Rapid and large-scale glycopeptides enrichment strategy based on chemical ligation

Abstract Protein glycosylation, the most universal post-translational modification, has been proposed to play a crucial role in regulating multiple essential cellular processes. However, the low abundance of glycoproteins and the heterogeneity of glycans complicate their comprehensive analysis. Here, we develop a rapid and large-scale glycopeptides enrichment strategy via bioorthogonal ligation and trypsin cleavage. The enrichment process is performed in one tube to minimize sample loss and time costs. This method combines convenience and practicality, identifying over 900 O-GlcNAc sites from 500 µg of sample. Surprisingly, it allows simultaneous identification of N-glycosites, O-GlcNAc sites, O-GalNAc sites, and N-glycans by a two-step enzymatic release strategy. Combined with quantitative analysis, it reveals the distinct O-GlcNAcylation patterns in different compartments during oxidative stress. In summary, our study offers a convenient and robust tool for glycoproteome and glycome profiling, facilitating in-depth analysis to elucidate the biological functions of glycosylation.