Site-Specific and Quantitative O‑GlcNAc Proteomics for Hepatocellular Carcinoma

O-linked β-N-acetylglucosamine
(O-GlcNAc)
modification (O-GlcNAcylation) underlies the pathogenesis of multiple
cancers, including hepatocellular carcinoma (HCC). However, comprehensive
and quantitative characterization of site-specific O-GlcNAcylation
at the proteome scale remains technically challenging. Here, we employed
an integrated workflow for the quantitative O-GlcNAc proteomics of
HCC and controls. Proteins from liver samples were subjected to chemoenzymatic
labeling, photocleavable alkyne-biotin-based enrichment, proteolytic
digestion, and isotopic labeling with tandem mass tags. The O-GlcNAc
peptides were analyzed by a nanoUPLC-MS/MS system in HCD product-dependent
EThcD (HCD-pd-EThcD) mode for site mapping and quantification. A total
of 440 O-GlcNAc peptides, representing 305 sites on 196 proteins,
were confidently identified. Differential analysis revealed 190 O-GlcNAc
peptides from 121 proteins significantly upregulated in HCC after
normalization to their corresponding protein abundance. Functional
enrichment and protein–protein interaction analyses indicate
that proteins with increased levels of O-GlcNAcylation are involved
in nuclear transport, transcriptional regulation, and ATP-dependent
chromatin remodeling. Our work provides quantitative proteomic insights
into O-GlcNAcylation in HCC, revealing global upregulation and functional
clustering of O-GlcNAc-modified proteins. These findings will help
elucidate the functional roles of O-GlcNAcylation in liver cancer,
facilitating the development of novel therapeutics and sensitive biomarkers.