Quantitative Glycan-Protein Cross-Linking Mass Spectrometry Using Enrichable Linkers Reveals Extensive Glycan-Mediated Protein Interaction Networks

Protein–protein interactions in the cell membrane are typically mediated by glycans, with terminal sialic acid often involved in these interactions. To probe the nature of the interactions, we developed quantitative cross-linking methods involving the glycans of the glycoproteins and the polypeptide moieties of proteins. We designed and synthesized biotinylated enrichable cross-linkers that were click-tagged to metabolically incorporate azido-sialic acid on cell surface glycans to allow cross-linking of the azido-glycans with lysine residues on proximal polypeptides. The glycopeptide–peptide cross-links (GPx) were enriched using biotin groups through affinity purification with streptavidin resin beads. Workflows using two linkers, one photocleavable and the other disulfide, were developed and applied to reveal the sialic acid-mediated cell-surface protein networks of PNT2 (prostate) cells. Glycopeptide–peptide pairs were identified, with 12000 GPx linked by sialylated glycoforms revealing interactions between source glycoproteins and nearly 700 target proteins. Protein–protein interactions were characterized by as many as 40 peptide pairs, and the extent of the interactions between proteins was prioritized by the number of GPx. Quantitation was performed by counting the number of GPx that identify the protein pairs. Abundant membrane proteins such as ITGB1 yielded an interactome consisting of around 400 other proteins, which were ranked from the most extensive interaction, having the largest number of GPx, to at least one. The interactome was further confirmed separately by published databases. This tool will enhance our understanding of glycosylation on protein–protein interactions and provide new potential targets for therapeutics.