 233-239. (22) Zaia, J.; McClellan, J. E.; Costello, C. E. Anal. Chem. 2001, 73, 6030-6039. (23) Schulz, B. L.; Packer, N. H.; Karlsson, N. G. Anal. Chem. 2002, 74, 60886097. (24) Desaire, H.; Leary, J. A. J. Am. Soc. Mass Spectrom. 2000, 11, 916-920. (25) Desaire, H.; Sirich, T. L.; Leary, J. A. Anal. Chem. 2001, 73, 3513-3520. (26) Saad, O. M.; Leary, J. A. Anal. Chem. 2003, 75, 2985-2995. (27) Oguma, T.; Toyoda, H.; Toida, T.; Imanari, T. Anal. Biochem. 2001, 290, 68-73.

Corp. Gal-β-1,4-GlcNAc was purchased from V-Labs, Inc. (Covington, LA). Sialic acid was purchased from Sigma (Product No. A9646) (St. Louis, MO); the specific isomer used here was 5-acetamido-3,5-dideoxy-D-glycero-D-galactononulosonic acid. Keratanase II (from Bacillus sp.), endo-β-galactosidase (from Escherichia freundii), and keratan sulfate (KS-I, from bovine cornea, purified by chromatography and chondroitinase ABC treatment, Na salt), were purchased from Seikagaku America (East Falmouth, MA). Ammonium acetate and ammonium sulfate were purchased from Fluka (St. Louis, MO). Solvents used were of HPLC grade and purchased from Fisher (Santa Clara, CA). Ultra free-MC Centrifugal Filter units (5000 NMWL, Millipore) were purchased from Fisher (Pittsburgh, PA). Sample Preparation. All KS oligosaccharide standards and sialic acid were diluted to 1 nmol/µL in water as original standard solutions and stored at -20 °C. For qualitative analysis, 10 µL of each standard solution was diluted by adding 70 µL of MeOH, 5 µL of 5 mM (pH 7.5) ammonium acetate buffer, 5 µL of 2 mM (NH4)2SO4, and 10 µL of water to make the solution 7:3 MeOH/ H2O and 100 pmol/µL of standard sample. The addition of (NH4)2SO4 was helpful in suppressing sodiated adducts.24 Enzyme Digestion of KS. For keratanase II digestion, 10 µL of bovine corneal KS (1µg/µL in water), 35 µL of 0.1 M ammonium acetate buffer (pH 6.0), and 5 µL of keratanase II (5 mU, in 0.01 M ammonium acetate buffer, pH 6.0) were mixed, and the solution mixture was incubated at 37 °C for 24 h. For endo-β-galactosidase digestion, 10 µL of bovine corneal KS (1 µg/µL in water), 37.5 µL of 0.05 M ammonium acetate buffer (pH 5.8), and 2.5 µL of endoβ-galactosidase (2.5 mU, in 0.01 M ammonium acetate buffer, pH 5.8) were mixed, and the solution mixture was incubated at 37 °C for 24 h. Enzymatic digestions were terminated by heating for Analytical Chemistry, Vol. 77, No. 3, February 1, 2005

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Figure 2. MS2 spectra of the two monosulfated isomeric disaccharides: (A) MS2 of m/z 462.0 f, Gal-β-1,4-GlcNAc(6S); (B) MS2 of m/z 461.9 f, GlcNAc(6S)-β-1,3-Gal.

Figure 3. MS2 and MS3 spectra of Gal(6S)-β-1,4-GlcNAc(6S): (A) MS2 of m/z 541.9 f; (B) MS3 of m/z 541.9 f 461.9 f; (C) MS2 of m/z 270.4 f; (D) MS3 of m/z 270.4 f 261.4 f.

10 min in boiling water.28 The digest was applied to Ultra freeMC Centrifugal Filter units (5000 NMWL, Millipore) and centrifuged at 3800 rcf for 30 min at 4 °C. The filtrate was used for MS analysis.27 (28) Plass, A. H. K.; West, L. A.; Midura R. J. Glycobiology 2001, 11, 779-790.

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Mass Spectrometry. Mass spectra were obtained using an electrospray ionization source on a quadrupole ion trap instrument (Bruker Daltonics Esquire 3000). The data acquisition software used was Bruker Daltonics Data Analysis 3.0. Mass spectra were obtained in negative ion mode. The spray voltage was 3.5 kV.

