Multistage Mass Spectrometric Sequencing of Keratan Sulfate-Related

Dec 27, 2005 - To establish a universal protocol for sequencing keratan sulfate (KS) using mass spectrometry (MS), systematic electrospray ionization-...
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Anal. Chem. 2006, 78, 891-900

Multistage Mass Spectrometric Sequencing of Keratan Sulfate-Related Oligosaccharides Toshikazu Minamisawa,†,‡ Kiyoshi Suzuki,‡ and Jun Hirabayashi*,†

Glycostructure Analysis Team, Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Central-2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan, and Central Research Laboratories, Seikagaku Corporation, 3-1253 Tateno, Higashi-yamato, Tokyo 207-0021, Japan

To establish a universal protocol for sequencing keratan sulfate (KS) using mass spectrometry (MS), systematic electrospray ionization-MSn fragmentation experiments were carried out for 10 KS-related oligosaccharides of defined structure. Under the experimental conditions employed, fully charged molecular-related ions were observed as dominant peaks in all MS1 spectra, which clearly reflected the number of sulfates and sialic acids in the oligosaccharide structures. In the subsequent MS2, almost all of the oligosaccharides gave fragment ions corresponding to their dehydrated molecular-related ions as well as 0,2Ar scission ions (according to the nomenclature developed by Domon and Costello, where “r” represents the reducing end in this study). Further fragmentation of the 0,2Ar ions in MS3 predominantly yielded the corresponding 2,4Ar ions. Finally, in MS4, these 2,4Ar ions were subjected to extensive glycosidic cleavage. Hence, the MS4 data of KS oligosaccharides provided sufficient information for their sequence determination. In addition, some important features of MSn fragmentation became evident. These findings should lead to the establishment of consensus rules applied for KS oligosaccharides, including those previously unidentified, and also accelerate functional studies on KS, i.e., KS-related glycosaminoglycomics. Recent advances in mass spectrometry (MS) have greatly facilitated the acquisition of valuable structural information on a variety of biomolecules. Developments in soft ionization methods, such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), have revolutionized the range of ionizable molecules while minimizing their decomposition. As a result, even complex oligosaccharides derived from biological sources have recently been a focus for structural analysis. In some cases, various isomers differing in linkages, anomeric configurations,1-4 and attachment sites of a particular functional group5,6 * Corresponding author. Phone: +81-29-861-3124. Fax: +81-29-861-3125. E-mail: [email protected]. † National Institute of Advanced Industrial Science and Technology. ‡ Seikagaku Corp. (1) Mulroney, B.; Traeger, J. C.; Stone, B. A. J. Mass Spectrom. 1995, 30, 12771283. (2) Chai, W.; Piskarev, V.; Lawson, A. M. Anal. Chem. 2001, 73, 651-657. (3) Pfenninger, A.; Karas M.; Finke, B.; Stahl, B. J. Am. Soc. Mass Spectrom. 2002, 13, 1331-1340. 10.1021/ac051359e CCC: $33.50 Published on Web 12/27/2005

© 2006 American Chemical Society

could be identified successfully by examination of their spectral patterns, intensities, or both of appropriate MSn fragment ions. However, such protocols have been studied exclusively on neutral and relatively simple oligosaccharides. Glycosaminoglycans (GAGs), a group of highly acidic, complex, and heterogeneous glycans, have been studied only poorly from a systematic viewpoint. Since ESI is generally recognized as a mild method for polar compounds and is known to cleave sulfate ester bonds to a lesser extent than does MALDI, it has been used extensively for MS analysis of GAGs.7 Successful ionization and detection of GAGderived oligosaccharides by ESI-MS has been widely reported: hyaluronan,8-13 chondroitin sulfate (CS),8,14-16 dermatan sulfate (DS),17 heparin/heparan sulfate (Hep/HS),18-21 N-acetylheparosan,22 acharan sulfate,23 and keratan sulfate (KS).24,25 Notably, most (4) Xue, J.; Song, L.; Khaja, S. D.; Locke, R. D.; West, C. M.; Laine, R. A.; Matta, K. L. Rapid Commun. Mass Spectrom. 2004, 18, 1947-1955. (5) Thomsson, K. A.; Karlsson, H.; Hansson, G. C. Anal. Chem. 2000, 72, 45434549. (6) Que´me´ner, B.; Pino, J. C. C.; Ralet, M. C.; Bonnin, E.; Thibault, J. F. J. Mass Spectrom. 2003, 38, 641-648. (7) Zaia, J. Mass Spectrom. Rev. 2004, 23, 161-227. (8) Takagaki, K.; Kojima, K.; Majima, M.; Nakamura, T.; Kato, I.; Endo, M. Glycoconjugate J. 1992, 9, 174-179. (9) Price, K. N.; Tuinman, A.; Baker, D. C.; Chisena, C.; Cysyk, R. L. Carbohydr. Res. 1997, 303, 303-311. (10) Mahoney, D. J.; Aplin, R. T.; Calabro, A.; Hascall, V. C.; Day, A. J. Glycobiology 2001, 11, 1025-1033. (11) Tawada, A.; Masa, T.; Oonuki, Y.; Watanabe, A.; Matsuzaki, Y.; Asari, A. Glycobiology 2002, 12, 421-426. (12) Ku ¨ hn, A. V.; Ru ¨ ttinger, H. H.; Neubert, R. H. H.; Raith, K. Rapid Commun. Mass Spectrom. 2003, 17, 576-582. (13) Ku ¨ hn, A. V.; Raith, K.; Sauerland, V.; Neubert, R. H. H. J. Pharm. Biomed. Anal. 2003, 30, 1531-1537. (14) Zaia, J.; McClellan, J. E.; Costello, C. E. Anal. Chem. 2001, 73, 6030-6039. (15) Zaia, J.; Li, X. Q.; Chan, S. Y.; Costello, C. E. J. Am. Soc. Mass Spectrom. 2003, 14, 1270-1281. (16) Zamfir, A.; Seidler, D. G.; Schonherr, E.; Kresse, H.; Peter-Katalinic, J. Electrophoresis 2004, 25, 2010-2016. (17) Yang, H. O.; Gunay, N. S.; Toida, T.; Kuberan, B.; Yu, G.; Kim, Y. S.; Linhardt, R. J. Glycobiology 2000, 10, 1033-1040. (18) Chai, W.; Luo, J.; Lim, C. K.; Lawson, A. M. Anal. Chem. 1998, 70, 20602066. (19) Pope, R. M.; Raska, C. S.; Thorp, S. C.; Liu, J. Glycobiology 2001, 11, 505513. (20) Thanawiroon, C.; Rice, K. G.; Toida, T.; Linhardt, R. J. J. Biol. Chem. 2004, 279, 2608-2615. (21) Henriksen, J.; Ringborg, L. H.; Roepstorrf, P. J. Mass Spectrom. 2004, 39, 1305-1312. (22) Kuberan, B.; Lech, M.; Zhang, L.; Wu, Z. L.; Beeler, D. L.; Rosenberg, R. D. J. Am. Chem. Soc. 2002, 124, 8707-8718. (23) Kim, Y. S.; Ahn, M. Y.; Wu, S. J.; Kim, D. H.; Toida, T.; Teesch, L. M.; Park, Y.; Yu, G.; Lin, J.; Linhardt, R. J. Glycobiology 1998, 8, 869-877.

