Fc Glycosylation Characterization of Human Immunoglobulins G Using Immunocapture and LC-MS

Immunoglobulins G (IgG) are proteins produced by the immune system of higher life forms that play a central role in the defense against microbial pathogens. IgG bind pathogens with the hypervariable Fab component and mediate a diversity of effector functions by binding to immune effector cells via their crystallizable (Fc) component. All IgG Fc carry a polymorphic N-glycan that regulates its binding properties and thereby its effector functions. The glycosylation profile of IgG Fc is modulated by physiological and pathological conditions, including infectious diseases and inflammatory disorders. Characterization of IgG Fc glycosylation profiles is a promising approach to understand the pathogenesis of diseases involving the immune system and to develop novel biomarkers of disease activity. Measuring the proportion of the different IgG Fc glycoforms remains an analytical challenge, that requires a sensitive and reproducible analytical approach.This chapter describes an optimized approach for the preparation and the analysis of Fc N-glycans from total serum or plasma IgG using magnetic beads, RapiFluor MS label©, and LC-MS.