Figure 4. MS2 spectra of Gal-β-1,4-GlcNAc(6S)-β-1,3-Gal(6S)-β-1,4-GlcNAc(6S): (A) MS2 of m/z 328.4 f; (B) MS2 of m/z 492.9 f. Table 1. Observed Molecular Ions in MS1 Spectra of Purified KS Oligosaccharide Standards KS oligosaccharide sequences Gal-β-1,4-GlcNAc Gal-β-1,4-GlcNAc(6S) Gal(6S)-β-1,4-GlcNAc(6S) Gal-β-1,4-GlcNAc(6S)-β-1,3Gal(6S)-β-1,4-GlcNAc(6S) Gal(6S)-β-1,4-GlcNAc(6S)-β1,3-Gal(6S)-β-1,4-GlcNAc(6S)

Sia-R-2,3-Gal-β-1,4GlcNAc(6S)-β-1,3-Gal(6S)β-1,4-GlcNAc(6S)

molecular ions

m/z values calcd obsd

[M-H][M-H][M-H][M-2H]2[HSO4][M-2H]2-

382.3 462.4 542.5 270.7 97.0 493.4

382.6 462.0 541.9 270.4 96.9 492.9

[M-3H]3[HSO4][Y3-2HH2O-SO3]2[M-3H-SO3]3[M-4H]4[HSO4][C2-H- 2H2O]-

328.6 97.0 362.8

328.4 96.9 362.3

328.6 266.2 97.0 433.3

328.4 266.0 96.9 432.8

[M-3H]3[M-4H]4[HSO4]-

425.7 319.0 97.0

425.3 318.9 96.9

Nitrogen dry gas flowed at 5.0 L/min and the drying temperature was 180 °C. These same conditions were used for all standards, mock mixtures, and digestion samples. RESULTS AND DISCUSSION Analysis of KS Oligosaccharides. Major MS1 molecular ions of purified KS oligosaccharide standards are shown in Figure 1 and summarized in Table 1. Established Domon and Costello nomenclature is used to describe various product ions.29 Gal-β1,4-GlcNAc and Gal-β-1,4-GlcNAc(6S) yield singly charged ion [M - H]- at m/z 382.6 (Figure 1A) and 462.0 (Figure 1B), respectively. In contrast, disulfated disaccharides and the other multiply sulfated KS oligosaccharides are ionized into multiple charge states. Gal(6S)-β-1,4-GlcNAc(6S) produces both doubly charged ion [M - 2H]2- at m/z 270.4 and singly charged ion [M - H]- at (29) Domon, B.; Costello, C. E. Glycoconjugate J. 1988, 5, 397-409.

m/z 541.9 (Figure 1C). For trisulfated tetrasaccharide, Gal-β-1,4GlcNAc(6S)-β-1,3-Gal(6S)-β-1,4-GlcNAc(6S), triply charged ion [M - 3H]3- at m/z 328.4 and double charged ion [M - 2H]2- at m/z 492.9 are observed (Figure 1D). For tetrasulfated tetrasaccharide, Gal(6S)-β-1,4-GlcNAc(6S)-β-1,3-Gal(6S)-β-1,4-GlcNAc(6S), quadruply charged ion [M - H]4- at m/z 266.0 is observed in addition to fragment ions [M - 3H - SO3]3- at m/z 328.4, [Y3 - 2H H2O - SO3]2- at m/z 362.3, and [HSO4]- at m/z 96.9 (Figure 1E). The MS1 spectra of Sia-R-2,3-Gal-β-1,4-GlcNAc(6S)-β-1,3-Gal(6S)β-1,4-GlcNAc(6S) shows quadruply charged ion [M - 4H]4- at m/z 318.9, triply charged ion [M - 3H]3- ion at m/z 425.3, and fragment ion [C2 - H - 2H2O]- at m/z 432.8 (Figure 1F). In general, the most abundant molecular ion in each spectrum corresponds to the [M - H]- ion for disaccharides with zero or one sulfate, the [M - 2H]2- ion for disulfated disaccharides, the [M - 3H]3- ion for the trisulfated tetrasaccharide, and the [M 4H]4- ion for both tetrasulfated tetrasaccharide and Sia-R-2,3-Galβ-1,4-GlcNAc(6S)-β-1,3-Gal (6S)-β-1,4-GlcNAc(6S). These mass spectra are considerably different from those generated by FABCID-MS/MS for the same oligosaccharides.17 Similar phenomena, with charge states in which the charge of the most abundant molecular ion equals the number of sulfates, have been observed with CS and HS oligosaccharides.30,31 Distinctions between KS Disaccharide Isomers. Nonsulfated KS disaccharides Gal-β-1,4-GlcNAc and GlcNAc-β-1,3-Gal are isomers. MS/MS under identical conditions can be used to differentiate these isomers. The different MS2 spectra fragment ions for Gal-β-1,4-GlcNAc and GlcNAc-β-1,3-Gal are listed in Table 2. MS2 for Gal-β-1,4-GlcNAc, using the MS1 m/z 382.6 as a previous ion (Figure 1A), shows mainly glycosidic bond cleavage ion C1 at m/z 178.8, together with ring cleavage ions at the reducing terminus. MS2 for GlcNAc-β-1,3-Gal, using the MS1 m/z 382.3 as a previous ion (Figure 9B), shows ring cleavage ion [0,2X1 - H]at the nonreducing terminus, together with glycosidic bond (30) McClellan, J. E.; Costello, C. E.; O’Connor, P. B, Zaia, J. Anal. Chem. 2002, 74, 3760-3771. (31) Pope, R. M.; Raska, C. S.; Thorp, S. C.; Liu, J. Glycobiology 2001, 11, 505513.

