Enzymatic Deacetylation as a Selective Strategy for <i>O</i> -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.