Anal. Chem. 2004, 76, 6560-6565
Hydrophilic Affinity Isolation and MALDI Multiple-Stage Tandem Mass Spectrometry of Glycopeptides for Glycoproteomics Yoshinao Wada,*,†,‡,§ Michiko Tajiri,‡ and Shumi Yoshida§
Department of Molecular Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health, 840 Murodo-cho Izumi, Osaka 594-1101, Japan, Innovation Plaza Osaka, Japan Science and Technology Agency, 3-1-10 Technostage, Izumi, Osaka 594-1144, Japan, and Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita 565-0871, Japan
In glycoproteomics, key structural issues, protein identification, locations of glycosylation sites, and evaluation of the glycosylation site microheterogeneity should be easily evaluated in a large number of glycoproteins, while mass spectrometry (MS) provides substantial information about individual purified glycoproteins. Considering that structural issues are elucidated by studying glycopeptides and that the tandem MS of a tryptic peptide composed of several amino acid residues is enough for protein identification, construction of an MS-based method handling tryptic glycopeptides would be of considerable benefit in research. To this end, a simple and efficient method, utilizing hydrophilic binding of carbohydrate matrixes such as cellulose and Sepharose to oligosaccharides, was successfully applied to the isolation of tryptic glycopeptides. Both peptide and oligosaccharide structures were elucidated by multiple-stage tandem MS (MSn) of the ions generated by matrix-assisted laser desorption/ionization (MALDI), as follows. The MALDI ion trap mass spectrum of a tryptic glycopeptide mixture from N-linked glycoproteins was composed of the [M + H]+ ions of component glycopeptides. Collision-induced dissociation (CID) of the glycopeptide [M + H]+ ion generated saccharide-spaced peaks, with an interval of, for example, 146, 162, and 203 Da, and their fragment ions corresponding to the peptide and peptide + N-acetylglucosamine (GlcNAc) species in the MS2 spectrum. The saccharide-spaced ladder served to outline oligosaccharide structures, which were then selected as precursors for subsequent MSn analyses. The peptide or peptide + GlcNAc ions in the MS2 spectrum or the corresponding ions abundant in the MS1 spectrum were subjected to CID for determination of peptide sequences, to identify proteins and their glycosylation sites. The strategy, isolation of glycopeptides followed by MSn analysis, efficiently characterized the structures of β2-glycoprotein I with four N-glycosylation * Corresponding author. Fax: +81 (725) 57 3021. E-mail:
[email protected]. osaka.jp. † Osaka Medical Center and Research Institute for Maternal and Child Health. ‡ Japan Science and Technology Agency. § Osaka University.
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sites and was applied to an analysis of total serum glycoproteins. Glycosylation is one of the major posttranslational modifications of more than half of secretory and cellular proteins and changes the physicochemical properties and biological activities of proteins.1-3 Indeed, several lines of evidence indicate that attachment of one monosaccharide to core glycans or branches changes glycoprotein function, and the resulting transformation of cellular phenotypes is suggested to be involved in cancer, infection, and reproduction.4-9 Glycosylation often determines the fate and activity of recombinant proteins in vivo. Based on this biological significance, the methodological development characteristic of glycoproteins is emerging as an essential issue in glycoproteomics, the combined field of proteomics and glycomics. Structural characterization of glycoproteins encompasses key analytical topics: protein identification in the database, locations of attachment sites, and evaluation of glycosylation site microheterogeneity. These issues are usually examined separately, by studying purified glycoproteins. Since a peptide of adequate length, e.g., 10 amino acid residues, allows a protein to be uniquely identified in the database, tryptic glycopeptides would be the optimal analytes for studying complex glycoprotein structures in a single mass spectrometric measurement. To date, several methods have been proposed for finding or isolating glycopeptides in peptide mixtures.10 For example, glycanspecific oxonium ions are formed by in-source fragmentation in electrospray ionization (ESI) mass spectrometry (MS) or by a similar technique of neutral loss scan using ESI-MS/MS, and these approaches are useful for recognizing glycopeptides especially in liquid chromatography (LC) of enzymatic digests.11-14 Porous (1) Varki, A. Glycobiology 1993, 3, 97-130. (2) Spiro, R. G. Glycobiology 2002, 12, 43R-56R. (3) Helenius, A.; Aebi, M. Annu. Rev. Biochem. 