Determination of Aberrant O-Glycosylation in the IgA1 Hinge Region by Electron Capture Dissociation Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry
2005; Elsevier BV; Volume: 280; Issue: 19 Linguagem: Inglês
10.1074/jbc.m411368200
ISSN1083-351X
AutoresMatthew B. Renfrow, Helen J. Cooper, Milan Tomana, Rose Kulhavy, Yoshiyuki Hiki, Kazunori Toma, Mark R. Emmett, Jiří Městecký, Alan G. Marshall, Jan Novák,
Tópico(s)Enzyme Structure and Function
ResumoIn a number of human diseases of chronic inflammatory or autoimmune character, immunoglobulin molecules display aberrant glycosylation patterns of N- or O-linked glycans. In IgA nephropathy, IgA1 molecules with incompletely galactosylated O-linked glycans in the hinge region (HR) are present in mesangial immunodeposits and in circulating immune complexes. It is not known whether the Gal deficiency in IgA1 proteins occurs randomly or preferentially at specific sites. To develop experimental approaches to address this question, the synthetic IgA1 hinge region and hinge region from a naturally Gal-deficient IgA1 myeloma protein have been analyzed by 9.4 tesla Fourier transform-ion cyclotron resonance mass spectrometry. Fourier transform-ion cyclotron resonance mass spectrometry offers two complementary fragmentation techniques for analysis of protein glycosylation by tandem mass spectrometry. Infrared multiphoton dissociation of isolated myeloma IgA1 hinge region peptides confirms the amino acid sequence of the de-glycosylated peptide and positively identifies a series of fragments differing in O-glycosylation. To localize sites of O-glycan attachment, synthetic IgA1 HR glycopeptides and HR from a naturally Gal-deficient polymeric IgA1 myeloma protein were analyzed by electron capture dissociation and activated ion-electron capture dissociation. Multiple sites of O-glycan attachment (including sites of Gal deficiency) in myeloma IgA1 HR glycoforms were identified (in all but one case uniquely). These results represent the first direct identification of multiple sites of O-glycan attachment in IgA1 hinge region by mass spectrometry, thereby enabling future characterization at the molecular level of aberrant glycosylation of IgA1 in diseases such as IgA nephropathy. In a number of human diseases of chronic inflammatory or autoimmune character, immunoglobulin molecules display aberrant glycosylation patterns of N- or O-linked glycans. In IgA nephropathy, IgA1 molecules with incompletely galactosylated O-linked glycans in the hinge region (HR) are present in mesangial immunodeposits and in circulating immune complexes. It is not known whether the Gal deficiency in IgA1 proteins occurs randomly or preferentially at specific sites. To develop experimental approaches to address this question, the synthetic IgA1 hinge region and hinge region from a naturally Gal-deficient IgA1 myeloma protein have been analyzed by 9.4 tesla Fourier transform-ion cyclotron resonance mass spectrometry. Fourier transform-ion cyclotron resonance mass spectrometry offers two complementary fragmentation techniques for analysis of protein glycosylation by tandem mass spectrometry. Infrared multiphoton dissociation of isolated myeloma IgA1 hinge region peptides confirms the amino acid sequence of the de-glycosylated peptide and positively identifies a series of fragments differing in O-glycosylation. To localize sites of O-glycan attachment, synthetic IgA1 HR glycopeptides and HR from a naturally Gal-deficient polymeric IgA1 myeloma protein were analyzed by electron capture dissociation and activated ion-electron capture dissociation. Multiple