Defining IgA1 O-glycan Heterogeneity by use of ECD and IgA Specific Proteases

摘要

The combination of bacterial IgA specific proteases and AI-ECD/ETD fragmentation provides complete site localization of the 5 most abundant IgA1 O-glycoforms. Each IgA1 HR fragment O-glycoform produces a unique set of ECD/ETD type fragments that appear to be charge state dependent. Complete analysis of O-glycoform microheterogeneity and site-localization can be accomplished with less than 3 (mu)g of isolated IgA1. This provides the opportunity to analyze the low abundance pathogenic forms of IgA1 that lead to the formation of circulating immune complexes and deposition of IgA1 in the kidney of IgAN patients. Analysis of clinical samples will be possible with standard collection amounts of serum from patients with IgA nephropathy. ECD analysis of the variety of IgA1 HR proteolytic fragments provides a practical set of guidelines for the analysis of clustered sites of O-glycosylation.