Simultaneous\nProtein\nQuantitation and Glycosylation\nProfiling of Antigen-Specific Immunoglobulin G1 in Large Clinical\nStudies

Antibodies have a\nkey role in the immune system, making their characterization\nessential to biomedical, biopharmaceutical, and clinical research\nquestions. Antibody effector functions are mainly controlled by quantity,\nsubclass, and Fc glycosylation. We describe an integrated method to\nmeasure these three critical dimensions simultaneously. The subclass-specific\nimmunoglobulin G (IgG) Fc glycosylation analysis combines immunosorbance\nwith glycopeptide-centered LC-MS detection. For integrated IgG1-specific\nquantitation, a commercial, stable isotope labeled IgG1 protein standard\nwas spiked into the immunosorbent eluates. Robust quantitation was\nachieved, relying on a combination of a proteotypic peptide and the\nmost abundant glycopeptides, generated through proteolytic cleavage\nfrom a mixture of natural IgG1 and the recombinant IgG1 standard.\nMethod performance was demonstrated in a large coronavirus vaccination\ncohort at a throughput of 100 samples/day. LC-MS-derived, anti-SARS-CoV-2\nspike protein IgG1 concentrations ranged from 100 to 10000 ng/mL and\ncorrelated well with a clinically relevant immunoassay. Technical\nvariation was 200 times lower than biological variation; intermediate\nprecision was 44%. In conclusion, we present a method capable of robustly\nand simultaneously assessing quantity, subclass, and Fc glycosylation\nof antigen-specific IgG in large clinical studies. This method will\nfacilitate a broader understanding of immune responses, especially\nthe important interplay among the three dimensions.