One-Step Chemoenzymatic Labeling and Oxime-Reversible Enrichment for O-GlcNAcylation Profiling under Oxidative Stress

Chemoenzymatic labeling strategies, relying on azide-modified sugar donors and subsequent Cu-based click chemistry to introduce enrichable handles, have greatly advanced the study of O-GlcNAcylation. However, multistep labeling and enrichment procedures often lead to substantial sample loss. While a recent one-step labeling approach using biotinylated UDP-GalNAc donors simplifies the procedures, the strong biotin–avidin interactions, as well as bulky tags, limited this strategy to protein-level identification rather than site-specific glycopeptide mapping. Herein, we developed an o xime-based r eversible O-GlcNAc enrichment ( ORO-GlcNAc ) approach employing a ketone-functionalized UDP-GalNAc analogue (UDP-GalNLeV) for one-step chemoenzymatic labeling with >96% efficiency, eliminating intermediate reactions and minimizing handling-induced loss. Crucially, oxime-based capture using hydroxylamine-functionalized beads, followed by reversible methoxyamine-mediated release, overcomes the limitations of conventional ketone-based enrichment strategies and eliminates the need for azide-based chemistry. Using the ORO-GlcNAc strategy, we identified 4129 potential O-GlcNAcylation sites in HeLa cells, of which 64.3% were annotated in the O-GlcNAc Atlas, demonstrating the high coverage and enrichment performance of ORO-GlcNAc. Functional studies suggested a potential role of O-GlcNAcylation in the nucleocytoplasmic redistribution of the chromatin remodeler EP400 during stress-granule assembly. This platform thus provides a powerful and versatile tool for advancing the O-GlcNAcylation research.