2004, 73, 1019-1049. (4) Hakomori, S. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 10231-10233. (5) Taniguchi, N.; Ihara, S.; Saito, T.; Miyoshi, E.; Ikeda, Y.; Honke, K. Glycoconjate J. 2001, 18, 859-865. (6) Rudd, P. M.; Elliott, T.; Cresswell, P.; Wilson, I. A.; Dwek, R. A. Science 2001, 291, 2370-2376. (7) Lowe, J. B. Cell 2001, 104, 809-812. (8) Carson, D. D. Front. Biosci. 2002, 7, d1535-d1544. (9) Elliott, S.; Chang, D.; Delorme, E.; Eris, T.; Lorenzini, T. J. Biol. Chem. 2004, 279, 16854-16862. (10) Harvey, D. J. Proteomics 2001, 1, 311-328. 10.1021/ac049062o CCC: $27.50
© 2004 American Chemical Society Published on Web 10/15/2004
graphitized carbon has an affinity for oligosaccharides and can be used to isolate glycopeptides with short amino acid sequences, but application to tryptic glycopeptides is limited.15 Capillary electrophoresis using a buffer system including anionic micelles and borate can separate peptides and glycopeptides but is used mainly for analytical purposes.16 Affinity chromatography with lectin is used to isolate glycopeptides but binding to specific glycan structures precludes its use for primary collection of glycopeptides with an unknown variety of glycan forms.17-19 MS is currently the most efficient and promising analytical tool for elucidating both peptide and glycan structures, and various relevant methods, with respect to ionization and fragmentation, have been reported.20,21 As described above, ESI with collisioninduced dissociation (CID) generates an abundance of ions that correspond to glycan fragmentation. On the other hand, peptide bond cleavage ions are not generally abundant especially for complex type N-linked oligosaccharides, making it difficult to analyze the oligosaccharide and peptide structure in an unknown glycopeptide in a single measurement. Fourier transform MS, which can be connected to an ESI source and holds promise in solving the problem, uses two fragmentation modes, electron capture dissociation for the peptide backbone and infrared multiphoton dissociation for the glycan.22,23 Matrix-assisted laser desorption/ionization (MALDI) causes, to some extent, metastable fragmentation mainly at the glycosidic bonds. Most recently, however, Wuhrer et al. have reported the determination of both oligosaccharide and peptide sequences using MALDI tandem time-of-flight (TOF) MS.24 Detailed characterization of oligosaccharides often requires multistage decomposition (MSn) to elucidate the branched structures.25,26 Moreover, the covalently linked peptide and oligosaccharide moieties of glycopeptides demand separate and systematic CID analysis for each group, making MSn technology highly desirable for samples that are complex mixtures of glycopeptides. In the present report, we described a simple method of isolating N-linked glycopeptides, based on the hydrophilic affinity principle, and the prepared glycopeptide mixture was analyzed by MALDI ion trap MSn for (11) Huddleston, M. J.; Bean, M. F.; Carr, S. A. Anal. Chem. 1993, 65, 877884. (12) Carr, S. A.; Huddleston, M. J.; Bean, M. F. Protein Sci. 1993, 2, 183-196. (13) Mazsaroff, I.; Yu, W.; Kelley, B. D.; Vath, J. E. Anal. Chem. 1997, 69, 25172524. (14) Medzihradszky, K. F.; Besman, M. J.; Burlingame, A. L. Anal. Chem. 1997, 69, 3986-3994. (15) Davies, M. J.; Smith, K. D.; Carruthers, R. A.; Chai, W.; Lawson, A. M.; Hounsell, E. F. J. Chromatogr. 1993, 646, 317-326. (16) Hunter, A. P.; Games, D. E. Rapid Commun. Mass Spectrom. 1995, 9, 4256. (17) Fu, D.; van Halbeek, H. Anal. Biochem. 1992, 206, 53-63. (18) Garcia, R.; Rodriguez, R.; Montesino, R.; Besada, V.; Gonzalez, J.; Cremata, J. A. Anal. Biochem. 1995, 231, 342-348. (19) Bunkenborg, J.; Pilch, B. J.; Podtelejnikov, A. V.; Wisniewski, J. R. Proteomics 2004, 4, 454-465. (20) Dell, A.; Morris, H. R. Science 2001, 291, 2351-2356. (21) Zaia, J. Mass Spectrom. Rev. 2004, 23, 161-227. (22) Håkansson, K.; Cooper, H. J.; Emmett, M. R.; Costello, C. E.; Marshall, A. G.; Nilsson, C. L. Anal. Chem. 2001, 73, 4530-4536. (23) Mormann, M.; Peter-Katalinic, J. Rapid Commun. Mass Spectrom. 2003, 17, 2208-2214. (24) Wuhrer, M.; Hokke, C. H.; Deelder A. M. Rapid Commun. Mass Spectrom. 2004, 18, 1741-1748. (25) Hirayama, K.; Yuji, R.; Yamada, N.; Kato, K.; Arata, Y.; Shimada, I. Anal. Chem. 1998, 70, 2718-2725. (26) Weiskopf, A. S.; Vouros, P.; Harvey, D. J. Anal. Chem. 1998, 70, 44414447.
