Site-Specific Profiling of Monoclonal IgG N-Glycosylation Reveals Dynamic Sialylation Changes Associated with Multiple Myeloma.

The glycosylation of monoclonal immunoglobulins (M-proteins) is implicated in the pathogenesis of multiple myeloma (MM), but a comprehensive, site-specific glycoproteomic characterization has been lacking. Here, we established an integrated workflow that couples serum protein electrophoresis with ZenoTOF mass spectrometry for the in-depth quantitative profiling of intact N-glycopeptides from purified serum M-proteins. This method was applied to a cohort including healthy controls (HCs) (n = 26), patients with IgG-MGUS (monoclonal gammopathy of undetermined significance) (n = 23), and IgG-MM patients (n = 32), with an independent validation set (n = 28). Longitudinal analysis was performed on samples (IgG-MM) from newly diagnosed MM (NDMM, n = 14), very good partial response (VGPR, n = 7), and complete response (CR, n = 5) patients. We identified that sialylated glycopeptides were significantly upregulated in the MM. Notably, the site-specific glycoform IgG1H4N4F1S1 demonstrated superior diagnostic performance for distinguishing MM from HCs (AUC = 0.902). Crucially, longitudinal tracking revealed dynamic changes in IgG1H4N4F1S1 abundance, which decreased significantly as patients achieved deeper clinical responses (NDMM to CR), and its levels correlated with key clinical parameters. Our study provides a robust glycoproteomic strategy and unveils specific, dynamic glycosylation signatures on M-proteins that offer novel insights into MM biology and serve as potential biomarkers for monitoring the therapeutic efficacy.