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Figure 5. MS2 and MS3 spectra of Gal(6S)-β-1,4-GlcNAc(6S)-β-1,3-Gal(6S)-β-1,4-GlcNAc(6S): (A) MS2 of m/z 266.0; (B) MS3 of m/z 266.0 f 261.3 f; (C) MS3 of m/z 266.0 f 240.7 f. Table 2. MS2 Fragment Ions from KS Nonsulfated Isomeric Disaccharides precursor ions m/z Gal-β-1,4GlcNAc GlcNAc-β1,3-Gala

382.6

382.3

fragment ions

m/z values calcd obsd

rel intens

C1

179.1

178.8

100

[0,2A2-H-H2O][0,2A2-H]Z1

263.2 281.2 163.1

262.8 280.9 162.9

92 52 37

[0,2X1-H][M-H2O]-

262.2 365.3

262.6 364.9

100 6

a The GlcNAc-β-1,3-Gal from endo-β-galactosidase-digested bovine cornea KS.

cleavage ion Z1. It is obvious that the MS2 patterns of fragment ions of these two nonsulfated isomeric disaccharides are different from each other. Therefore, the MS2 spectra can be used to differentiate KS nonsulfated disaccharide isomers. Monosulfated KS disaccharide Gal-β-1,4-GlcNAc(6S) also has a molecular isomer, GlcNAc(6S)-β-1,3-Gal. For Gal-β-1,4-GlcNAc(6S), using m/z 462.0 as a precursor ion (Figure 1B), product ions are observed at m/z 444.0 corresponding to [M - H - H2O]-, m/z 360.9 corresponding to 0,2A2, m/z 183.8 corresponding to [Z1 - H2O - SO3]-, and m/z 138.8 corresponding to [OCHCH2OSO3]906

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(Figure 2A). For GlcNAc(6S)-β-1,3-Gal, using m/z 461.9 as a precursor ion (Figure 9B), product ions are observed at m/z 371.8 corresponding to 0,4A2, m/z 299.8 corresponding to C1, m/z 281.8 corresponding to [C1 - H2O]-, m/z 198.7 corresponding to 0,2A1, and m/z 138.7 corresponding to [OCHCH2OSO3]- (Figure 2B). Comparing the MS2 product ions of these two monosulfated disaccharide isomers, Gal-β-1,4-GlcNAc(6S) shows strong ring cleavage at the reducing terminal glycoside, which produces 0,2A2 as the most abundant ion. In contrast, GlcNAc(6S)-β-1,3-Gal exhibits mainly glycosidic cleavage, which produces ion [C1 H2O]- at m/z 281.8 as the most abundant ion. Thus, MS2 fragment ions at m/z 360.9 and 281.8 can be used as diagnostic ions for Gal-β-1,4-GlcNAc(6S) and GlcNAc(6S)-β-1,3-Gal, respectively. MS2 and MS3 of Multiple Charge-State KS Oligosaccharides. For Gal(6S)-β-1,4-GlcNAc(6S), using m/z 541.9 (Figure 1C) as a precursor ion, fragment ions are observed at m/z 461.9 corresponding to [M - H - SO3]- and m/z 299.9 corresponding to Y1, which represents glycosidic cleavage. However, no product ion involving ring cleavage is generated (Figure 3A). Using MS2 fragment ion m/z 461.9 as a precursor ion, an MS3 spectrum is generated, where product ions include [OCHCH2OSO3]- at m/z 138.8, 2,4X0/ [C1 - H2O]- at m/z 240.9, Y1 at m/z 300.0, 0,2A2 at m/z 360.9, and [M - H - H2O - SO3]- at m/z 443.9 (Figure

