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One-Step Chemoenzymatic Labeling and Oxime-Reversible Enrichment for O-GlcNAcylation Profiling under Oxidative Stress
Li Liu1,2, Zhuo Zhang3,4, Xuyang Yue1,2
1State Key Laboratory of Medical Proteomics, National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
Analytical Chemistry
|June 30, 2026
Summary
A new oxime-based reversible O-GlcNAc (ORO-GlcNAc) enrichment method enables efficient, one-step chemoenzymatic labeling for O-GlcNAcylation studies. This approach minimizes sample loss and advances site-specific glycopeptide mapping.
Area of Science:
- Biochemistry
- Glycobiology
- Chemical Biology
Background:
- Chemoenzymatic labeling using azide-modified sugars and click chemistry advances O-GlcNAcylation research.
- Multistep procedures cause sample loss, and existing one-step methods have limitations for site-specific mapping.
Purpose of the Study:
- To develop a novel, efficient, one-step chemoenzymatic labeling and enrichment strategy for O-GlcNAcylation analysis.
- To overcome limitations of existing methods, enabling site-specific glycopeptide mapping with reduced sample loss.
Main Methods:
- Developed an oxime-based reversible O-GlcNAc (ORO-GlcNAc) enrichment approach.
- Utilized a ketone-functionalized UDP-GalNAc analogue (UDP-GalNLeV) for one-step labeling (>96% efficiency).
- Employed oxime-based capture and reversible methoxyamine-mediated release for enrichment.
Main Results:
- The ORO-GlcNAc strategy achieved efficient one-step labeling, minimizing handling-induced loss.
- Identified 4129 potential O-GlcNAcylation sites in HeLa cells, with 64.3% matching the O-GlcNAc Atlas.
- Demonstrated high coverage and enrichment performance, surpassing conventional methods.
Conclusions:
- The ORO-GlcNAc platform provides a powerful and versatile tool for O-GlcNAcylation research.
- The method facilitates site-specific glycopeptide mapping and functional studies.
- Potential role of O-GlcNAcylation in EP400 redistribution during stress-granule assembly was suggested.
