Comprehensive evaluation of cleavable bioorthogonal probes for site-specific O-GlcNAc proteomics

ABSTRACT: O-linked β-N-acetylglucosamine (O-GlcNAc) modification (i.e., O-GlcNAcylation) on proteins is an essential modification in physiology and pathology. Although O-GlcNAcylation is functionally critical, its analysis has never been an easy task. Despite the existence of a number of methods developed in the past years, which one(s) might have the best performance is largely unclear. To that end, we proposed to conduct rigorous comparison of several cleavable bioorthogonal biotin-alkyne probes which showed promise for sensitive O-GlcNAc proteomics. In brief, we developed chemoenzymatic labeling/click chemistry-based analytical workflows for O-GlcNAc proteomics, by utilizing four cleavable bioorthogonal probes including photocleavabe-biotin-alkyne (PC-biotin-alkyne), dialkoxydiphenylsilane-biotin-alkyne (DADPS-biotin-alkyne); 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl-biotin-alkyne (Dde-biotin-alkyne), and diazobenzene-biotin-alkyne (Diazo-biotin-alkyne). The analytical performance of the probes was evaluated with synthetic O-GlcNAc peptides and then benchmarked by mouse brain lysates for O-GlcNAc proteomics. Besides providing valuable technical insights to O-GlcNAc proteomics methods, our work yielded an unprecedented O-GlcNAc proteome depth of mouse brain. In total, 2,906 O-GlcNAc sites were unambiguously assigned on 878 proteins. Among them, 1,611 sites were newly identified, including 138 O-GlcNAcylated tyrosine residues. Our work will not only help guide selection/development of O-GlcNAc proteomics methods for future studies but also provide an invaluable resource for functional elucidation of protein O-GlcNAcylation in brain biology as well as critical insights into tyrosine O-GlcNAcylation.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain

SUBMITTER: Chunyan Hou  

LAB HEAD: Junfeng Ma

PROVIDER: PXD063995 | Pride | 2025-10-20

REPOSITORIES: Pride