Chemically Cleavable Enrichment Enables Site-Specific Profiling of Cell-Surface N-Glycan Microheterogeneity Dynamics.

Cell-surface glycans exhibit extensive microheterogeneity, yet their site-specific dynamic behavior remains largely uncharacterized due to the lack of effective analytical methods. Here, we report a chemical enrichment strategy for profiling the temporal dynamics of intact cell-surface sialylated N-glycopeptides at site-specific and single-glycoform resolution. This method integrates metabolic labeling of sialylated glycans with azide reporters, copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) using a photocleavable biotin probe for glycan labeling in living cells, and affinity enrichment of intact glycopeptides. Coupling this strategy with pulse-chase analysis enables quantitative tracking of glycan turnover across multiple structural levels, including glycoproteins, glycosylation sites, glycans, and site-specific glycoforms. The results reveal distinct turnover kinetics among glycoforms at the same site and show that glycan-level dynamics can differ substantially from protein-level stability. This work provides a robust analytical platform for investigating cell-surface glycan dynamics with high structural resolution.