Figure 6. Mass spectra of sialic acid (5-acetamido-3,5-dideoxy-D-glycero-D-galactononulosonic acid): (A) MS1 of sialic acid; (B) MS2 of m/z 308.0 f; (C) MS3 of m/z 308.0 f 289.9 f.

3B). Both glycosidic cleavage (Y1 at m/z 300.0) and ring cleavage (0,2A2 at m/z 360.9) are observed. Using double charge-state ion m/z 270.4 (Figure 1C) as a precursor ion, the MS2 product ions include [HSO4]- at m/z 96.9, [OCHCH2OSO3]- at m/z 138.8, [Y1 - SO3]- at m/z 219.8, and [M - 2H - H2O]2- at m/z 261.4 (Figure 3C). MS3 spectra of Gal(6S)-β-1,4-GlcNAc(6S) obtained using the MS2 ion at m/z 261.4 as a precursor ion display a profile of fragment ions that include 0,2A1 at m/z 198.7 and [Z1 - H]- at m/z 281.9, indicating major cleavage in the ring (Figure 3D). Therefore, a combination of MS2 and MS3 spectra can provide a fingerprint to unequivocally identify disulfated disaccharides of KS. Figure 4 shows the MS2 spectra of the trisulfated KS tetrasaccharide. Using [M - 3H]3- at m/z 328.4 as a precursor ion (Figure 1D), MS2 product ions are observed at m/z 96.9 corresponding to [HSO4]-, m/z 138.8 corresponding to [OCHCH2OSO3]-, m/z 294.7 to [0,2A4 - 3H]3-, m/z 322.4 corresponding to [M - 3H H2O]3-, m/z 351.5 corresponding to [C3 - 2H]2-, and m/z 372.5 corresponding to [2,4X2 - 2H - H2O]2- (Figure 4A). Using [M 2H]2- at m/z 492.9 as a precursor ion (Figure 1D), product ions are observed at m/z 299.8 corresponding to Y1, m/z 452.9 corresponding to [M - 2H - SO3]2-, m/z 541.8 corresponding to Y2, and m/z 605.9 corresponding to [0,2X2 - H - H2O]- (Figure 4B). For precursor ion m/z 328.4 (Figure 1D), the [0,2A4 - 3H]3ion is the most abundant, and [C3 - 2H]2- indicates that a glycosidic cleavage occurs at the reducing terminal glycoside to give a disulfated trisaccharide. For precursor ion m/z 492.9 (Figure 1D), Y1 and Y2 ions reveal that glycosidic cleavages occur at the reducing terminal sugar, to give an GlcNAc(6S) and a disulfated disaccharide, respectively. These MS2 fragment ions