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of the current studies have utilized tandem MS instruments for detailed structural characterization. For disaccharide isomers differing in sulfation sites (i.e., positional isomers), both CS26-28 and Hep/HS29-31 were successfully differentiated by detecting appropriate “diagnostic” fragment ions in their MSn spectra. Extension of the techniques by combination with conventional sequential enzymatic digestion and quantification has led to the development of a CS sequencing methodology.28 Careful examination of collision-induced dissociation (CID) fragmentation patterns and relative abundances of particular fragment ions allowed the identification of much longer isomers of CS- and DS-related oligosaccharides.14,15,32 Furthermore, an innovative algorithm named “HOST” (heparin oligosaccharide sequencing tool) was most recently provided, which generates all possible sequences from MSn data of a Hep/HS oligosaccharide.33 These targets, i.e., CS and Hep/HS, are relevant considering their relatively clear biological functions. On the other hand, no practical way for sequencing has yet been established for KS, the remaining group of GAGs. KS is a unique member of GAGs predominantly found in mammalian cornea, cartilage, and brain.34 It has a linear backbone of a repeating disaccharide unit, -3Gal-β-1-4GlcNAc-β-1- (Gal, galactose; GlcNAc, N-acetylglucosamine), i.e., poly(N-acetyllactosamine). In the structure, most of the hydroxyl groups at C-6 of the N-acetylglucosamines are sulfated, and the extent of sulfation at the C-6 hydroxyls of galactose significantly varies depending on origin. Further modifications with sialic acid at the nonreducing end as well as with fucose are also evident. For instance, human cartilage KS is known to undergo such modifications during maturation.35 KS alterations in response to some human diseases have also been described: for example, the lack and reduction of sulfation in corneal KS have been closely associated with type I and type II macular corneal dystrophies, respectively.36 Other examples include the change in sulfation contents in synovial fluid KS, which is clearly correlated with the severity of osteoarthritis.37 Thus, structural changes in KS can be more extensively associated with various pathological states, i.e., type and severity, possibly in all tissues containing KS. An approach to investigate KS structures using tandem MS was first reported using a fast-atom bombardment-MS instrument.38 More recently, tandem ESI-MS was applied for structural (24) Oguma, T.; Toyoda, H.; Toida, T.; Imanari, T. Anal. Biochem. 2001, 290, 68-73. (25) Zhang, Y.; Kariya, Y.; Conrad, A. H.; Tasheva, E. S.; Conrad, G. W. Anal. Chem. 2005, 77, 902-910. (26) Desaire, H.; Leary, J. A. J. Am. Soc. Mass Spectrom. 2000, 11, 916-920. (27) Zaia, J.; Costello, C. E. Anal. Chem. 2001, 73, 233-239. (28) Desaire, H.; Sirich, T. L.; Leary, J. A. Anal. Chem. 2001, 73, 3513-3520. (29) Ruiz-Calero, V.; Moyano, E.; Puignou, L.; Galceran, M. T. J. Chromatogr., A 2001, 914, 277-291. (30) Saad, O. M.; Leary, J. A. Anal. Chem. 2003, 75, 2985-2995. (31) Saad, O. M.; Leary, J. A. J. Am. Soc. Mass Spectrom. 2004, 15, 1274-1286. (32) McClellan, J. E.; Costello, C. E.; O’Connor, P. B.; Zaia, J. Anal. Chem. 2002, 74, 3760-3771. (33) Saad, O. M.; Leary, J. A. Anal. Chem. 2005, 77, 5902-5911. (34) Funderburgh, J. L. Glycobiology 2000, 10, 951-958. (35) Brown, G. M.; Huckerby, T. N.; Bayliss, M. T.; Nieduszynski, I. A. J. Biol. Chem. 1998, 273, 26408-26414. (36) Plaas, A. H.; West, L. A.; Thonar, E. J. A.; Karcioglu, Z. A.; Smith, C. J.; Klintworth, G. K.; Hascall, V. C. J. Biol. Chem. 2001, 276, 39788-39796. (37) Yamada, H.; Miyauchi, S.; Morita, M.; Yoshida, Y.; Yoshihara, Y.; Kikuchi, T.; Washimi, O.; Washimi, Y.; Terada, N.; Seki, T.; Fujikawa, K. J. Rheumatol. 2000, 27, 1721-1724.