sites of O-glycan attachment (including sites of Gal deficiency) in myeloma IgA1 HR glycoforms were identified (in all but one case uniquely). These results represent the first direct identification of multiple sites of O-glycan attachment in IgA1 hinge region by mass spectrometry, thereby enabling future characterization at the molecular level of aberrant glycosylation of IgA1 in diseases such as IgA nephropathy. Several human diseases of autoimmune or chronic inflammatory character exhibit abnormal glycosylation of serum proteins, including immunoglobulins (1Rudd P. Elliott T. Cresswell P. Wilson I.A. Dwek R.A. Science. 2001; 291: 2370-2376Crossref PubMed Scopus (1394) Google Scholar, 2Dwek R.A. Science. 1995; 269: 1234-1235Crossref PubMed Scopus (107) Google Scholar, 3Mullinax F. Mullinax G.L. Arthritis Rheum. 1975; 18: 417-418Google Scholar, 4Mestecky J. Novak J. Julian B.A. Tomana M. Nephrology. 2002; 7: S92-S99Crossref Google Scholar, 5Mestecky J. Tomana M. Matousovic K. Konecny K. Julian B.A. Nephrology. 1997; 3: 85-89Crossref Scopus (13) Google Scholar, 6Tomana M. Montreuil J. Vliegenhart J.F.G. Schachter H. Glycoproteins in Disease. Elsevier, Amsterdam1996: 291-298Google Scholar, 7Kobata A. Glycoconj. J. 1998; 15: 323-331Crossref PubMed Scopus (65) Google Scholar). The distinctive carbohydrate side chains of IgA1 molecules play a pivotal role in the pathogenesis of IgA nephropathy (IgAN) 1The abbreviations used are: IgAN, IgA nephropathy; HR, hinge region; FT-ICR, Fourier transform-ion cyclotron resonance; MS, mass spectrometry; MS/MS, tandem mass spectrometry; IRMPD, infrared multiphoton dissociation; ECD, electron capture dissociation; AI-ECD, activated ion-ECD; ESI, electrospray ionization; SWIFT, stored waveform inverse Fourier transform; W, watt; CID, collision-induced dissociation. (8Mestecky J. Tomana M. Crowley-Nowick P.A. Moldoveanu Z. Julian B.A. Jackson S. Contrib. Nephrol. 1993; 104: 172-182Crossref PubMed Google Scholar, 9Novak J. Julian B.A. Tomana M. Mestecky J. J. Clin. Immunol. 2001; 21: 310-327Crossref PubMed Scopus (100) Google Scholar, 10Smith A.C. Feehally J. Springer Semin. Immunopathol. 2003; 24: 477-493Crossref PubMed Scopus (30) Google Scholar). IgA1 contains a hinge region (HR) between the first and second heavy chain constant region domains with a high content of proline (Pro), serine (Ser), and threonine (Thr) (Fig. 1). Several groups have identified three to five O-linked glycan chains within the IgA1 HR (11Baenziger J. Kornfeld S. J. Biol. Chem. 1974; 249: 7270-7281Abstract Full Text PDF PubMed Google Scholar, 12Field M.C. Dwek R.A. Edge C.J. Rademacher T.W. Biochem. Soc. Trans. 1989; 17: 1034-1035Crossref PubMed Scopus (81) Google Scholar, 13Iwase H. Tanaka A. Hiki Y. Kokubo T. Karakasa-Ishii I. Kobayashi Y. Hotta K. J. Biochem. (Tokyo). 1996; 120: 393-397Crossref PubMed Scopus (50) Google Scholar, 14Mattu T.S. Pleass R.J. Willis A.C. Kilian M. Wormald M.R. Lellouch A.C. Rudd P.M. Woof J.M. Dwek R.A. J. Biol. Chem. 1998; 273: 2260-2272Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, 15Novak J. Tomana M. Kilian M. Coward L. Kulhavy R. Barnes S. Mestecky J. Mol. Immunol. 