Precise mapping of O-glycosylation in the hinge region of serum IgA1 via site-specific O-glycoprotease coupled with ultra-high-resolution mass spectrometry.

Characterizing immunoglobulin A1 (IgA1) hinge-region O-glycosylation remains challenging due to its high glycosite density and extensive microheterogeneity. Here, we developed an integrated workflow combining dual-enzyme digestion-using the immunomodulating metalloprotease of Pseudomonas aeruginosa (IMPa) and trypsin-with complementary ultra-high-resolution mass spectrometry (MS) workflows utilizing higher-energy collisional dissociation (HCD) and HCD product-dependent electron-transfer/higher-energy collisional dissociation (HCD-pd-EThcD). This strategy generated short, site-specific O-glycopeptides, thereby reducing analytical complexity while preserving intact glycan structures. Using this approach, we characterized 103 O-glycopeptides and 13 O-glycan compositions, seven of which were previously unreported. Five O-glycosylation sites were confidently localized at single-residue resolution via site-determining fragment ions, while three additional sites received tentative assignments supported by IMPa cleavage specificity and complementary glycopeptide evidence. Glycan structural isomers and amino acid positional isomers were distinguished through characteristic chromatographic and fragmentation features, and a reproducible proline-directed fragmentation pattern was observed, providing complementary support for peptide and glycopeptide characterization. Collectively, these findings provide a robust analytical framework for future structure-informed biomarker studies in IgA-related diseases.