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characterization of KS disaccharides24 and di- to hexasaccharides.25 However, no universal protocol based on a set of fragmentation rules has been proposed for KS oligosaccharide sequencing. In the present study, systematic MSn fragmentation experiments for 10 KS-related oligosaccharides with defined structures were carried out using ESI-MS equipped with an ion trap (IT) analyzer. Most of the observed fragment ions were successfully assigned. As a result of close examination of the data, some useful rules of fragmentation became evident, which should provide us with keys to respective structural elements including sulfation patterns. Moreover, the MS4 spectra of individual compounds, which were obtained by sequential isolation and activation of the target precursor ions, have proved to include extensive and abundant glycoside-cleaved fragment ions essential for oligosaccharide sequencing. Taken together, a tight combination of these useful findings established a rapid and precise sequencing procedure of KS-related oligosaccharides. EXPERIMENTAL SECTION Materials. SL1 (NeuAc-R-2-3-Gal-β-1-4GlcNAc) was commercially obtained from Funakoshi Corp. (Tokyo, Japan). All the other oligosaccharides analyzed were prepared in Seikagaku Corp. and National Institute of Advanced Industrial Science and Technology (AIST, Ibaraki, Japan). L2 (Gal-β-1-4GlcNAc6S) and L2L2 (Gal-β-1-4GlcNAc6S-β-1-3Gal-β-1-4GlcNAc6S) were purified from keratanase II digest of bovine corneal KS. L4 (Gal6S-β-14-GlcNAc6S), L4L4 (Gal6S-β-1-4-GlcNAc6S-β-1-3-Gal6S-β-1-4GlcNAc6S), and SL2L4 (NeuAc-R-2-3-Gal-β-1-4-GlcNAc6S-β-13-Gal6S-β-1-4-GlcNAc6S) were purified from similar digest of shark cartilageous KS. L2L4 (Gal-β-1-4-GlcNAc6S-β-1-3Gal6Sβ-1-4-GlcNAc6S) was obtained from SL2L4 by acidic desialylation. G4L2 (GlcNAc6S-β-1-3-Gal-β-1-4-GlcNAc6S) and G4L4 (GlcNAc6Sβ-1-3Gal6S-β-1-4-GlcNAc6S) were prepared from L2L2 and L2L4 respectively, by β-galactosidase (Lactase, K. I. Chemical Industry Corp., Shizuoka, Japan) treatment. L3 (Gal6S-β-1-4-GlcNAc) was a chemically desulfated product of L4, using a methanolhydrochloric acid system. 18O-Labeled L4 (18O-L4) was prepared by incubation of L4 in H218O (Nippon Sanso Corp., Tokyo, Japan) for 48 h at 37 °C.39 Methanol (spectrophotometric grade, 99.9%) was purchased from Aldrich Chemical Co. (Milwaukee, WI), and acetic acid was an analytical grade from Wako Pure Chemical Industries (Osaka, Japan). Water as a solvent was purified using a Milli-Q filtration apparatus (Millipore Co., Bedford, MA). Mass Spectrometry. ESI-IT-MS experiments in the negative ion mode were carried out on a Bruker Daltonik GmbH (Bremen, Germany) Esquire 3000 plus instrument. In general, oligosaccharides were dissolved at 10 pmol/µL in 50% methanol containing 1.0 mM acetic acid and infused into the mass spectrometer directly by using a syringe pump at a flow rate of 360 µL/h. The acetic acid concentration was changed to 0.01 mM to minimize desialylation, when sialylated oligosaccharides were analyzed. The instrument was operated with a capillary voltage of -3.8 kV and an end plate offset of -500 V. Dry nitrogen gas was delivered at 4.0 L/min, and the drying temperature was set at 300 °C. A capillary exit voltage was set at -99.4 V, and a skimmer voltage (38) Kubota, M.; Yoshida, K.; Tawada, A.; Ohashi, M. Eur. J. Mass Spectrom. 2000, 6, 193-203. (39) Minamisawa, T.; Hirabayashi, J. Rapid Commun. Mass Spectrom. 2005, 19, 1788-1796.

Figure 1. Structures of the KS-related oligosaccharides analyzed in this study (molecular mass in parentheses).

was at -40.0 V. For CID MSn experiments, the particular ion of interest was isolated using an isolation width of 1 Da and excited for 40 ms. The fragmentation amplitude was adjusted at 1.0 V. For each experiment, the mass range scanned was m/z 50-1500, setting the scan resolution at 1650 m/z/s. The only difference in the conditions was the use of H218O instead of H216O in the solvent when 18O-L4 was measured. Reproducibility of MSn spectra was confirmed by measuring each fresh sample at least twice under the same conditions. RESULTS AND DISCUSSION To establish a novel and practical sequencing methodology for KS, 10 standard KS-related oligosaccharides, having Nacetylglucosamine at their reducing ends, were analyzed by negative ion ESI-MSn. They comprised di- to pentasaccharides with zero to four sulfate groups, two of which had an R-2-3-linked N-acetylneuraminic acid (sialic acid) at their nonreducing ends (Figure 1). Under the experimental conditions employed (see Experimental Section), all 10 samples afforded sufficient reproducibility in their MSn (n ) 1-4) spectra to suggest the possibility of developing consensus rules of fragmentation. Table 1 summarizes the observed m/z values, charge states, and relative abundances (in parentheses), as well as structural assignments for the major fragment ions observed (the nomenclature system is that according to Domon and Costello40). Relative abundances of the ions in each spectrum are expressed as a percentage, where the intensity of the base peak was taken as 100%. MS1 Spectra. Acquisition of an accurate and distinct MS1 spectrum is critical not only to determine the molecular weight (40) Domon, B.; Costello, C. E. Glycoconjugate J. 1988, 5, 397-409.

of each oligosaccharide but also to achieve reliable MSn fragmentation patterns. For this, any destructive event such as glycosidic cleavage, desulfation, and desialylation should be avoided during the ionization and detection processes. Paying attention to these requirements, the overall ESI-MS conditions were first optimized for disulfated disaccharide L4, as described in the Experimental Section, and then applied to the other oligosaccharides. The solvent was determined by varying the kinds and amounts of organic cosolvents and additives. The use of acetic acid as a cosolvent attained the highest sensitivity with minimal formation of the sodium salt. However, when molecular-related ions carrying sodium were detected in abundance, even under the above optimized conditions, sample solutions were treated with extensively washed, highly purified strong cation exchanger (H+-type, Muromac, Muromachi Technos Corp., Tokyo, Japan). After the final optimization, MS1 spectra were obtained for all of the compounds as shown in Figure 2. In all cases of sulfated di- to tetrasaccharides, strong peaks of molecular-related ions were obsereved, in which charge states were identical to the number of sulfate groups. In fact, no apparent signals corresponding to ions with an excess charge or desulfation were detected except for the case of L4L4 (described below). Therefore, any negative charge seems to be exclusively located on a sulfate group during ionization. Among the samples, L2 and L3 are the sole pair of positional isomers as regards sulfation that were not distinguished from each other in MS1. In the case of L4L4, a monodesulfated ion was seen in 10% relative abundance, unexpectedly. This observation is attributable to significant electric repulsion between adjacent sulfate groups, which may make their ester bonds more fragile during the ionization processes. This Analytical Chemistry, Vol. 78, No. 3, February 1, 2006