elucidation of the glycan and peptide backbone structures of individual glycopeptides. Finally, the usefulness of this combination in glycoproteomics was examined in an analysis of whole serum. EXPERIMENTAL SECTION Materials. Human transferrin, iodoacetamide, lysylendopeptidase (Achromobacter protease I), and recrystalized 2,5-dihydroxybenzoic acid (DHB) were purchased from Wako Pure Chemical Ind. (Osaka, Japan). Immunoglobulin G (IgG) was purified from adult human serum by protein G affinity chromatography, and β2-glycoprotein I (β2GPI) was prepared as described previously.27 Cellulose microcrystalline and Sepharose CL-4B were purchased from Merck (Darmstadt, Germany) and Amersham Bioscience (Piscataway, NJ), respectively. Modified trypsin was obtained from Promega (Madison, WI). Ethyleneimine was acquired from Tokyo Kasei Kogyo Ltd. (Tokyo, Japan). Human serum was obtained from a healthy volunteer. Hydrophilic Affinity Separation of Glycopeptides. Transferrin, IgG, and serum proteins were reduced with dithiothreitol and carbamidomethylated, and β2GPI was aminoethylated with ethyleneimine after reduction. The alkylated sample was digested with an enzyme mixture of trypsin and lysylendopeptidase.28 In-gel digestion was carried out on 1 µg of transferrin electrophoresed on a 6% acrylamide gel according to the method of Shevchenko et al.29 Affinity separation was carried out by partitioning with cellulose or Sepharose in a microcentrifuge tube as follows. Typically, a 100-µg digest was mixed with 1 mg of cellulose or a 15-µL packed volume of Sepharose in 1 mL of an organic solvent of 1-butanol/ ethanol/H2O (4:1:1, v/v). After gentle shaking for 45 min, the gel was washed twice with the organic solvent. The gel was then incubated with an aqueous solvent of ethanol/H2O (1:1, v/v) for 30 min, and the solution phase was recovered and dried using a Speedvac concentrator. High-performance LC was carried out on a C18 column (250 × 2.0 mm, 200 Å) with a linear grandient elution of acetonitrile (5-50%, v/v) in 0.1% (v/v) trifluoroacetic acid (TFA). Mass Spectrometry. Linear TOF measurements were carried out on a Voyager DE Pro MALDI-TOF mass spectrometer with a nitrogen pulsed laser (337 nm) (Applied Biosystems, Foster City, CA). Mass spectrometric peptide fragmentation and sequencing were performed on an Axima MALDI-QIT-TOF MS instrument (Shimadzu Corp., Kyoto, Japan) with a nitrogen pulsed laser (337 nm).30 For CID, argon was used as the collision gas. For both MALDI linear and QIT measurements, 0.1-1 pmol of a glycopeptide mixture was dissolved in 1 µL of 10 mg/mL DHB dissolved in a 0.1% (v/v) TFA and 50% (v/v) acetonitrile solution on a MALDI sample target and dried. The measurements were carried out in positive ion mode. Mascot (Matrix Science Ltd., London, U.K.) was used for protein identification based on MS/MS product ions. RESULTS AND DISCUSSION Hydrophilic Affinity Separation of Tryptic Glycopeptides. A method of isolating oligosaccharides by cellulose column (27) Wurm, H. Int. J. Biochem. 1984, 16, 511-515. (28) Wada, Y.; Kadoya, M. J. Mass Spectrom. 2003, 38, 117-118. (29) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Anal. Chem. 1996, 68, 850-858. (30) Koy, C.; Mikkat, S.; Raptakis, E.; Sutton, C.; Resch, M.; Tanaka, K.; Glocker, M. O. Proteomics 2003, 3, 851-858.
