Glycosylation profiling of immunoglobulin G (IgG) subclasses from human serum

Abstract All four subclasses of human serum IgG contain a single N ‐glycosylation site in the constant region of their heavy chain, which is occupied by biantennary, largely core‐fucosylated and partially truncated oligosaccharides, that may carry a bisecting N ‐acetylglucosamine and sialic acid residues. IgG glycosylation has been shown to be altered under various physiological and pathological circumstances. IgG N ‐glycan profiles vary with age, and galactosylation for example is enhanced during pregnancy. Several diseases including rheumatoid arthritis are associated with a reduction in galactosylation of the IgG N ‐glycans. Here, we describe a robust method for the isolation of IgG subclasses using protein A (binds IgG1, IgG2, and IgG4) and protein G (binds additionally IgG3) at the 96‐well plate level, which is suitable for automation. Isolated IgGs were digested with trypsin, and obtained glycopeptides were analyzed by nano‐LC‐MS. Glycopeptides were characterized by CID as well as electron transfer dissociation (ETD). The method provided glycosylation profiles for IgG1, IgG2, IgG3, and IgG4 and revealed distinct differences in N ‐glycosylation between the four IgG subclasses. The changes in galactosylation associated with rheumatoid arthritis could readily be monitored. This method is suitable for the subclass‐specific analysis of IgG glycosylation from clinical samples.