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Table 1. m/z Values, Charge States and Relative Abundances (in Parentheses), and Assignments of the Major Ions Observed compds [MW]

MS1

MS2 (on [M-nH]n-)

MS3 (on [M - H2O - nH]n-)

MS3 (on 0,2Ar)

MS4 (on 2,4Ar)

L2 461.9 (1-, 100) 343.0 (1-, 5.0) [0,2Ar-H2O]360.9 (1-, 92.9) 0,2Ar [463.1] [M-H]443.8 (1-, 100) [M-H2O-H]-

183.9 (1-, 100) [Y1-H2O-OSO3H]263.8 (1-, 57.4) [Z1-H2O]-

138.9 (1-, 50.0) [OCHCH2OSO3]- 342.8 (1-, 100) [0,2Ar-H2O]-

L3 461.9 (1-, 100) 241.0 (1-, 12.0) B1 [463.1] [M-H]258.9 (1-, 6.9) C1 300.9 (1-, 31.9) 2,4Ar 360.9 (1-, 100) 0,2Ar 443.9 (1-, 48.4) [M-H2O-H]-

198.8 (1-, 19.0) 0,2A1 240.9 (1-, 27.7) B1 258.8 (1-, 100) C1

300.8 (1-, 100) 2,4Ar

96.9 (1-, 58.6) [OSO3H]138.8 (1-, 19.1) [OCHCH2OSO3]168.8 (1-, 7.2) 0,3A1 183.9 (1-, 37.5) [Y1-H2O-OSO3H]198.8 (1-, 100) 0,2A1 240.8 (1-, 63.7) B1 383.9 (1-, 11.7) [M-H2OOCHCH2OSO3H]-

138.8 (1-, 65.6) [OCHCH2OSO3]- 96.9 (1-, 70.2) [OSO3H]210.8 (2-, 22.2) [0,2Ar-H2O]2240.8 (1-, 100) B1 240.8 (1-, 10.7) B1 2,4 300.8 (1-, 100) Ar

L4 270.4 (2-, 100) [543.1] [M-2H]2-

220.0 (2-, 39.3) 0,2Ar 261.4 (2-, 100) [M-H2O-2H]2-

G4L2 371.9 (2-, 100) 138.8 (1-, 13.5) [OCHCH2OSO3][746.1] [M-2H]2312.3 (2-, 10.0) [0,2Ar-H2O]2321.3 (2-, 83.5) 0,2Ar 362.9 (2-, 100) [M-H2O-2H]2461.8 (1-, 12.8) C2 and/or Y2 503.8 (1-, 17.1) 2,4Ar

183.9 (1-, 19.2) [Y1-H2O-OSO3H]- 138.8 (1-, 32.5) [OCHCH2OSO3]281.8 (1-, 16.5) B1 and/or [Y1-H2O]- 312.3 (2-, 100) [0,2Ar-H2O]2443.9 (1-, 29.2) [C2-H2O]503.8 (1-, 89.4) 2,4Ar 461.8 (1-, 100) C2

96.9 (1-, 44.9) [OSO3H]138.8 (1-, 6.9) [OCHCH2OSO3]240.8 (1-, 100) B1

180.9 (1-, 11.0) [0,2A1-H2O]198.8 (1-, 10.0) 0,2A1 281.8 (1-, 100) B1 299.8 (1-, 14.6) C1 427.9 (1-, 19.0) [B2-H2O]444.0 (1-, 17.5) B2

96.7 (1-, 100) [OSO3H]183.7 (1-, 36.3) [Y1-H2O-OSO3H]198.6 (1-, 38.9) 0,2A1 253.2 (2-, 37.4) [Z2-H2O]261.2 (2-, 88.0) [Y2-H2O]281.8 (1-, 14.9) B1/[Y1-H2O]299.7 (1-, 37.1) C1 332.7 (1-, 23.1) [M-H2OOCHCH2OSO3H]371.7 (1-, 34.9) 1,4A2

138.7 (1-, 34.0) [OCHCH2OSO3]- 96.7 (1-, 57.9) [OSO3H]291.2 (2-, 100) 2,4Ar 138.7 (1-, 9.6) [OCHCH2OSO3]198.6 (1-, 11.1) 0,2A1 240.6 (1-, 14.8) B2/Y2 and/or C2/Z2 261.2 (2-, 100) B2 281.7 (1-, 54.2) B1 300.7 (1-, 31.4) Y2 485.7 (1-, 11.1) [2,4Ar-OSO3H]-

SL1 673.1 (1-, 100) 290.0 (1-, 100) B1 469.9 (1-, 5.1) C2 [674.2] [M-H]572.0 (1-, 26.1) 0,2Ar 655.0 (1-, 16.2) [M-H2O-H]-

289.9 (1-, 100) B1 470.0 (1-, 44.8) C2

289.9 (1-, 100) B1

-

L2L2 453.0 (2-, 100) 393.3 (2-, 10.8) [0,2Ar-H2O]2[908.2] [M-2H]2402.4 (2-, 61.2) 0,2Ar 443.9 (2-, 100) [M-H2O-2H]2623.8 (1-, 17.0) C3 665.9 (1-, 10.0) 2,4Ar

183.9 (1-, 17.1) [Y1-H2O-OSO3H]362.9 (2-, 12.1) [Y3-H2O]605.8 (1-, 22.4) [C3-H2O]624.0 (1-, 100) C3

138.8 (1-, 25.1) [OCHCH2OSO3]393.3 (2-, 100) [0,2Ar-H2O]2461.8 (1-, 11.1) C2 605.9 (1-, 18.3) B3 623.9 (1-, 13.6) C3 665.9 (1-, 63.9) 2,4Ar