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Figure 1. Reversed-phase chromatography of tryptic peptides from transferrin. (a) Total digest; (b) Sepharose-binding peptides. The Sepharose-binding fraction from the tryptic digest of carbamidomethylated human transferrin (200 µg) was recovered and analyzed.
chromatography was reported by Shimizu et al.31 This method is based on the hydrophilic affinity of cellulose for carbohydrate hydroxyls and was not intended for application to glycopeptides, especially those with a peptide of several amino acids that are typically generated by tryptic digestion. Aiming at small-scale preparation, affinity separation was carried out by partitioning in a microcentrifuge tube rather than by column chromatography. Human transferrin is cleaved into more than 50 peptides by tryptic digestion. With cellulose, the tryptic glycopeptides from human transferrin with two glycosylation sites were isolated with some contamination by other peptides (data not shown). Subsequently, the digest (200 µg) was incubated with a 30-µL packed volume of Sepharose CL-4B in an organic solvent, and peptides eluted with 50% ethanol were analyzed by reversed-phase LC. As shown in Figure 1, two peptides were clearly isolated from the others, while a few small signals were observed earlier in the LC chromatogram. The recovery of this batch separation in a microcentrifuge tube was 30-50%. The Sepharose-binding fraction was dried and analyzed by MALDI linear TOF MS in order to examine whether the bound
Figure 2. MALDI linear TOF mass spectra of the Sepharose-binding glycopeptides from tryptic digests of human transferrin. The binding peptides were eluted with 50% ethanol and dried for MS. (a) Preparation from in-solution digest; (b) Preparation from in-gel digest. Transferrin has two N-glycosylation sites, and the derived glycopeptides are numbered herein from the N-terminal as gp1 (402-414) and gp2 (603-623). Glycopeptide [M + H]+ ions were observed. A marked sole contamination is indicated by an asterisk. 9, N-acetylglucosamine; O, mannose; b, galactose; [, fucose; 2, N-acetylneuraminic acid. 6562 Analytical Chemistry, Vol. 76, No. 22, November 15, 2004
Figure 3. MALDI linear TOF mass spectra of Sepharose-binding glycopeptides from a tryptic digest of human IgG. The binding peptides were eluted with 50% ethanol and dried for MS. Symbols are as described in the legend to Figure 2.
peptides were the glycosylated ones. As shown in Figure 2a, two glycopeptides, gp1 (positions 402-414) and gp2 (positions 603623), were identified. The only other peptide observed in the mass spectrum was an unglycosylated one with the sequence SDNCEDTPEAGYFAVAVVK (positions 415-433; Mr 2073) of transferrin, indicating that peptides rich in serine, threonine, aspartic acid, and glutamic acid residues were likely to be in the glycopeptide fraction. This method was applied to the in-gel digest from an electrophoresed gel piece containing 1 µg of human transferrin and recovered the same glycopeptide species of 3-6 kDa as those from the in-solution digestion (Figure 2b). Håkansson et al. analyzed an in-gel digest of R1-antitrypsin (50 kDa) by ESI-MS and characterized the glycan structures attached to a glycopeptide of more than 6 kDa.32 This demonstrates that large glycopeptides of several kilodaltons can be extracted from the polyacrylamide gel and thus that the gel-based strategy is indeed feasible in glycoproteomics. To demonstrate the binding to glycopeptides with various N-linked glycan forms, other glycoproteins were digested and subjected to glycopeptide isolation. In the case of human immunoglobulin G with a single N-glycosylation site in the heavy chain, the glycopeptide with bisecting N-acetylglucosamine, which has low affinity for a typical lectin, concanavalin A, was normally recovered (Figure 3). A peptide with triantennary oligosaccharides was also identified, and the distribution of glycopeptide [M + H]+ ions observed in the MALDI linear TOF mass spectrum faithfully represented the microheterogeneity of this molecule.33 A glycopeptide with high-mannose type oligosaccharides from ribonuclease B was also recovered (data not shown). The affinity isolation of glycopeptides by cellulose or Sepharose is obviously dependent upon hydrogen bonding between the hydroxyl group of carbohydrates in the medium and glycopeptides. Binding is thus universal as regards the oligosaccharide structures including various N-linked structures, such as sialic acid-bearing, fucosylated, and high-mannose types, and a large (31) Shimizu, Y.; Nakata, M.; Kuroda, Y.; Tsutsumi, F.; Kojima, N.; Mizuochi, T. Carbohydr. Res. 2001, 332, 381-388. (32) Håkansson, K.; Emmett, M. R.; Marshall, A. G.; Davidsson, P.; Nilsson, C. L. J. Proteome Res. 2003, 2, 581-588. (33) Takahashi, N.; Ishii, I.; Ishihara, H.; Mori, M.; Tejima, S.; Jefferis, R.; Endo, S.; Arata, Y. Biochemistry 1987, 26, 1137-1144.
