Simultaneous Protein Quantitation and Glycosylation Profiling of Antigen-Specific Immunoglobulin G1 in Large Clinical Studies

Steinar Gijze, Anna Wasynczuk, Leanne van Leeuwen, Marloes Grobben, Marit J. van Gils, Jan Nouta, Wenjun Wang, Virgil A. S. H. Dalm, Hetty Jolink, Manfred Wuhrer, David Falck

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Abstract

Antibodies have a key role in the immune system, making their characterization essential to biomedical, biopharmaceutical, and clinical research questions. Antibody effector functions are mainly controlled by quantity, subclass, and Fc glycosylation. We describe an integrated method to measure these three critical dimensions simultaneously. The subclass-specific immunoglobulin G (IgG) Fc glycosylation analysis combines immunosorbance with glycopeptide-centered LC-MS detection. For integrated IgG1-specific quantitation, a commercial, stable isotope labeled IgG1 protein standard was spiked into the immunosorbent eluates. Robust quantitation was achieved, relying on a combination of a proteotypic peptide and the most abundant glycopeptides, generated through proteolytic cleavage from a mixture of natural IgG1 and the recombinant IgG1 standard. Method performance was demonstrated in a large coronavirus vaccination cohort at a throughput of 100 samples/day. LC-MS-derived, anti-SARS-CoV-2 spike protein IgG1 concentrations ranged from 100 to 10000 ng/mL and correlated well with a clinically relevant immunoassay. Technical variation was 200 times lower than biological variation; intermediate precision was 44%. In conclusion, we present a method capable of robustly and simultaneously assessing quantity, subclass, and Fc glycosylation of antigen-specific IgG in large clinical studies. This method will facilitate a broader understanding of immune responses, especially the important interplay among the three dimensions.
Original languageEnglish
Pages (from-to)5600-5605
Number of pages6
JournalJournal of Proteome Research
Volume23
Issue number12
DOIs
Publication statusPublished - 13 Nov 2024

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© 2024 The Authors. Published by American Chemical Society.

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