Relative Quantitation of Subclass-Specific Murine IgG Fc N-Glycoforms by Multiple Reaction Monitoring

Abstract

N-Linked glycosylation of the fragment crystallizable (Fc) domain of immunoglobulin G (IgG) is considered a significant modulator of antibody functions, which is known to be subclass-specific. As mice are the most widely used model organisms in immunological research, determining the variation in Fc glycosylation among each murine IgG subclass in different physiological or pathological statuses is beneficial for studying how the IgG subclass effector function is affected by Fc glycosylation. In this study, we established a method to quantify murine IgG Fc glycoforms normalized to the protein abundance at a subclass-specific level for various mouse strains using multiple reaction monitoring. The glycoform level was normalized to the subclass protein abundance (subclass-specific peptide intensity) in each IgG subclass to eliminate the contribution from the subclass protein abundance. Both good linearity and high repeatability of the method were validated by investigating a mixed mouse serum sample. The method was applied to quantify the differences in subclass-specific IgG Fc N-glycoforms between systemic sclerosis (SSc) mice and healthy control mice. The results demonstrated that each IgG subclass had its own characteristic-altered glycosylation, implying the close association of subclass-specific IgG Fc glycosylation with SSc in mice. This report demonstrates a method with great reliability and practicality that has promising potential for the relative quantitation of subclass-specific IgG Fc N-glycoforms in multiple mouse models.