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.