can be used as a fingerprint to identify molecular ions at m/z 328.4 and 492.9. For quadruply sulfated tetrasaccharide (Figure 1E), using [M - 4H]4- at m/z 266.0 as a precursor ion, the MS2 spectrum is shown in Figure 5A. Major fragment ions at m/z 240.7 and 261.3 are observed. The MS3 spectrum of the ion at m/z 261.3 (Figure 5B) displays a fragment ion composition similar to that of the MS3 spectrum of the precursor ion at m/z 261.3 (Figure 3D). This implies that the fragment ion at m/z 261.3 (Figure 5A) corresponds to [Y2 - 2H - H2O]2-, and that the major glycosidic cleavage occurs at the middle glycosidic cleavage to give a disulfated disaccharide. The MS3 spectrum for precursor ion m/z 240.7 is shown in Figure 5C. Prominent fragment ions are observed at m/z 274.5 corresponding to [0,2A4 - 3H - OCHCH2OSO3H]3- and m/z 261.3 corresponding to [C2 - 2H - H2O]2-. This indicates that the molecular ion at m/z 240.7 (Figure 5A) represents [0,2A4 - 4H],4- and that the major ring cleavage occurs at the reducing terminus. The MS2 fragment ions at m/z 240.7 and 261.3, therefore, can be used as diagnostic ions for the KS tetrasulfated tetrasaccharide. To identify the sialylate in Sia-R-2,3-Gal-β-1,4-GlcNAc(6S)-β-1,3Gal (6S)-β-1,4-GlcNAc(6S), we analyzed sialic acid (5-acetamido3,5-dideoxy-D-glycero-D-galactononulosonic acid) using ESI-MS/ MS. The molecular ion at m/z 308.0 corresponds to [M - H](Figure 6A). Using m/z 308.0 as a precursor ion, MS2 product ions are observed at m/z 87.0, 118.9, 169.8, 219.8, and 289.9 (Figure 6B). Using the ion at m/z 289.9 as a precursor ion, a very simple MS3 spectrum for sialic acid is obtained (Figure 6C). The fragment ion at m/z 169.8 corresponds to ring cleavage mainly at Analytical Chemistry, Vol. 77, No. 3, February 1, 2005

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Figure 7. MS2 and MS3 spectra of Sia-R-2,3-Gal-β-1,4-GlcNAc(6S)-β-1,3-Gal(6S)-β-1,4-GlcNAc(6S): (A) MS2 of m/z 318.9 f; (B) MS2 of m/z 425.3 f; (C) MS3 of m/z 425.3 f 289.9 f.

the 0, 4 position. Therefore, the fragment ions in MS2 and MS3 spectra can be used as fingerprint ions to identify sialic acid. Figure 7 shows the MS2 and MS3 spectra of molecular ions m/z 318.9 and 425.3 obtained in MS1 of Sia-R-2,3-Gal-β-1,4-GlcNAc(6S)-β-1,3-Gal (6S)-β-1,4-GlcNAc(6S) (Figure 1F). Using the MS1 ion at m/z 318.9 as a precursor ion, MS2 product ions are observed at m/z 294.6 corresponding to [Y4 - 3H - 0,2X0]3-, m/z 328.4 corresponding to [Y4 - 3H]3-, m/z 351.7 corresponding to [Y4 2H - Z1]2-, and m/z 372.4 corresponding to [2,4X2 - 2H]2- (Figure 7A). The presence of the most abundant ion at m/z 328.4 indicates that primary glycosidic cleavage occurs at the nonreducing terminus. Using the MS1 molecular ion at m/z 425.3 as a precursor ion, fragment ions are observed at m/z 289.9 corresponding to [C1 - H2O]-, m/z 328.4 corresponding to [Y4 - 3H]3-, m/z 452.9 corresponding to [Y4 - 2H - SO3]2-, and m/z 492.9 corresponding to [Y4 - 2H]2- (Figure 7B). The pattern of these fragment ions shows that glycosidic cleavages occur at both reducing and nonreducing termini. Neither middle glycosidic cleavage nor ring cleavage is observed. Using the fragment ion at m/z 289.9 (Figure 7B) as a precursor ion, MS3 product ions at m/z 169.8 and 97.9 are observed (Figure 7C). Comparing Figure 7C with Figure 6C, the fragment ion m/z 289.9 therefore can be used as a diagnostic ion for the sialylated KS oligosaccharides. Identifying KS Oligosaccharides in a Mixture. Figure 8A and Table 3 show the MS1 spectra generated from a mixture of 908 Analytical Chemistry, Vol. 77, No. 3, February 1, 2005

