Analysis of O-glycoforms of the IgA1 hinge region by sequential deglycosylation

Abstract

Abstract A common renal disease, immunoglobulin A (IgA) nephropathy (IgAN), is associated with glomerular deposition of IgA1-containing immune complexes. IgA1 hinge region (HR) has up to six clustered O -glycans consisting of Ser/Thr-linked N -acetylgalactosamine with β1,3-linked galactose and variable sialylation. IgA1 glycoforms with some galactose-deficient (Gd) HR O -glycans play a key role in IgAN pathogenesis. The clustered and variable O -glycans make the IgA1 glycomic analysis challenging and better approaches are needed. Here, we report a comprehensive analytical workflow for IgA1 HR O -glycoform analysis. We combined an automated quantitative analysis of the HR O -glycopeptide profiles with sequential deglycosylation to remove all but Gd O -glycans from the HR. The workflow was tested using serum IgA1 from healthy subjects. Twelve variants of glycopeptides corresponding to the HR with three to six O -glycans were detected; nine glycopeptides carried up to three Gd O -glycans. Sites with Gd O -glycans were unambiguously identified by electron-transfer/higher-energy collision dissociation tandem mass spectrometry. Extracted ion chromatograms of isomeric glycoforms enabled quantitative assignment of Gd sites. The most frequent Gd site was T 236 , followed by S 230 , T 233 , T 228 , and S 232 . The new workflow for quantitative profiling of IgA1 HR O -glycoforms with site-specific resolution will enable identification of pathogenic IgA1 HR O -glycoforms in IgAN.