2000; 37: 1047-1056Crossref PubMed Scopus (61) Google Scholar). In normal human serum IgA1, glycosylated sites have been localized to Ser/Thr residues 225, 228, 230, 232, and 236 by N-terminal sequencing methods (14Mattu T.S. Pleass R.J. Willis A.C. Kilian M. Wormald M.R. Lellouch A.C. Rudd P.M. Woof J.M. Dwek R.A. J. Biol. Chem. 1998; 273: 2260-2272Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar) showing that Ser/Thr residues 228, 230, and 232 were occupied in most IgA1 molecules (14Mattu T.S. Pleass R.J. Willis A.C. Kilian M. Wormald M.R. Lellouch A.C. Rudd P.M. Woof J.M. Dwek R.A. J. Biol. Chem. 1998; 273: 2260-2272Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). IgA1 O-linked glycans consist of GalNAc with a β1,3-linked Gal (11Baenziger J. Kornfeld S. J. Biol. Chem. 1974; 249: 7270-7281Abstract Full Text PDF PubMed Google Scholar, 14Mattu T.S. Pleass R.J. Willis A.C. Kilian M. Wormald M.R. Lellouch A.C. Rudd P.M. Woof J.M. Dwek R.A. J. Biol. Chem. 1998; 273: 2260-2272Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, 16Field M.C. Amatayakul-Chantler S. Rademacher T.W. Rudd P.M. Dwek R.A. Biochem. J. 1994; 299: 261-275Crossref PubMed Scopus (87) Google Scholar). Sialic acid (NeuAc) may be attached to GalNAc by an α2,6-linkage or to Gal by an α2,3-linkage (11Baenziger J. Kornfeld S. J. Biol. Chem. 1974; 249: 7270-7281Abstract Full Text PDF PubMed Google Scholar, 14Mattu T.S. Pleass R.J. Willis A.C. Kilian M. Wormald M.R. Lellouch A.C. Rudd P.M. Woof J.M. Dwek R.A. J. Biol. Chem. 1998; 273: 2260-2272Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, 16Field M.C. Amatayakul-Chantler S. Rademacher T.W. Rudd P.M. Dwek R.A. Biochem. J. 1994; 299: 261-275Crossref PubMed Scopus (87) Google Scholar). Carbohydrate composition of O-linked glycans in the HR of normal human serum IgA1 is variable, and the prevailing forms include Gal-GalNAc disaccharide and its mono- and di-sialylated forms (11Baenziger J. Kornfeld S. J. Biol. Chem. 1974; 249: 7270-7281Abstract Full Text PDF PubMed Google Scholar, 12Field M.C. Dwek R.A. Edge C.J. Rademacher T.W. Biochem. Soc. Trans. 1989; 17: 1034-1035Crossref PubMed Scopus (81) Google Scholar, 14Mattu T.S. Pleass R.J. Willis A.C. Kilian M. Wormald M.R. Lellouch A.C. Rudd P.M. Woof J.M. Dwek R.A. J. Biol. Chem. 1998; 273: 2260-2272Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). A variant with terminal GalNAc or sialylated GalNAc is rare for normal serum IgA1 (12Field M.C. Dwek R.A. Edge C.J. Rademacher T.W. Biochem. Soc. Trans. 1989; 17: 1034-1035Crossref PubMed Scopus (81) Google Scholar, 14Mattu T.S. Pleass R.J. Willis A.C. Kilian M. Wormald M.R. Lellouch A.C. Rudd P.M. Woof J.M. Dwek R.A. J. Biol. Chem. 1998; 273: 2260-2272Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar) but is more common in IgAN patients (8Mestecky J. Tomana M. Crowley-Nowick P.A. Moldoveanu Z. Julian B.A. Jackson S. Contrib. Nephrol. 1993; 104: 172-182Crossref PubMed Google Scholar, 10Smith A.C. Feehally J. Springer Semin. Immunopathol. 2003; 24: 477-493Crossref PubMed Scopus (30) Google Scholar, 17Allen A.C. Harper S.J. Feehally J. Clin. Exp. Immunol. 1995; 100: 470-474Crossref PubMed Scopus (268) Google Scholar, 18Hiki Y. Horii A. Iwase H. Tanaka A. Toda Y. Hotta K. Kobayashi Y. Contrib. Nephrol. 1995; 111: 73-84Crossref PubMed Google Scholar, 19Tomana M. Matousovic K. Julian B.A. Radl J. Konecny K. Mestecky J. Kidney Int. 1997; 52: 509-516Abstract Full Text PDF PubMed Scopus (282) Google Scholar, 20Tomana M. Novak J. Julian B.A. Matousovic K. Konecny K. Mestecky J. J. Clin. Investig. 1999; 104: 73-81Crossref PubMed Scopus (399) Google Scholar). Recent reports from several laboratories support the earlier findings that O-linked glycans in the HR of some IgA1 molecules in the circulation of IgAN patients are deficiently galactosylated (8Mestecky J. Tomana M. Crowley-Nowick P.A. Moldoveanu Z. Julian B.A. Jackson S. Contrib. Nephrol. 1993; 104: 172-182Crossref PubMed Google Scholar, 17Allen A.C. Harper S.J. Feehally J. Clin. Exp. Immunol. 