One-Step Chemoenzymatic Labeling and Oxime-Reversible Enrichment for O-GlcNAc Profiling in Oxidative Stress

ABSTRACT: Chemoenzymatic labeling has greatly advanced O-GlcNAc proteomics, yet existing strategies using azide-modified donors (e.g., UDP-GalNAz) require multi-step click-based workflows that introduce substantial sample loss and limit sensitivity toward low-abundance targets. While recent one-step labeling with biotinylated UDP-GalNAc donors streamlines workflows, the irreversible avidin–biotin interaction prevents glycopeptide recovery and restricts site-specific analysis. To address these challenges, we developed a ketone-reversible O-GlcNAc enrichment (KRO-GlcNAc) strategy employing a ketone-functionalized UDP-GalNAc analog (UDP-GalNLev) for one-step enzymatic tagging. GalT1(Y289L) transfers the GalNLev moiety with >96% efficiency, eliminating intermediate reactions and minimizing handling-induced loss. Crucially, reversible oxime-based capture on hydroxylamine-functionalized beads followed by mild methoxyamine release not only overcomes the irreversible nature of reported ketone-hydroxylamine-biotin enrichment, but fundamentally address the broader limitations that originally necessitated the transition from ketone-based labeling to azide chemistry. Application of KRO-GlcNAc to HeLa cells identified 4,129 potential O-GlcNAcylation sites across 740 proteins, with ~30% being novel. Functional studies revealed that O-GlcNAcylation regulates nuclear-cytoplasmic shuttling of chromatin remodeler EP400, linking this modification to stress granule assembly. This platform thus provides a powerful and versatile tool for advancing O-GlcNAcylation research.

ORGANISM(S): Homo Sapiens

SUBMITTER: Mingliang Ye  

PROVIDER: PXD072723 | iProX | Tue Jan 06 00:00:00 GMT 2026

REPOSITORIES: iProX