An integrated approach for comprehensive profiling and quantitation of IgG-Fc glycopeptides with application to...

Glycosylation plays an important role in the maintenance of the structure and function of glycoproteins, while aberrant protein glycosylation is correlated with various diseases. Human immunoglobulin G (IgG), which is composed of four subclasses (IgG1, IgG2, IgG3 and IgG4), is one of the most dominant and significant glycoprotein in human serum. The glycosylation on IgG-Fc moiety is known to be alternated with various physiological and pathological states. We herein report an integrated approach for comprehensive profiling and quantitation of IgG-Fc glycopeptides. Firstly, IgG N-glycans were profiled by using mAb-Glyco chip quadrupole time-of-flight mass spectrometry (Q-TOF-MS), resulting characterization of 87 N‑glycans originating from 29 different oligosaccharide compositions. Secondly, subclass-specific glycopeptides were identified on the basis of high-resolution MS and MS/MS data by using ultra high performance liquid chromatography (UHPLC) coupled to Q-TOF-MS. As a result, 83 IgG-Fc glycopeptides from human serum, including 17 sialylated glycopeptides, were identified. In addition, a quantitation method with high sensitivity and repeatability was established by using UHPLC triple quadrupole (QQQ) MS. We applied this approach to carry out quantitative analysis of IgG glycosylation in RA patients. Finally, 36 potential glycopeptide biomarkers, including 13 species from IgG1, 12 species from IgG2/3 and 11 species from IgG4 were identified.