Abstract

Cell surface glycoproteins are vital mediators of intercellular communication and primary targets for therapeutic intervention. However, the comprehensive profiling of the “surface glycome” on living cells remains hindered by the low abundance of these proteins and the inherent complexity of glycosylation. Here, we present GlycoCSP, a site-specific glycoproteomics strategy that utilizes alkyne-functionalized DNA scaffolds to achieve high-resolution GlycoBarcoding of cell surface proteins. By leveraging the spatial reach of extended DNA chains and a high-density alkyne array, GlycoCSP ensures specific labeling and robust covalent capture of surface glycoproteins. This platform integrates protein-level enrichment with an orthogonal tandem release proteolysis, enabling the precise mapping of N-glycosylation signatures at the site level. Applying GlycoCSP to live cells, we identified 2,016 extracellular N-glycosylation sites across 1,420 proteins, validated by deamidation mass shifts and the canonical N-X-S/T/C motif. Comparative analysis across breast cancer cell lines revealed that site-specific glycosylation occupancy provides a distinct layer of surfaceome heterogeneity that is independent of protein abundance. By providing a mass spectrometry-readable framework for decoding the surface glycode, GlycoCSP enables the unbiased discovery of glycosylation-dependent biomarkers and therapeutic targets previously inaccessible to conventional proteomics.