Enzymatic Deacetylation as a Selective Strategy for O-GlcNAc Identification

The unambiguous identification of protein O-GlcNAcylation remains challenging because O-GlcNAc and O-GalNAc are isobaric and produce nearly indistinguishable fragmentation patterns under standard higher-energy collisional dissociation (HCD) conditions. Here, we report a chemoenzymatic strategy that enables selective detection of O-GlcNAc through enzymatic deacetylation. We show that the Cyclobacterium marinum deacetylase CmCBDA uniquely and efficiently converts beta-O-GlcNAc, but not alpha-O-GlcNAc, alpha-O-GalNAc, or N-glycans, into the corresponding beta-O-GlcN glycoform. This transformation introduces a non-native primary amine that fundamentally alters glycopeptide ionization and fragmentation behavior, generating characteristic diagnostic ions under HCD-MS/MS. The resulting O-GlcN modification serves as a selective chemical marker for beta-O-GlcNAc, enabling confident site localization using widely available mass spectrometers. Application of this workflow to O-GlcNAcylated model proteins and complex cell lysates demonstrates robust discrimination of O-GlcNAc sites without enrichment or specialized fragmentation methods. This deacetylation-assisted approach provides a general and practical solution to a long-standing analytical challenge in O-GlcNAc proteomics.