Quantitative determination of human IgA subclasses and their Fc‐glycosylation patterns in plasma by using a peptide analogue internal standard and ultra‐high‐performance liquid chromatography/triple quadrupole mass spectrometry

Rationale Glycosylation on immunoglobulins is important for the immune function. In this study, we developed and validated a method for the absolute quantification of IgA subclasses and relative quantification of IgA‐Fc glycopeptides by using affinity purification and ultrahigh‐performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS). Only micro‐volumes of plasma were required from each sample and we also applied the method to discover IgA and IgA‐glycopeptide profiles in patients with chronic kidney diseases and IgA nephropathy. Methods Peptide M affinity beads were used to purify IgA, and a cost‐effective peptide analogue was added as internal standard. With an efficient on‐bead digestion process, purified samples were analyzed by UHPLC/MS/MS in multiple reaction monitoring mode. Results Correlation coefficients were greater than 0.999 for the IgA1 and IgA2 calibration curves and greater than 0.994 for glycopeptide regression curves. Intraday and interday precisions for IgA1 and IgA2 were