Click‐iG: Simultaneous Enrichment and Profiling of Intact N‐linked, O‐GalNAc, and O‐GlcNAcylated Glycopeptides

Abstract Proteins are ubiquitously modified with glycans of varied chemical structures through distinct glycosidic linkages, making the landscape protein glycosylation challenging to map. Profiling intact glycopeptides mass spectrometry (MS) has recently emerged as a powerful tool for revealing matched information sites and attached (i.e., glycosites), but is largely limited individual types. Herein, we describe Click‐iG, which integrates metabolic labeling clickable unnatural sugars, an optimized MS method, tailored version pGlyco3 software enable simultaneous enrichment profiling three types glycopeptides: N‐linked, mucin‐type O‐linked, O‐GlcNAcylated glycopeptides. We demonstrate utility Click‐iG by identification thousands glycosites in cell lines living mice. From mouse lung, heart, spleen, total 2053 N‐glycosites, 262 O‐GalNAc glycosites, 1947 O‐GlcNAcylation were identified. Click‐iG‐enabled comprehensive coverage lays foundation interrogating crosstalk between different pathways.