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

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.

Keywords: Glycoproteomics; LC-MS; antibody glycosylation; antibody quantitation; antigen-specific antibody responses; glycopeptides; immunoglobulin G; liquid chromatography−mass spectrometry; stable isotope labeled protein standard.

Figure 1

Workflow for simultaneous quantitation and Fc glycosylation analysis of antigen-specific IgG. For correction of measurement variability, a stable-isotope labeled (SIL) human recombinant IgG1 (rhIgG1), commercially available as SILuMAB, was used.

Figure 2

Zoomed-in view of a sum spectrum of the IgG1 elution range (48 to 62 s). The isotopologue pattern of the heavy labeled IgG1-G1F glycopeptide of SILuMAB is well-separated from that of the natural glycopeptide. Importantly, it also does not interfere with the far-less abundant IgG1-G2 glycopeptide. R* = heavy labeled arginine.

Figure 3

GlYcoLISA anti-S IgG1 concentrations showed a high correlation with anti-S IgG Luminex levels. Only samples yielding a value in both methods were included (Figure S1). Spearman rank correlation r = 0.83 (p < 0.001). n = 844. BAU = Binding Antibody Units.

Figure 4

Glycosylation profiles of anti-S IgG1 from replicate measurements of the pooled sample in several batches of the EMC hospital samples (N = 19). The low variability and the good fit with expectation values underline that SILuMAB addition does not disturb glycosylation measurements. Shown are mean and standard deviation. Cartoons show tentative glycan structures of selected, abundant glycoform.