biostudies-literatureUnknown24(10)Hou CO-linked β-N-acetylglucosamine (O-GlcNAc) modification (i.e., O-GlcNAcylation) on proteins is an essential modification in physiology and pathology. Although O-GlcNAcylation is functionally critical, its analysis has been challenging. Despite the existence of a number of methods developed in the past years, which one(s) might have the best performance is largely unclear. To that end, we conducted a rigorous comparison of several cleavable bioorthogonal biotin-alkyne probes which showed promise for sensitive O-GlcNAc proteomics. In brief, we developed chemoenzymatic labeling/click chemistry-based analytical workflows for O-GlcNAc proteomics by utilizing four cleavable bioorthogonal probes, including photocleavabe-biotin-alkyne (PC-biotin-alkyne), dialkoxydiphenylsilane-biotin-alkyne (DADPS-Molecular & cellular proteomics : MCP101064https://www.ebi.ac.uk/biostudies/studies/S-EPMC12506516biostudies-literatureComprehensive Evaluation of Cleavable Bioorthogonal Probes for Site-Specific O-GlcNAc Proteomics.PMC12506516Pak DTSZhang HHart GWDeng JByers SPei HMa JHou CWang XMoremen KWLevi MfalseComprehensive Evaluation of Cleavable Bioorthogonal Probes for Site-Specific O-GlcNAc Proteomics.O-linked β-N-acetylglucosamine (O-GlcNAc) modification (i.e., O-GlcNAcylation) on proteins is an essential modification in physiology and pathology. Although O-GlcNAcylation is functionally critical, its analysis has been challenging. Despite the existence of a number of methods developed in the past years, which one(s) might have the best performance is largely unclear. To that end, we conducted a rigorous comparison of several cleavable bioorthogonal biotin-alkyne probes which showed promise for sensitive O-GlcNAc proteomics. In brief, we developed chemoenzymatic labeling/click chemistry-based analytical workflows for O-GlcNAc proteomics by utilizing four cleavable bioorthogonal probes, including photocleavabe-biotin-alkyne (PC-biotin-alkyne), dialkoxydiphenylsilane-biotin-alkyne (DADPS-2025-01-01T00:00:00Z2025 Aug2026-06-04T09:01:06.437Z2026-06-01T03:06:31.032ZS-EPMC125065164088548210.1016/j.mcpro.2025.101064