Comprehensive Immunoglobulin G, A, and M Glycopeptide Profiling for Large-Scale Biomedical Research

• DOI: 10.1016/j.mcpro.2025.100928
• Publication Date: 2025-02-19T17:44:56Z
• Authors: Bianca D.M. van Tol; Anna M. Wasynczuk; Steinar Gijze; Oleg A. Mayboroda; Jan Nouta; Radboud J.E.M. Do...; Manfred Wuhrer; David Falck

Glycosylation of immunoglobulin G (IgG) is recognized as a key modulator of cellular effector functions. At the same time, an increasing body of evidence underlines the importance of other antibody isotypes, especially IgA and IgM, in pathophysiological conditions. Therefore, methods to efficiently study the complex interplay between isotypes, subclasses, and glycosylation of antibodies during acute and chronic states of inflammation are needed. As a solution, we present an integrated and comprehensive method combining simultaneous affinity enrichment of IgG, IgA, and IgM with a single measurement, glycopeptide-centered LC-MS analysis of all isotypes which provides protein-specific (isotype and subclass), and site-specific N- and O-glycosylation quantitation. A two-protease approach provided individual peptides for each glycosylation site, allowing unambiguous compositional assignment and relative quantitation of glycoforms on the MS1 level as well as structural confirmation and partial isomer assignment on the MS/MS level. We demonstrate that our methodology can be efficiently applied to large clinical studies revealing differences in antibody glycosylation in women during and after pregnancy, as well as between healthy donors and patients with rheumatoid arthritis. In addition, this showcased the advantages of our method in comprehensiveness and resolution of isotypes, subclasses, and glycosylation sites as well as its precision and robustness.