Figure 4. Amino acid sequence of human β2GPI (Swiss-Prot Entry P02749). Cysteine is converted into aminoethylcysteine (small letter “c”), and tryptic cleavage sites are indicated by boldface type. Four N-glycosylation sites are marked by boldface type and italics.
Figure 5. MALDI QIT TOF mass spectrum of tryptic glycopeptides from human β2GPI. The Sepharose-binding peptides from a tryptic digest of aminoethylated β2GPI were eluted with 50% ethanol and dried for MS. All four glycopeptides, gp1 (139-148), gp2 (149-169), gp3 (170-177), and gp4 (232-241), were identified as a pair of peptide and peptide + HexNAc ions except for gp1, which generated a O,2X1 ion instead of the peptide one, as indicated in the lower mass region. Two other glycopeptides, gp1′ (136-148) and gp4′ (232242), were derived from missed cleavage. For signals corresponding to the glycopeptides assigned in the higher mass region, compositions of the N-glycans are given by the following abbreviations: Hex, hexose; HexNAc, N-acetylhexosamine; Pent, pentose (Man3GlcNAc2).
N-linked glycopeptide with 44 amino acid residues and the O-linked structures can be recovered (our unpublished data). It is also noteworthy that the mass spectrum is clear for the sample from this simple preparation, for which a desalting procedure is not necessary. The simple and efficient method of glycopeptide Analytical Chemistry, Vol. 76, No. 22, November 15, 2004
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Figure 6. MALDI QIT MSn analyses of glycopeptide [M + H]+ ions. (a) MS/MS, or MS2, spectrum of the ion at m/z2933.9 in Figure 4. (b) MS/MS/MS, or MS3, spectrum of the peptide + HexNAc ion at m/z 1148.2 in the MS2 spectrum. Symbols are as described in the legend to Figure 2.
isolation described herein will facilitate the study of large glycoproteins that generate numerous peptides by enzymatic digestion. The procedure can be carried out in a Sepharose-loaded pipet tip for microscale preparation, which would improve recovery of small sample quantities, such as an in-gel digest, and make possible a robotic system for high-throughput analysis. MALDI MSn Analysis of Glycopeptide Mixtures. The goal of MS of glycopeptides is to obtain as much information as possible on the glycan and peptide structures, in the simplest manner possible. In the present study, MALDI with ion trap MSn was assessed, because glycopeptide analysis with this type of instrument has not been thoroughly studied and may be useful for mixture samples. The Sepharose-binding fraction of tryptic peptides of human β2GPI, a serum phospholipid-binding protein composed of 326 amino acids and with four glycosylation sites (Figure 4), was analyzed by MS. A number of signals were observed in the region over m/z 2000, presumably the [M + H]+ ions of glycopeptides (Figure 5). Then, each ion was selected as a precursor for CID analysis. As shown in an MS/MS spectrum of the ion at m/z2933.9 (Figure 6a), product ions at m/z2771.7, 2568.5, 2406.4, 2203.2, and 2041.0 were generated, and the spaces between these ladderlike signals indicated that the precursor was indeed a glycopeptide. In addition, prominent signals representing the peptide and its HexNAc-bearing species (corresponding to the oligosaccharide Y1 ion) were identified at m/z 945.0 and 1148.2, respectively. In addition, a cross-ring cleavage product, corresponding to the 6564 Analytical Chemistry, Vol. 76, No. 22, November 15, 2004
Figure 7. MALDI QIT TOF mass spectra of an LC fraction of a complex mixture of glycopeptides derived from human serum. The proteins in serum from a healthy individual were carbamidomethylated and digested with trypsin. The Sepharose-binding glycopeptides were separated by reversed-phase LC, and one faction of the eluent was collected and analyzed by MS. (a) MS spectrum; (b) MS/MS spectrum of the ion at m/z 3419.6; (c) MS/MS spectrum of the peptide ion at m/z 1796.1. The haptoglobin β-chain was identified from the product ions. Symbols are as described in the legend to Figure 2.
