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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

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Project description:Over the past decades, protein O-GlcNAcylation has been found to play a fundamental role in cell cycle control, metabolism, transcriptional regulation, and cellular signaling. Nevertheless, quantitative approaches to determine in vivo GlcNAc dynamics at a large-scale are still not readily available. Here, we have developed an approach to isotopically label O-GlcNAc modifications on proteins by producing 13C-labeled UDP-GlcNAc from 13C6-glucose via the hexosamine biosynthetic pathway. This metabolic labeling was combined with quantitative mass spectrometry-based proteomics to determine site-specific protein O-GlcNAcylation turnover rates. First, an efficient enrichment method for O-GlcNAc peptides was developed with the use of phenylboronic acid solid-phase extraction and anhydrous DMSO. The near stoichiometry reaction between the diol of GlcNAc and boronic acid dramatically improved the enrichment efficiency. Additionally, our kinetic model for turnover rates integrates both metabolomic and proteomic data, which increase the accuracy of the turnover rate estimation. Other advantages of this metabolic labeling method include in vivo application, direct labeling of the O-GlcNAc sites and higher confidence for site identification. Concentrating only on nuclear localized GlcNAc modified proteins, we are able to identify 159 O-GlcNAc sites on 74 proteins and determine turnover rates of 24 O-GlcNAc peptides from 21 proteins extracted from HeLa nuclei. In general, we found O-GlcNAcylation turnover rates are slower than those published for phosphorylation or acetylation. Nevertheless, the rates widely varied depending on both the protein and the residue modified. We believe this methodology can be broadly applied to reveal turnovers/dynamics of protein O-GlcNAcylation from different biological states and will provide more information on the significance of site-specific O-GlcNAcylation, enabling us to study the temporal dynamics of this critical modification in a site-specific manner for the first time.

Dataset Information

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

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