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 Emmett, Jiri Mestecky, Alan G. Marshall, Jan Novak

Research output: Contribution to journalArticle

115 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)19136-19145
Number of pages10
JournalJournal of Biological Chemistry
Volume280
Issue number19
DOIs
StatePublished - May 13 2005
Externally publishedYes

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Glycosylation
Cyclotrons
Cyclotron resonance
Fourier Analysis
Hinges
Immunoglobulin A
Mass spectrometry
Mass Spectrometry
Fourier transforms
Electrons
Ions
Polysaccharides
Myeloma Proteins
Peptides
Molecules
Glycopeptides
Tandem Mass Spectrometry
Antigen-Antibody Complex
Autoimmune Diseases
Immunoglobulins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Determination of aberrant O-glycosylation in the IgA1 hinge region by electron capture dissociation Fourier transform-ion cyclotron resonance mass spectrometry. / Renfrow, Matthew B.; Cooper, Helen J.; Tomana, Milan; Kulhavy, Rose; Hiki, Yoshiyuki; Toma, Kazunori; Emmett, Mark; Mestecky, Jiri; Marshall, Alan G.; Novak, Jan.

In: Journal of Biological Chemistry, Vol. 280, No. 19, 13.05.2005, p. 19136-19145.

Research output: Contribution to journalArticle

Renfrow, Matthew B. ; Cooper, Helen J. ; Tomana, Milan ; Kulhavy, Rose ; Hiki, Yoshiyuki ; Toma, Kazunori ; Emmett, Mark ; Mestecky, Jiri ; Marshall, Alan G. ; Novak, Jan. / Determination of aberrant O-glycosylation in the IgA1 hinge region by electron capture dissociation Fourier transform-ion cyclotron resonance mass spectrometry. In: Journal of Biological Chemistry. 2005 ; Vol. 280, No. 19. pp. 19136-19145.
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AU - Cooper, Helen J.

AU - Tomana, Milan

AU - Kulhavy, Rose

AU - Hiki, Yoshiyuki

AU - Toma, Kazunori

AU - Emmett, Mark

AU - Mestecky, Jiri

AU - Marshall, Alan G.

AU - Novak, Jan

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