1995; 100: 470-474Crossref PubMed Scopus (268) Google Scholar, 18Hiki Y. Horii A. Iwase H. Tanaka A. Toda Y. Hotta K. Kobayashi Y. Contrib. Nephrol. 1995; 111: 73-84Crossref PubMed Google Scholar, 19Tomana M. Matousovic K. Julian B.A. Radl J. Konecny K. Mestecky J. Kidney Int. 1997; 52: 509-516Abstract Full Text PDF PubMed Scopus (282) Google Scholar, 20Tomana M. Novak J. Julian B.A. Matousovic K. Konecny K. Mestecky J. J. Clin. Investig. 1999; 104: 73-81Crossref PubMed Scopus (399) Google Scholar, 21Andre P.M. Le Pogamp P. Chevet D. J. Clin. Lab. Anal. 1990; 4: 115-119Crossref PubMed Scopus (98) Google Scholar, 22Coppo R. Amore A. Kidney Int. 2004; 65: 1544-1547Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). The Gal-deficient IgA1 in the circulation is exclusively present in circulating immune complexes and is mostly a J-chain-containing polymer (20Tomana M. Novak J. Julian B.A. Matousovic K. Konecny K. Mestecky J. J. Clin. Investig. 1999; 104: 73-81Crossref PubMed Scopus (399) Google Scholar). In the absence of Gal, the terminal sugar is GalNAc (19Tomana M. Matousovic K. Julian B.A. Radl J. Konecny K. Mestecky J. Kidney Int. 1997; 52: 509-516Abstract Full Text PDF PubMed Scopus (282) Google Scholar, 20Tomana M. Novak J. Julian B.A. Matousovic K. Konecny K. Mestecky J. J. Clin. Investig. 1999; 104: 73-81Crossref PubMed Scopus (399) Google Scholar). Subsequently, these aberrant O-glycans or HR glycopeptides (23Kokubo T. Hiki Y. Iwase H. Tanaka A. Nishikido J. Hotta K. Kobayashi Y. Nephrol. Dial. Transplant. 1999; 14: 81-85Crossref PubMed Scopus (27) Google Scholar, 24Kokubo T. Hashizume K. Iwase H. Arai K. Tanaka A. Toma K. Hotta K. Kobayashi Y. Nephrol. Dial. Transplant. 2000; 15: 28-33Crossref PubMed Scopus (41) Google Scholar) are recognized by naturally occurring antibodies with anti-glycan or anti-HR peptide specificities (20Tomana M. Novak J. Julian B.A. Matousovic K. Konecny K. Mestecky J. J. Clin. Investig. 1999; 104: 73-81Crossref PubMed Scopus (399) Google Scholar, 24Kokubo T. Hashizume K. Iwase H. Arai K. Tanaka A. Toma K. Hotta K. Kobayashi Y. Nephrol. Dial. Transplant. 2000; 15: 28-33Crossref PubMed Scopus (41) Google Scholar), and thus circulating immune complexes are formed (25Novak J. Vu H.L. Novak L. Julian B.A. Mestecky J. Tomana M. Kidney Int. 2002; 62: 465-475Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). It is hypothesized that these Gal-deficient IgA1-containing circulating immune complexes are not efficiently cleared in IgAN patients and thus deposit in the mesangium where they bind to and activate the resident mesangial cells, inducing cellular proliferation and matrix overproduction (9Novak J. Julian B.A. Tomana M. Mestecky J. J. Clin. Immunol. 2001; 21: 310-327Crossref PubMed Scopus (100) Google Scholar, 26Julian B.A. Novak J. Curr. Opin. Nephrol. Hypertens. 2004; 13: 171-179Crossref PubMed Scopus (86) Google Scholar). In fact, these Gal-deficient IgA1-containing complexes bind to mesangial cells more efficiently than uncomplexed IgA1 or similarly sized IgA1-containing complexes from healthy controls (25Novak J. Vu H.L. Novak L. Julian B.A. Mestecky J. Tomana M. Kidney Int. 2002; 62: 465-475Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Furthermore, Gal-deficient IgA1 was detected in glomerular immune deposits (27Allen A.C. Bailey E.M. Brenchley P.E.C. Buck K.S. Barrat J. Feehally J. Kidney Int. 2001; 60: 969-973Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 28Hiki Y. Odani H. Takahashi M. Yasuda Y. Nishimoto A. Iwase H. Shinzato T. Kobayashi Y. Maeda K. Kidney Int. 2001; 59: 1077-1085Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). These observations support the hypothesis that aberrantly glycosylated IgA1-containing immune complexes participate in the pathogenesis of IgAN (4Mestecky J. Novak J. Julian B.A. Tomana M. Nephrology. 