unknown oligosaccharides from shark cartilage KS. Molecular ions at m/z 266.0 and 328.3 are observed. Based on each pattern of MS2 fragment ions of m/z 266.0 and 328.3, there are tetrasulfated KS tetrasaccharide and trisulfated KS tetrasaccharide in the KS oligosaccharide mixture. The MS1 molecular ion at m/z 301.6, corresponding to [M - 5H],5- suggests that the original KS oligosaccharides mixture also contains a hexasaccharide compound of molecular weight 1513.0, which has five sulfates. There are many possible isomers for this hexasaccharide, corresponding to the many different possible arrays of five sulfates in the backbone. However, the MS2 spectrum for m/z 301.6 (Figure 8B) yields a fragment ion [Y4 - 3H]3- at m/z 328.3, which indicates that a trisulfated tetrasaccharide unit is in glycosidic linkage with the hexasaccharide. The MS2 fragment ion at m/z 261.3 corresponds to [C2 - 2H - H2O]2-/[Y2 - 2H - 2H2O]2-, m/z 281.4 corresponds to [Y1 - H2O]-, m/z 317.3 corresponds to [Y5 4H],4- m/z 322.3 corresponds to [Y4 - 3H - H2O]3-, and m/z 342.9 corresponds to [0,2A2 - H2O]-. On the basis of the MS2 pattern of fragment ions, the sequence of this hexasaccharide is likely to be that shown in Figure 8C. To demonstrate the general utility of the present ESI-MS/MS method, an enzyme digest of bovine corneal KS was analyzed directly. Spectrta A and B of Figure 9 show the MS1 spectra of keratanase II-digested KS and endo-β-galactosidase-digested KS from bovine cornea, respectively. Based on the fingerprint maps

Figure 8. Mass spectra of an oligosaccharide mixture from shark cartilage KS: (A) MS1; (B) MS2 301.6 f; (C) sequences of a KS hexasaccharide. Table 3. Observed Molecular Ions in MS1 Spectra of an Oligosaccharide Mixture from Shark Cartilage KS molecular ions

calcd m/z

obsd m/z

[M-4H]4[2,4A2-H-H2O][M-5H]5[M-3H]3[Y3-2H-H2O-SO3]2-

266.2 283.2 301.8 328.6 362.8

266.0 283.2 301.6 328.3 362.3

of KS oligosaccharide standards, the major molecular ions in keratanase II-digested KS (Figure 9A) can be identified. The molecular ion at m/z 462.0 corresponds to Gal-β-1,4-GlcNAc(6S). Both ions at m/z 270.5 and 541.9 suggest that Gal (6S)-β-1,4GlcNAc(6S) is in the keratanase II digest. Molecular ion at m/z 328.4 suggests that Gal-β-1,4-GlcNAc(6S)-β-1,3-Gal (6S)-β-1,4GlcNAc(6S) is in the keratanase II digest, and m/z 453.0 is consistent with the presence of Gal-β-1,4-GlcNAc(6S)-β-1,3-Gal-β1,4-GlcNAc(6S) in the keratanase II digest. Figure 9B represents the mass spectra of endo-β-galactosidase-digested KS. Endo-βgalactosidase (from E. freundii) cleaves at an unsulfated galactose adjacent to an unsulfated N-acetylglucosamine residue.28 There-

fore, the molecular ion at m/z 461.9 represents GlcNAc(6S)-β1,3-Gal, and m/z 382.3 corresponds to GlcNAc-β-1,3-Gal. Their MS2 spectra fragment ions are shown in Figure 2B and Table 2, respectively. CONCLUSIONS Characterization of oligosaccharides derived enzymically from KS is an important initial step toward complete understanding of the relation between its chemical structure and biological functions. In this study, ESI-MS/MS has been used directly to identify keratan sulfate oligosaccharides. Two nonsulfated disaccharide isomers and two monosulfated disaccharide isomers can be distinguished through MS/MS. In MS1 spectra of multiply sulfated KS oligosaccharides, the charge state of the most abundant molecular ion equals the number of sulfates it carries. Subsequent MS2 and MS3 spectra of mono-, di-, tri-, and tetrasulfated KS oligosaccharides and the sialylated tetrasaccharide reveal diagnostic ions that can be used as fingerprint maps to identify unknown KS oligosaccharides. Based on the pattern of fragment ions, the detailed structural compositions of an oligosaccharide mixture from shark cartilage KS and from two different enzyme Analytical Chemistry, Vol. 77, No. 3, February 1, 2005

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Figure 9. Mass spectra of enzyme digests of bovine cornea: (A) keratanase II digestion; (B) endo-β-galactosidase digestion.

digests of bovine corneal KS were determined. This work presents the first evidence that ESI-MS/MS can be used to distinguish between KS disaccharide isomers and characterize KS oligosaccharide sequences. Our method permits simple and reproducible

ACKNOWLEDGMENT This research was supported by NIH Grants EY 00952 and EY13379. The authors gratefully acknowledge Seikagaku Corp. for kindly providing KS oligosaccharides.

identification of the components of KS oligosaccharide mixtures

Received for review April 23, 2004. Accepted November 10, 2004.

without prior purification or chromatographic separation.

AC040074J

910

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