Comprehensive Evaluation of Cleavable Bioorthogonal Probes for Site-Specific O-GlcNAc Proteomics

Area of Science:

• Biochemistry
• Proteomics
• Chemical Biology

Background:

• O-linked β-N-acetylglucosamine (O-GlcNAc) modification is crucial in cellular processes but challenging to analyze.
• Existing methods for O-GlcNAc analysis lack clear performance benchmarks, hindering progress in the field.
• Sensitive and reliable O-GlcNAc proteomics is essential for understanding its physiological and pathological roles.

Purpose of the Study:

• To rigorously compare the performance of four cleavable bioorthogonal biotin-alkyne probes for O-GlcNAc proteomics.
• To develop and validate chemoenzymatic labeling and click chemistry workflows for sensitive O-GlcNAc analysis.
• To identify the most effective probes and methods for deep O-GlcNAc proteome profiling.

Main Methods:

• Development of chemoenzymatic labeling and click chemistry-based analytical workflows.
• Utilization of four distinct cleavable bioorthogonal probes: photocleavable-biotin-alkyne (PC-biotin-alkyne), dialkoxydiphenylsilane-biotin-alkyne (DADPS-biotin-alkyne), Dde-biotin-alkyne, and diazobenzene-biotin-alkyne.
• Evaluation of probe performance using synthetic O-GlcNAc peptides and benchmarking with mouse brain lysates.

Main Results:

• Successful application of the developed workflows for sensitive O-GlcNAc proteomics, achieving unprecedented depth.
• Unambiguous assignment of 2,906 O-GlcNAc sites on 878 proteins in mouse brain lysates.
• Identification of 1,611 novel O-GlcNAc sites, including 138 on tyrosine residues, expanding the known O-GlcNAcylome.

Conclusions:

• The study provides critical technical insights to guide the selection and development of O-GlcNAc proteomics methods.
• The identified probes and workflows enable deep profiling of the O-GlcNAc proteome, particularly in brain tissue.
• The findings offer a valuable resource for understanding protein O-GlcNAcylation in brain biology and highlight the significance of tyrosine O-GlcNAcylation.