title: Clustered O-glycans of IgA1: defining macro- and microheterogeneity by use of electron capture/transfer dissociation.
abstract: IgA nephropathy (IgAN) is the most common primary glomerulonephritis in the world. Aberrantly glycosylated IgA1, with galactose (Gal)-deficient hinge region (HR) O-glycans, plays a pivotal role in the pathogenesis of the disease. It is not known whether the glycosylation defect occurs randomly or preferentially at specific sites. We have described the utility of activated ion-electron capture dissociation (AI-ECD) mass spectrometric analysis of IgA1 O-glycosylation. However, locating and characterizing the entire range of O-glycan attachment sites are analytically challenging due to the clustered serine and threonine residues in the HR of IgA1 heavy chain. To address this problem, we analyzed all glycoforms of the HR glycopeptides of a Gal-deficient IgA1 myeloma protein, mimicking the aberrant IgA1 in patients with IgAN, by use of a combination of IgA-specific proteases + trypsin and AI-ECD Fourier transform ion cyclotron resonance (FT-ICR) tandem mass spectrometry (MS/MS). The IgA-specific proteases provided a variety of IgA1 HR fragments that allowed unambiguous localization of all O-glycosylation sites in the six most abundant glycoforms, including the sites deficient in Gal. Additionally, this protocol was adapted for on-line liquid chromatography (LC)-AI-ECD MS/MS and LC-electron transfer dissociation MS/MS analysis. Our results thus represent a new clinically relevant approach that requires ECD/electron transfer dissociation-type fragmentation to define the molecular events leading to pathogenesis of a chronic kidney disease. Furthermore, this work offers generally applicable principles for the analysis of clustered sites of O-glycosylation.
authors.affiliation: Biomedical FT-ICR MS Laboratory, Department of Biochemistry and Molecular Genetics, University of Alabama, Birmingham, Alabama 35294-0005, USA.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Takahashi
authors.forename: Stephanie B
authors.initials: SB
authors.lastname: Wall
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Suzuki
authors.forename: Archer D
authors.initials: AD
authors.lastname: Smith
authors.forename: Stacy
authors.initials: S
authors.lastname: Hall
authors.forename: Knud
authors.initials: K
authors.lastname: Poulsen
authors.forename: Mogens
authors.initials: M
authors.lastname: Kilian
authors.forename: James A
authors.initials: JA
authors.lastname: Mobley
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Julian
authors.forename: Jiri
authors.initials: J
authors.lastname: Mestecky
authors.forename: Jan
authors.initials: J
authors.lastname: Novak
authors.forename: Matthew B
authors.initials: MB
authors.lastname: Renfrow
mesh_terms: D000595:Amino Acid Sequence; D004583:Electrons; D005583:Fourier Analysis; D005922:Glomerulonephritis, IGA / Q000378:metabolism; D006020:Glycopeptides / Q000032:analysis; D006031:Glycosylation; D006801:Humans; D007070:Immunoglobulin A / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry*
chemical_list: D006020:Glycopeptides; D007070:Immunoglobulin A; D011134:Polysaccharides
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