Quantitative Targeted Analysis of Antibody Fc Glycosylation by Glyco-MRM

The N-glycoforms of Fc domain critically regulate binding affinity of IgG1 to Fcγ receptor IIIA (FcγRIIIa), Fcγ receptor IIb (FcγRIIb), and complement components. Quantifying antigen-specific IgG1 glycopeptides may provide precise insights into the pathogenesis of severe viral infections and autoantibody-mediated diseases. Here, we developed a liquid chromatography (LC) coupled with mass spectrometry (MS) by the multiple reaction monitoring (MRM) method to analyze IgG1 glycosylation profiles. Calibration curves were generated for six glycopeptides with integrated isotope-labeled internal standards, yielding lower limits of quantification (LLOQ) of G2 (200 pg/mL, 70.92 pM), G0F (500 pg/mL, 189.39 pM), G0NF (40 pg/mL, 14.05 pM), G2S (2.5 ng/mL, 802.87 pM), G1 (500 pg/mL, 187.98 pM), and G1N (100 pg/mL, 34.93 pM). For absolute IgG1 quantification, the LLOQ was determined as 1.26 μg/mL (8.43 nM). Application of calibration curve-based assays to influenza and COVID-19 infected individuals (within 3 months after infection) revealed distinct glycosylation profiles: influenza infected individuals exhibited significantly reduced core-fucosylation (28%), while both disease groups showed elevated galactosylation levels. This methodology provides a platform for laboratory-developed tests to track glycosylation alterations using widely accessible liquid chromatography-mass spectrometry (LC-MS) equipment.