Dataset Information

Quantitative O-GlcNAc Proteomics for Hepatocellular Carcinoma

ABSTRACT: O-linked beta-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation), a post-translational modification on proteins, underlies the pathogenesis of multiple types of cancers including hepatocellular carcinoma (HCC). Arguably, detailed molecular characterization of O-GlcNAcylated proteins holds promise to delineate the roles of O-GlcNAcylation in cancer biology. However, due to technical hurdles, site-specific characterization of O-GlcNAcylated proteins particularly at the proteome scale is lacking. In this study, we employed an integrated workflow for O-GlcNAc proteomics of HCC. In brief, proteins from liver tissue samples were subjected to GalT1(Y289L)-mediated chemoenzymatic labeling, photocleavable alkyne-biotin probe-based click chemistry, proteolytic digestion, and neutravidin chromatography followed by 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 O-GlcNAc site mapping and quantification. A total of 440 O-GlcNAc peptides, representing 305 sites on 196 proteins, were identified with high confidence. 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 network of proteins with increased O-GlcNAcylation highlighted their roles in nuclear transport, transcription regulation, ATP-dependent chromatin remodeling, among others. 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 functional roles of O-GlcNAcylation in liver cancer, facilitating the development of novel therapeutics.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Junfeng Ma