281.8 (1-, 20.4) Y3/B2 299.8 (1-, 10.0) Y3/C2 360.9 (1-, 10.0) 0,2A2 443.9 (1-, 100) B2 461.8 (1-, 19.7) C2 503.8 (1-, 78.8) Y3

L2L4 328.1 (3-, 100) 294.6 (3-, 100) 0,2Ar [988.2] [M-3H]3322.2 (3-, 9.7) [M-H2O-3H]3342.4 (2-, 5.9) B3 351.4 (2-, 23.6) C3 372.4 (2-, 10.8) 2,4Ar

183.9 (1-, 20.8) [Y1-H2O-OSO3H]264.0 (1-, 14.5) [Z1-H2O]268.2 (3-, 24.6) [Y3-H2O]281.7 (1-, 20.8) [Y1-H2O]342.5 (2-, 30.4) B3 351.4 (2-, 100) C3 461.9 (1-, 30.4) C2

138.8 (1-, 16.1) [OCHCH2OSO3]- 261.4 (2-, 26.4) Y3/B3 372.4 (2-, 100) 2,4Ar 291.4 (2-, 100) Y3 300.8 (1-, 28.7) Y2 312.3 (2-, 10.3) 0,2X3 342.3 (2-, 36.3) B3 443.8 (1-, 26.7) B2 647.9 (1-, 11.3) [2,4Ar-OSO3H]-

G4L4 274.3 (3-, 100) [826.1] [M-3H]3422.8 (2-, 14.3) [M+Na-3H]2-

240.3 (3-, 100) 0,2Ar 261.1 (2-, 10.8) B2 and/or Y2 268.0 (3-, 51.3) [M-H2O-3H]3291.2 (2-, 13.9) 2,4Ar

L4L4 265.8 (4-, 100) 240.7 (4-, 100) 0,2Ar [1068.1] [M-4H]4260.5 (3-, 18.5) C3 328.1 (3-, 10.0) 274.5 (3-, 12.8) 2,4Ar 3[M-SO3-3H] 362.2 (3-, 16.2) [M+Na-4H]3SL2L4 318.8 (4-, 100) [1279.2] [M-4H]4425.2 (3-, 11.6) [M-3H]3432.6 (3-, 19.1) [M+Na-4H]3-

294.6 (3-, 59.3) Y4/0,2Ar 328.3 (3-, 100) Y4 351.6 (2-, 40.2) Y4/C4 372.4 (2-, 11.0) Y4/2,4Ar 415.4 (2-, 15.3) unassigned 443.8 (2-, 11.0) [Y4-OSO3H]2-

can be a characteristic feature for oligosaccharides having a significantly high sulfate density. With respect to sialylated oligosaccharides, the acetic acid concentration (1.0 mM) optimized for L4 was found to be quite inappropriate in terms of stability. When measuring SL2L4 at the acetic acid concentration of 1.0 or 0.1 mM, in addition to a triply 894

138.9 (1-, 12.6) [OCHCH2OSO3]- 96.9 (1-, 16.8) [OSO3H]274.6 (3-, 100) 2,4Ar 240.8 (1-, 19.9) B1 291.3 (2-, 14.7) Y3/2,4Ar 254.5 (3-, 30.8) B3 261.3 (2-, 100) B2 291.4 (2-, 52.4) Y3 300.8 (1-, 33.1) Y2 363.3 (2-, 14.8) [2,4Ar-OSO3H]2-

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charged molecular-related ion, [M - 3H]3-, several other ions were observed, which corresponded to desialylation, desulfation, or both (spectra not shown). When the acetic acid concentration was lowered to 0.01 mM, both desialylated and desulfated species completely disappeared, and the spectra were much improved, showing a predominant [M - 4H]4- ion as well as very minor

Figure 2. Negative ion MS1 spectra of the KS-related oligosaccharides. Acetic acid concentration was adjusted at 0.01 mM for SL1 and SL2L4 and 1.0 mM for the others, respectively. Impurities are indicated by asterisks.

Figure 3. MS2 spectra of the KS-related oligosaccharides. Fully deprotonated molecular-related ion ([M - nH]n-) was chosen as a precursor ion.

[M - 3H]3- and [M + Na - 4H]3- ions. The [M - 4H]4- ion most probably resulted from deprotonation of both the carboxylic acid in sialic acid and the three sulfate groups in the remaining structure. When this diluted acid condition was applied to SL1, only an [M - H]- ion was observed, with no sign of desialylation. Thus lowering the concentration of acetic acid is effective from a practical viewpoint for the analyses of sialic acid-containing oligosaccharides. Thus, in MS1, the fully charged molecular-related ions for all the KS-related oligosaccharides used in this study were detected with a strong intensity. By using the developed procedure, neither desulfation nor glycoside cleavage was observed to a significant extent. Moreover, the exact molecular weight and the number of ionizable acidic groups, i.e., sulfate groups and sialic acid, could be determined by estimating the charge state of the fully deprotonated molecular-related ion. The analytical conditions optimized here are also applicable for longer and more highly sulfated oligosaccharides.

MS2 Spectra: [M - nH]n- as a Precursor Ion. MS2 experiments on the [M - nH]n- ions obtained in MS1 were performed. Few fragment ions corresponding to glycosidic cleavages were observed (Figure 3 and Table 1). This is in contrast to previous reports for CS oligosaccharides.14,15 Intense fragment ions explained by cross-ring cleavage 0,2Ar (subscript “r” represents the reducing end throughout this study), as well as loss of a water molecule, were commonly observed. In fact, 9 out of the 10 compounds analyzed yielded 0,2Ar ions, although there was no apparent correlation between their structures and relative abundances. Formation of this 0,2Ar ion is also common to Hep/HS constituent disaccharides.31 Since their reducing terminus saccharides bear no substituent at the C-3 hydroxyl, they should, in principle, undergo 0,2Ar fragmentation via a retro-Diels-Alder rearrangement mechanism (Figure 4).31,41,42 Only SL2L4 did not (41) Dell, A. Adv. Carbohydr. Chem. Biochem. 1987, 45, 19-72. (42) Carroll, J. A.; Willard, D.; Lebrilla, C. B. Anal. Chim. Acta 1995, 307, 431447.