2002; 7: S92-S99Crossref Google Scholar, 9Novak J. Julian B.A. Tomana M. Mestecky J. J. Clin. Immunol. 2001; 21: 310-327Crossref PubMed Scopus (100) Google Scholar, 22Coppo R. Amore A. Kidney Int. 2004; 65: 1544-1547Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 26Julian B.A. Novak J. Curr. Opin. Nephrol. Hypertens. 2004; 13: 171-179Crossref PubMed Scopus (86) Google Scholar). Available techniques for analysis of IgA1 O-glycosylation, such as lectin binding assays, can identify the presence of Gal-deficient O-glycan chains (27Allen A.C. Bailey E.M. Brenchley P.E.C. Buck K.S. Barrat J. Feehally J. Kidney Int. 2001; 60: 969-973Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 28Hiki Y. Odani H. Takahashi M. Yasuda Y. Nishimoto A. Iwase H. Shinzato T. Kobayashi Y. Maeda K. Kidney Int. 2001; 59: 1077-1085Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 29Hiki Y. Kokubo T. Iwase H. Tanaka A. Toma K. Hotta K. Kobayashi Y. 8th International IgA Nephropathy Symposium, Noordwijkerhout, Netherlands, May 10-13. 1998; Google Scholar, 30Hiki Y. Tanaka A. Kokubo T. Iwase H. Nishikido J. Hotta K. Kobayashi Y. J. Am. Soc. Nephrol. 1998; 9: 577-582Crossref PubMed Google Scholar, 31Allen A. Feehally J. Adv. Exp. Med. Biol. 1998; 435: 175-183Crossref PubMed Scopus (15) Google Scholar). However, it is not known whether the Gal deficiency in IgAN patients occurs randomly or preferentially at specific sites. Mass spectrometric analysis of IgA1 HR O-glycosylation has characterized the heterogeneity of glycoforms (15Novak J. Tomana M. Kilian M. Coward L. Kulhavy R. Barnes S. Mestecky J. Mol. Immunol. 2000; 37: 1047-1056Crossref PubMed Scopus (61) Google Scholar, 28Hiki Y. Odani H. Takahashi M. Yasuda Y. Nishimoto A. Iwase H. Shinzato T. Kobayashi Y. Maeda K. Kidney Int. 2001; 59: 1077-1085Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 30Hiki Y. Tanaka A. Kokubo T. Iwase H. Nishikido J. Hotta K. Kobayashi Y. J. Am. Soc. Nephrol. 1998; 9: 577-582Crossref PubMed Google Scholar) but has not localized specific sites of O-glycosylation. Tandem mass spectrometry (MS/MS) has become a standard tool for the structural analysis of carbohydrates (for a review of complex carbohydrate mass spectrometry see Ref. 32Zaia J. Mass Spectrom. 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Marshall A.G. Nilsson C.L. Anal. Chem. 2001; 73: 4530-4536Crossref PubMed Scopus (343) Google Scholar, 47Håkansson K. Chalmers M.J. Quinn J.P. McFarland M.A. Hendrickson C.L. Marshall A.G. Anal. Chem. 2003; 75: 3256-3262Crossref PubMed Scopus (223) Google Scholar) implemented both of these fragmentation techniques in a single FT-ICR mass spectrometer configuration and successfully demonstrated their use in analysis of glycoproteins. These studies suggest ECD FT-ICR MS could be a valuable tool for the analysis of Gal-deficient IgA1 HR. Here we analyze O-glycans of synthetic IgA1 HR and a naturally Gal-deficient polymeric (p) IgA1 myeloma protein. We have shown previously (20Tomana M. Novak J. Julian B.A. Matousovic K. Konecny K. Mestecky J. J. Clin. Investig. 1999; 104: 73-81Crossref PubMed Scopus (399) Google Scholar) that this IgA1 can inhibit re-association of IgA1-containing immune complexes, suggesting that this IgA1 protein has properties similar to the aberrantly glycosylated IgA1 present in the circulation of IgA nephropathy patients. We observed a heterogeneous population of glycosylated IgA1 HR as in prior studies (15Novak J. Tomana M. Kilian M. Coward L. Kulhavy R. Barnes S. Mestecky J. Mol. Immunol. 