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J. Biol. Chem., Vol. 280, Issue 19, 19136-19145, May 13, 2005

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Determination of Aberrant O-Glycosylation in the IgA1 Hinge Region by Electron Capture Dissociation Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry^*

Matthew B. Renfrow¶¶, Helen J. Cooper, Milan Tomana¶, Rose Kulhavy¶, Yoshiyuki Hiki||, Kazunori Toma**, Mark R. Emmett, Jiri Mestecky¶, Alan G. Marshall, and Jan Novak¶

From the National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida 32310-4005, ^¶Departments of Microbiology and Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294, ^||Division of Nephrology, Department of Medicine, Fujita Health University, School of Medicine, Toyoake, 470-1192 Japan, ^**Research Department, The Noguchi Institute, Tokyo, 173-0003 Japan, and the Department of Chemistry, Florida State University, Tallahassee, Florida 32306

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.

Received for publication, October 5, 2004 , and in revised form, January 28, 2005.

^* This work was supported in part by National Science Foundation Grant CHE-99-09502, Florida State University, the National High Magnetic Field Laboratory in Tallahassee, FL, and by National Institutes of Health Grants DK57750, DK61525, and DE13694. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶¶ Present address: Dept. of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294.

Supported by the Wellcome Trust. Present address: School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.

To whom correspondence should be addressed: Dept. of Microbiology, University of Alabama, 845 19th St. S, BBRB 734 (Box 1), Birmingham, AL 35294. Tel.: 205-934-4480; Fax: 205-934-3894; E-mail: Jan_Novak{at}microbio.uab.edu.

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