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Figure 4. Putative fragmentation mechanisms for generation of an

0,2A

Figure 5. MS3 spectra of the KS-related oligosaccharides. An

ion produced in MS2 was chosen as a precursor ion.

0,2A

give 0,2Ar but Y4/0,2Ar ion; i.e., not only 0,2Ar scission but also loss of the sialic acid should have occurred. Another characteristic feature at this stage was the observation of a dehydrated ion (described in detail later), generation of which seemed to compete with the above 0,2Ar fragmentation. Eight out of 10 compounds gave [M - H2O - nH]n- ions in MS2, while none gave ions with loss of more water molecules. Such dehydrated ions were not detected for L4L4 and SL2L4. When focusing on the sialylated oligosaccharides, intense fragment ions explained by desialylation were observed. In the case of SL1, deprotonated sialic acid itself (i.e., as a B1 fragment) was detected. On the contrary, SL2L4 gave multiple fragment ions lacking sialic acid in significant abundance. Unexpectedly, B1 ion, i.e., deprotonated sialic acid, was not detected in this case, despite the fully deprotonated [M - 4H]4- ion having been subjected to the fragmentation. When [M - H]- ions of the isomeric pair L2 and L3 were subjected to MS2, distinct fragmentation patterns were observed (Figure 3) though the difference is relatively small. Therefore, clear discrimination between these isomers would be difficult if they were to occur as a mixture. Since the subsequent MS3 of these fragment ions gave completely different fragmentation patterns (Figure 5 and Table 1), MS3 measurement is more favorable for definite identification of such an isomeric structure. MS3 Spectra: [M - H2O - nH]n- as a Precursor Ion. Eight oligosaccharides gave [M - H2O - nH]n- ions in MS2, and all provided distinct MS3 spectra (spectra not shown). The observed fragment ions were structurally assigned on the assumption that the dehydration in the precursor [M - H2O 896 Analytical Chemistry, Vol. 78, No. 3, February 1, 2006

r

r

ion in MS2 and an

2,4A

r

ion in MS3.

nH]n- occurred at the reducing end GlcNAc (described later). As a result, it was found that most of the fragmentations were associated with glycosidic cleavages (Table 1). For example, among the fragment ions, the one at m/z 241 can be diagnostic for Gal6S located at the nonreducing end. The ion at m/z 282 derived from G4L2 and G4L4 is difficult to assign definitely, since the m/z value can correspond to both B1 and [Y1 - H2O]- ions, i.e., GlcNAc6S at the nonreducing end and dehydrated GlcNAc6S at the reducing end, respectively, on the basis of the reported “mass shift rule”.43 Another diagnostic ion depicted at this stage is [Y1 - H2O OSO3H]- at m/z 184, which was commonly observed for six oligosaccharides having GlcNAc6S at the reducing end. Thus. MS3 fragmentation targeting the [M - H2O - nH]n- ion should provide useful information on both reducing and nonreducing terminus structures of KS-related oligosaccharides. MS3 Spectra: 0,2Ar as a Precursor Ion. MS3 fragmentation targeting the 0,2Ar ions was performed for the nine oligosaccharides (Figure 5 and Table 1). At this stage, three interesting features were observed. First, an intense peak corresponding to 2,4Ar ion was observed for all of the oligosaccharides except for L2 and SL1. This is the most characteristic feature in MS3 targeting 0,2Ar. A putative mechanism for production of this 2,4Ar ion is shown in Figure 4. In this context, the fact that MS3 fragmentation of Y4/0,2Ar ion, derived from MS2 of SL2L4, yielded Y4/2,4Ar ion was to be expected. In the case of L2, the absence of a sulfate group in its (43) Nakata, H. Eur. J. Mass Spectrom. 1999, 5, 411-418.

Figure 6. MS4 spectra of the KS-related oligosaccharides. An glycosidic cleavages observed were also shown. 2,4A r

2,4A

structure should have made the structure undetectable, even though such a cleavage had occurred. In the case of SL1, the cleavage should have exclusively occurred at the more fragile sialic acid. Second, the [OCHCH2OSO3]- ion at m/z 139, though not so intense but significant, was observed except for L3 and SL1. Considering the fragmentation mechanism for the formation of 2,4A ion (Figure 4), simultaneous production of this fragment r containing C-5, C-6, and the ring oxygen at the reducing end GlcNAc is quite reasonable. The two exceptions, compounds L3 and SL1, cannot yield such an ion because of the absence of a sulfate at the reducing end GlcNAc. That is, appearance of the [OCHCH2OSO3]- ion at this stage is indicative for sulfation at the reducing end C-6. Third, the most intense ion commonly derived from L2, G4L2 and L2L2 is the dehydrated fragment ion [0,2Ar - H2O]n-. Notably, these three compounds have L2 disaccharide unit at their reducing ends, while the others do not. Although the unsulfated C-6 hydroxyl of Gal in this unit appears to contribute to the dehydration, the precise mechanism is not known. Anyway, the appearance of this fragment ion is clear indication that reducing terminal disaccharide is L2, i.e., Gal-β-1-4GlcNAc6S. MS4 Spectra: 2,4Ar as a Precursor Ion. MS4 experiments were performed targeting the 2,4Ar ions derived above. Unlike the previous stages, completely distinct features were observed for this stage in that extensive glycosidic cleavages occurred (Figure 6 and Table 1). They were found to give abundant information regarding oligosaccharide sequence. For example, all oligosaccharides having a sulfate at the nonreducing end (i.e., L3, L4, G4L2, G4L4, L4L4) yielded B1 ion,