2000; 37: 1047-1056Crossref PubMed Scopus (61) Google Scholar, 28Hiki Y. Odani H. Takahashi M. Yasuda Y. Nishimoto A. Iwase H. Shinzato T. Kobayashi Y. Maeda K. Kidney Int. 2001; 59: 1077-1085Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 30Hiki Y. Tanaka A. Kokubo T. Iwase H. Nishikido J. Hotta K. Kobayashi Y. J. Am. Soc. Nephrol. 1998; 9: 577-582Crossref PubMed Google Scholar). FT-ICR MS/MS experiments localized sites of O-glycosylation in the IgA1 HR. Four and five glycan chains composed of either a GalNAc-Gal disaccharide or a GalNAc monosaccharide were localized to 5 of the 10 possible sites of O-glycosylation in the myeloma IgA1 HR. These analytical approaches will be used in the future for characterization of O-glycans in IgA1 from IgAN patients. Synthesis of IgA1 HR Peptide and Glycopeptide Variants—A panel of 19-mer hinge region peptide and glycopeptides, with an α-O-linked GalNAc residue, corresponding to the amino acid sequence of the human IgA1 HR were synthesized by and purchased from the Peptide Institute Inc. (Osaka, Japan). Purity and molecular weight of the preparations were confirmed by high pressure liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The following HR peptide and glycopeptides used: HP, VPSTPPTPSPSTPPTPSPS; 4-HP (GalNAc attached to Thr4 and so on), VPS-(GalNAc)TPPTPSPSTPPTPSPS; 7-HP, VPSTPP-(GalNAc)TPSPSTPPTPSPS; 9-HP, VPSTPPTP-(GalNAc)SPSTPPTPSPS; 11-HP, VPSTPPTPSP-(GalNAc)STPPTPSPS; 15-HP, VPSTPPTPSPSTPP-(GalNAc)TPSPS; 4-15-HP, VPS-(GalNAc)TPP-(GalNAc)TP-(GalNAc)SP-(GalNAc)STPP-(GalNAc)TPSPS. Isolation of IgA1 HR Glycopeptide from a Myeloma IgA1 (Mce)—HR from pIgA1 myeloma protein (Mce) was isolated as a tryptic-peptic fragment following the procedure described by Wolfenstein-Todel and co-workers (48Wolfenstein-Todel C. Frangione B. Franklin E.C. Biochemistry. 1972; 11: 3971-3975Crossref PubMed Scopus (16) Google Scholar, 49Frangione B. Wolfenstein-Todel C. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 3673-3676Crossref PubMed Scopus (74) Google Scholar). The resultant preparation was lyophilized and dissolved in water before analyses. Monosaccharide Compositional Analysis—The monosaccharides from purified myeloma IgA1 HR were determined as trifluoroacetates of methylglycosides by gas chromatography as described previously (50Tomana M. Prchal J.T. Garner L.C. Skalka H.W. Barker S.A. J. Lab. Clin. Med. 1984; 103: 137-142PubMed Google Scholar). The analyses were performed with a gas chromatograph (model 5890, Hewlett-Packard, Sacramento, CA, equipped with a 25-m fused silica (0.22 mm inner diameter) OV-1701 WCOT column (Chrompack, Bridge-water, NJ) electron capture detector and a Hewlett-Packard model 3396 integrator. About 10 μg of HR glycopeptide was used for the analysis. Sample Preparation—For electrospray ionization (ESI) experiments, synthetic IgA1 HR samples (5 mm in 1:1 water/methanol, 2% acetic acid) were micro-electrosprayed (51Emmett M.R. Caprioli R.M. J. Am. Soc. Mass Spectrom. 