oligosaccharide O,2X1 ion, of the HexNAc-bearing peptide was identified at m/z 1028.1. The mass of the oligosaccharide, which could be deduced from the mass-to-charge ratios for the peptide and glycopeptide ions, as well as the spaces of the ladder, suggested the glycopeptide to have a triantennary oligosaccharide. Subsequently, an MS3 of the peptide + HexNAc ion at m/z 1148.2 was carried out (Figure 6b), and the product ions determined the amino acid sequence TTHGNWTK, where N was the third glycosylation site of β2GPI. While the peptide + HexNAc ion was utilized for CID as reported before,34 the same result was obtained by choosing the peptide ion at m/z 945.0 as a precursor. Since the ions used for these CID analyses in the MS2 were traced in the MS1 spectrum shown in Figure 5, the amino acid sequence could be elucidated by the MS2 of these ions. In the same way, glycosylation at other sites was characterized as summarized in Figure 5; gp1 and gp3 had a triantennary oligosaccharide as well as a biantennary one. No sialylated species were observed under the current instrument conditions, while sialylation of β2GPI was demonstrated by MALDI linear TOF MS (data not shown). Finally, to evaluate the applicability of affinity separation followed by MSn to a large number of glycoproteins, glycopeptide (34) Bateman, K. P.; White, R. L.; Yaguchi, M.; Thibault, P. J. Chromotogr., A 1998, 794, 327-344.
mixtures derived from tryptic digestion of whole serum proteins were analyzed by LC off-line MALDI-MS. After isolation by Sepharose, the glycopeptides were separated by reversed-phase LC, and a fraction was collected and subjected to MS (Figure 7a). When an ion at m/z 3419.6 was taken as a precursor for CID, a saccharide-spaced ladder, with an interval of 162 or 203 Da, and the ions for peptide (m/z 1796.4) and peptide + HexNAc (m/z 1999.3) were generated (Figure 7b). The mass spectrum indicated that the peptide had a biantennary N-linked oligosaccharide. Since the peptide ion at m/z 1796.1 was observed in the MS1 spectrum, it was selected as a precursor for sequence determination (Figure 7c). The MS/MS ion search with the product ions specified the sequence VVLHPNYSQVDIGLIK, which was derived from the haptoglobin β-chain and contained an N-glycosylation site. In this analysis of whole serum glycoproteins, the hydrophilic affinity preparation of glycopeptides was fractionated by LC before MS measurement. Alternatively, the complexity of analytes can be reduced by, for example, segmental fractionation of a onedimensional electrophoresis gel of the starting protein sample, since glycopeptide purification can be applied to the in-gel digest. CONCLUSIONS Glycopeptides of adequate length for protein identification by MS/MS product ion search were used as analytes for glycoproteomics, and a simple strategy composed of isolation of tryptic
glycopeptides followed by MS for protein identification, locating glycosylation sites, and evaluating glycosylation site microheterogeneity was described. The hydrophilic affinity partitioning of glycopeptides with Sepharose is expected to effective irrespective of glycan structures, and an additional benefit was that the isolated glycopeptides did not need to be desalted for MS measurements of glycopeptide [M + H]+ ions. Using the collected glycopeptide mixtures, protein identification and elucidation of the glycan structure outlines were achieved by MALDI multiple-stage tandem MS. This method is applicable to in-gel digests of electrophoresed proteins and to protein mixtures obtained by crude separation by LC and has the potential to make glycoproteomics for extremely complex samples a reality. ACKNOWLEDGMENT The authors thank Shimadzu Corporation for instrument support, and the corporate members, K. Tanaka, S. Sekiya, and Y. Fukuyama, for helpful comments. This work was supported by The 21st Century Center of Excellence (COE) Program, Osaka University. Received for review June 27, 2004. Accepted September 8, 2004. AC049062O
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