r

ion produced in MS3 was chosen as a precursor ion. Schemes of the

i.e., m/z 241 for Gal6S and m/z 282 for GlcNAc6S. Similarly, singly charged B2 ion (m/z 444) appeared if a disaccharide at the nonreducing end was monosulfated (i.e., G4L2, L2L2, L2L4), while doubly charged B2 ion (m/z 261.5 in theory) appeared if it was disulfated (G4L4, L4L4). On the other hand, oligosaccharides having L4 sequence at the reducing end, except for L4 itself (i.e., G4L4, L2L4, L4L4), necessarily yielded Y2 ion at m/z 301. Thus, several ions detected have turned out to be valuable for searching for particular structures. Taken together, by combining the fragment information, any oligosaccharide sequence can be satisfactorily determined, even if it was unknown previously. By performing up to MS4 experiments, which were carried out by sequential selection and activation of specified precursor ions, abundant and valuable sequence information was obtained, for the first time, for KS-related oligosaccharides. Another characteristic feature in MS4 on 2,4Ar is that oligosaccharides containing Gal6S at either positions in their sequences always gave fragment ions attributable to desulfation, i.e., [OSO3H]at m/z 97 or [2,4Ar - OSO3H]n-. This finding is consistent with the previous report that a sulfate on Gal rather than on GlcNAc in L4L4 was more susceptible to chemical desulfation.44 Confirmation of a Dehydration Site in MS2. As mentioned, MS2 fragmentation of the molecular-related ions of eight oligosaccharides yielded a monodehydrated species, i.e., [M - H2O - nH]n-. Since all of these compounds have lost a single water molecule, the dehydration site is considered to be common and specific. Thus, the hydroxyl group (16OH) at the reducing C-1 of L4 was labeled with H218O to generate 18OH,31,39 and its MSn (44) Kariya, Y.; Watabe, S.; Mochizuki, H.; Imai, K.; Kikuchi, H.; Suzuki, K.; Kyogashima, M.; Ishii, T. Carbohydr. Res. 2003, 338, 1133-1138.

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897

Figure 7. Comparison of the MSn spectra between (A) L4 and (B)

spectra were compared with those derived for nonlabeled L4 in detail (Figure 7). It is evident that both L4 and labeled L4 (18O-L4) provided closely similar fragmentation patterns including signal intensities. This observation indicates that isotopic labeling does not significantly influence fragmentation patterns. In MS1 of both compounds, the doubly charged molecular-related ion, [M - 2H]2-, was solely observed with 1 m/z difference from each other. This ensures quantitative formation of the 18O-L4. MS2 fragmentation of the [M - 2H]2- ion yielded [M - H2O - 2H]2- as well as 0,2Ar ions. Interestingly, the former maintained the 1 mass unit difference, while the latter lost it. This result indicates that 18O was incorporated into the reducing end hydroxyl, and then the dehydration occurred in the region excluding the reducing end C-1, whereas the 0,2Ar scission having occurred at this site yielded completely identical fragment ions. Both 0,2Ar ions, having the same m/z value (m/z 220), were fragmented in MS3 to give closely similar spectra (Figure 7, MS3 (m/z 270.4/219.8) for L4 and MS3 (m/z 271.4/219.9) for 18O-L4). On the other hand, MS3 targeting on the [M - H2O - 2H]2ion yielded many (>7) fragment ions. Among them, singly charged ions, i.e., [Y1 - H2O - OSO3H]- and [M - H2O OCHCH2OSO3H]-, showed a 2 mass unit difference, which indicates that the dehydration site includes the C-3 hydroxyl of the reducing terminal GlcNAc. Further fragmentation in MS4 targeting the [Y1 - H2O - OSO3H]- ion yielded [3,5Xr - H2O]and [Y1 - 2H2O - OSO3H]- ions, again both retaining the mass difference. The presence of the former fragment, having the dehydration site as well as the C-1 hydroxyl, could finally define the dehydration site between C-2 and C-3 of the reducing end GlcNAc. 898 Analytical Chemistry, Vol. 78, No. 3, February 1, 2006

18O-L4.

Thus, the dehydration site in the [M - H2O - 2H]2- derived from L4 was unambiguously determined by an isotopic labeling method. As expected, this result was consistent with the finding for MS2 dehydration of GlcNAc6S,39 which had also occurred between C-2 and C-3. Though dehydration sites in other oligosaccharides remain to be determined, the same results are assumed considering that the loss of only a single water molecule took place in all of them. All the MS3 fragments derived from their [M - H2O - nH]n- ions were successfully assigned on this assumption. Toward the Establishment of a Universal Sequencing Protocol for KS. Structural analysis of GAGs at present requires depolymerization of target samples into small fragments, for which the following two-step MS approaches are taken. The first step is data-matching identification called “profiling” or “fingerprinting”, for which the construction of a comprehensive MSn spectral database of structurally defined standard oligosaccharides is essential. The more structures the database covers, the better it should be. However, the size and quality of the database will not necessarily be ideal. To overcome this difficulty, development of “universal rules” is necessary in the second step, for the method to be applicable to unidentified structures. For this development, inspection of MSn fragmentation data including structural assignments is required. In this context, the most significant finding of the present study, targeting KS, is the extensive glycosidic cleavage at the MS4 stage, which affords valuable sequence information. For the achievement, there are several requirements as follows: In MS1, all the acidic groups in an oligosaccharide should be ionized. When anionic oligosaccharides are analyzed, the more acidic groups, e.g., sulfate or carboxylate, are ionized initially. It

Table 2. Summary of the Fragmentation Rules and Diagnostic m/z Values MSn stage

fragmentation (ionization) rule

diagnostic m/z

MS1

Fully deprotonated molecular related ion is given without considerable loss of sulfate. The number of sulfate and/or carboxylate (n), as well as molecular weight (M) is determinable.

MS2 (on [M-nH]n-)

Dehydration ([M-H2O-nH]n-) and 0,2Ar cleavage occur in most oligosaccharides. The dehydration is ascertained to take place between C-2 and C-3 of reducing GlcNAc. Desialylation occurs preferentially in sialylated oligosaccharides.

m/z 290

MS3 (on [MH2O-nH]n-)

[Y1 - H2O - OSO3H]- ion is detected for GlcNAc6S at reducing end. B1 ion is detected for Gal6S at nonreducing end.

m/z 184 m/z 241

MS3 (on 0,2Ar)

2,4A

MS4 (on 2,4Ar)

Extensive fragment ions resulting from glycosidic cleavages are obtained, which give valuable information for sequence determination. B1 ion is detected for GlcNAc6S or Ga16S at nonreducing end.

