Intact glycopeptide-based N-glycoproteomic analysis reveals disease signatures of altered glycoforms and site-specific glycan modifications in hepatocellular carcinoma tissues

Abnormal glycosylation has been regarded as a hallmark of cancer. Although glycoproteomic analysis on hepatocellular carcinoma (HCC) have been performed using patient serum, tissues and cell lines, systematic studies of the global changes in the N-glycosylated proteins, especially their site-specific glycan alterations in HCC tissues remain limited. In this study, we carried out an intact glycopeptide-based quantitative N-glycoproteomic study using tumor and non-tumor liver tissues of HCC patients via a liquid chromatography mass spectrometry approach. In total, we identified 5375 intact N-glycopeptides, corresponding to 5118 N-glycoforms with 258 distinct N-glycan compositions at 985 N-glycosites in 575 N-glycoproteins. Our data provided previously unknown disease signatures of altered glycoforms and site-specific glycan modifications in HCC tumor tissue, including increased sialylation, paucimannosylation, tri-antennary, tetra-antennary and highly branched or elongated glycans and reduced mono-antennary or hybrid glycans. Importantly, the overall glycosylation was revealed to be significantly reduced in HCC tumor tissues and we uncovered previously unknown glycan features of key subunits of oligosaccharyltransferase (OST), such as STT3A, STT3B and RPN1 in HCC. Furthermore, our data showed the prominent dysregulated glycan modification-affected processes and pathways, including ECM receptor interaction, focal adhesion, PI3K-Akt signaling pathway, and lysosome, which have been known to play important roles in cancer pathophysiology. Overall, our study provided a proteome-wide landscape of N-glycoproteome alterations in HCC, including the overall glycan features, site-specific glycan signatures and various pathways affected by glycosylation. Importantly, the identified disease-associated key glycoforms and pathways provide attractive targets for HCC biomarker and therapeutic target discovery.