Integrating Multiple MS Techniques for in-Depth Antibody Glycosylation Analysis: Revealing Glycosylation-Dependent Structural and Functional Properties

Glycosylation plays a critical role in modulating protein structure, stability, and binding properties, yet comprehensive tools to systematically characterize these effects are scarce. Here, we integrated multiple mass spectrometry (MS) techniques, including high-resolution nanoelectrospray ionization MS (nESI-MS), cross-linking matrix-assisted laser desorption/ionization time-of-flight MS (XL-MALDI-MS), native MS, ion mobility mass spectrometry (IM-MS), together with collision-induced unfolding and a temperature-controlled nESI source to comprehensively investigate glycosylation-dependent changes in protein structural and functional properties. Applying this integrated platform to human IgG Fc, we uncovered how glycosylation alterations in hospitalized COVID-19 patients impact Fc conformation, stability, and receptor binding. nESI-MS profiling revealed a loss of core fucosylation, galactosylation, and sialylation in patient samples. These changes in glycosylation, particularly the loss of fucosylation (afucosylation), correlate with enhanced FcγRIIIa binding, a more open conformation, and reduced stability. These findings highlight glycosylation as a key factor in immune dysregulation during severe COVID-19, and demonstrate the power of integrating multiple MS techniques to uncover the structural and functional consequences of glycan variation. This integrated MS platform is broadly applicable to other glycoprotein systems, including quality control in glycoengineering and research on infectious diseases.