1994; 5: 605-613Crossref PubMed Scopus (480) Google Scholar) from an emitter consisting of a 50-μm inner diameter fused silica capillary that had been mechanically ground to a uniform thin walled tip (52Quinn J.P. Emmett M.R. Marshall A.G. 46th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, FL, May 31-June 4, 1998. American Society for Mass Spectrometry, Santa Fe, NM1998: 1388Google Scholar) at a flow rate of 300 nl/min. For separated myeloma IgA1 HR glycopeptides, samples were desalted with a C18 ZipTip (Millipore, City, MA) into 30 μl of a 4:1 acetonitrile/water solution containing 0.1% formic acid. Desalted samples were then microelectrosprayed as above at a flow rate of 200-400 nl/min or from a chip-based electrospray interface (NanoMate, Advion, Ithaca, NY). FT-ICR MS of IgA1 HR Glycopeptides—IgA1 HR glycopeptides were analyzed with a home built 9.4 tesla ESI Q FT-ICR mass spectrometer (47Håkansson K. Chalmers M.J. Quinn J.P. McFarland M.A. Hendrickson C.L. Marshall A.G. Anal. Chem. 2003; 75: 3256-3262Crossref PubMed Scopus (223) Google Scholar, 53Senko M.W. Hendrickson C.L. Pasa-Tolic L. Marto J.A. White F.M. Guan S. Marshall A.G. Rapid Commun. Mass Spectrom. 1996; 10: 1824-1828Crossref PubMed Scopus (277) Google Scholar) under the control of a modular ICR data acquisition system (54Senko M.W. Canterbury J.D. Guan S. Marshall A.G. Rapid Commun. Mass Spectrom. 1996; 10: 1839-1844Crossref PubMed Scopus (310) Google Scholar, 55Blakney G.T. van der Rest G. Johnson J.R. Freitas M.A. Drader J.J. Shi S. D.H. Hendrickson C.L. Kelleher N.L. Marshall A. 49th ASMS Conference on Mass Spectrometry and Allied Topics, Chicago, May 27-31, 2001. American Society for Mass Spectrometry, Santa Fe, NM2001Google Scholar). Ions were transported through a Chait-style atmosphere-to-vacuum interface (56Chowdhury S.K. Katta V. Chait B.T. Rapid Commun. 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Elsevier, Amsterdam1990Google Scholar). Frequency domain spectra were frequency to m/z calibrated (60Ledford Jr., E.B. Rempel D.L. Gross M.L. Anal. Chem. 1984; 56: 2744-2748Crossref PubMed Scopus (444) Google Scholar, 61Shi S.D.-H. Drader J.J. Freitas M.A. Hendrickson C.L. Marshall A.G. Int. J. Mass Spectrom. 2000; 195: 591-598Crossref Scopus (207) Google Scholar) externally from the measured ICR frequencies of Agilent calibration mixture ions. Each displayed spectrum represents a sum of 25-50 time-domain transients. Masses and m/z values were calculated with Isopro 3.1 (MS/MS software, members.aol.com/msmssoft/). FT-ICR MS/MS of IgA1 HR Glycopeptides—Precursor ions were mass-selectively accumulated externally for 5-15 s (62Hendrickson C.L. Quinn J.P. Emmett M.R. Marshall A.G. 48th ASMS Conference on Mass Spectrometry and Allied Topics, Long Beach, CA, June 11-15, 2000. American Society for Mass Spectrometry, Santa Fe, NM2000Google Scholar, 63Belov M.E. Nikolaev E.N. Anderson G.A. Udseth H.R. Conrads T.P. Veenstra T.D. Masselon C.D. Gorshkov M.V. Smith R.D. Anal. Chem. 2001; 73: 253-261Crossref PubMed Scopus (92) Google Scholar). Following transfer to the ICR cell (1.0-1.4 ms), stored waveform inverse Fourier transform (SWIFT) (64Marshall A.G. Wang T.-C.L. Ricca T.L. J. Am. Chem. Soc. 1985; 107: 7893-7897Crossref Scopus (601) Google Scholar, 65Guan S. Marshall A.G. Int. J. Mass Spectrom. Ion Processes. 1996; 157: 5-37Crossref Scopus (347) Google Scholar) ejection was applied for increased m/z selectivity. An indirectly heated 10-mm diameter dispenser cathode (1109; Heat Wave, Watsonville, CA) mounted on the central axis of the system provided the electrons for ECD; IRMPD was performed with a 40-W, 10.6-μm, CO2 laser (Synrad, Mukilteo, WA), fitted with a 2.5× beam expander. The laser beam is directed to the center of the cell through an off-axis BaF2 window (47Håkansson K. Chalmers M.J. Quinn J.P. McFarland M.A. Hendrickson C.L. Marshall A.G. Anal. Chem. 2003; 75: 3256-3262Crossref PubMed Scopus (223) Google Scholar). Activated ion (AI)-ECD was performed wit
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