([M-nH]n-)

r ion appears intensely (except for L2 and SL1). [OCHCH2OSO3]- ion is detected for 6S at reducing end. 0,2 [ Ar-H2O]n- ion is detected for Gal-GlcNAc6S (L2) sequence at reducing end.

B2 ion is detected for monosulfated disaccharide sequence at nonreducing end. Doubly charged B2 ion is detected for disulfated disaccharide sequence at nonreducing end. Y2 ion is detected for Gal6S-GlcNAc6S (L4) sequence at reducing end. [OSO3H]- and/or [2,4Ar-OSO3H]n- ions are detected for Gal6S at any position in the sequence.

is generally considered that, the more charges involved, the more information is afforded by MSn fragmentation. For instance, L4 provided predominantly a singly charged molecular-related ion [M - H]- when ammonium formate was used as a solvent instead of acetic acid. However, it gave in MS2 only an [M - SO3]- ion, from which useful fragment ions were no longer generated (data not shown). This observation clearly demonstrates the critical importance of generating a fully deprotonated molecular-related ion at the first step of MS. The second requisite is related to sequential isolation and activation of the relevant precursor ions. This is necessary to lead us to the final stage of MS4, which provides extensive glycosidic cleavages. To summarize the procedures of up to MS4; in MS1, a fully deprotonated [M - nH]n- ion is generated; in MS2, the [M - nH]n- ion is fragmented to generate a 0,2Ar ion; in MS3, the 0,2Ar ion is further fragmented into 2,4Ar ion; and finally in MS4, the 2,4Ar ion is subjected to glycosidic cleavages, which provide sequence information. These procedures can be explained by the above-mentioned fragmentation mechanisms (Figure 4), where it is assumed that the most active sites to trigger fragmentations in [M - nH]n- (in MS2) and 0,2Ar (in MS3) are the C-1 hemiacetal and the C-3 aldehyde group, respectively. Thus, the 2,4Ar ion detected in MS3 should have been derived by a cross-ring cleavage of the precursor ion 0,2Ar, as long as some negative charge (i.e., sulfate and/or carboxylate) remains in its 2,4Ar portion. Since the 2,4Ar ion produced can no longer promote further cross-ring cleavage in MS4, glycosidic bonds, which are secondarily reactive, are able to undergo extensive cleavages. This finally results in the acquisition of sequence information. It is reported that sequence information on CS is derived at the MS2 step but not at the MS4.15 The MS2 fragmentation of CS oligosaccharides mostly takes place at the β-1-3 uronides. In this respect, cross-ring 0,2Ar fragmentation is unlikely to occur in CS oligosaccharides, of which C-3 hydroxyl of the reducing end sugar

m/z 139

m/z 282 (GlcNAc6S) m/z 241 (Gal6S) m/z 444 m/z 261.5 m/z 301 m/z 97

is involved in a glycosidic bond.31 As a result, a series of β-1-3 uronides is subjected to glycosidic cleavages as an alternative approach in CS oligosaccharides. In contrast, KS oligosaccharides, of which the C-3 hydroxyl of the reducing end sugar (GlcNAc) remains intact, were found to undergo 0,2Ar fragmentation at the reducing end in preference to glycosidic cleavages. However, we cannot exclude the possibility that CS oligosaccharides containing uronic acid might undergo glycosidic cleavages in preference to cross-ring cleavages. We also note that our protocol targets only nonlabeled reducing oligosaccharides. In fact, this is the third requirement for applying the universal rules. As mentioned, 0,2Ar fragmentation is considered as a specific event for a reducing sugar, and it accounts for the necessity to analyze up to MS4 to cause glycosidic cleavages for KS oligosaccharides. Certainly it is supposed that, when the reducing end of a target oligosaccharide is labeled by an appropriate procedure (which abolishes hemiacetal structure), abundant glycosidically cleaved ions may arise in MS2 with an absence of cross-ring cleavages. CONCLUDING REMARKS Table 2 summarizes the fragmentation rules and diagnostic ions identified in this study. By making the best use of these findings, not only previously known KS oligosaccharides but also a much wider range of unknown structures, even if their sulfation patterns are unclear, can be determined. The present study for the first time demonstrates that MSn experiments using an advanced ESI-IT-MS technology are highly promising for structural analysis of KS-related oligosaccharides, especially for sequence determination. The methodology reported herein is expected to be widely applicable for KS oligosaccharides isolated from biological samples that include normal or diseaseaffected body fluids and tissues, as well as chemically or enzymatically derivatized KS-related compounds.44,45 The strategy should also contribute to the promotion of fundamental studies of not Analytical Chemistry, Vol. 78, No. 3, February 1, 2006

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only KS but also other GAGs by creating a novel paradigm for GAG functions, i.e., glycosaminoglycomics. ACKNOWLEDGMENT Most of the KS-related oligosaccharides analyzed in this study were prepared by the members skilled in preparing GAG-derived oligosaccharides in Seikagaku Corp., to whom the authors express our highest gratitude: Keiichi Yoshida, Akira Tawada, Hiroshi Kikuchi, Satoshi Miyauchi, Yutaka Kariya, Hideo Mochizuki, and Kyoko Watanabe-Imai. All the notations for the KS-related oligosaccharides were produced by Keiichi Yoshida, Akira Tawada, (45) Habuchi, O.; Suzuki, Y.; Fukuta, M. Glycobiology 1997, 7, 405-412.

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and Jin-ichi Inokuchi. This article is dedicated to them with respect. The authors are indebted to Akihiko Kameyama and Ko Hayama in the National Institute of Advanced Industrial Science and Technology, and Takatoshi Kubo and Atsushi Watanabe of Seikagaku Corp, for their helpful advice for ESI-MS measurements. This work was supported in part by the New Energy and Industrial Technology Development Organization (NEDO) in Japan.

Received for review July 29, 2005. Accepted October 24, 2005. AC051359E