Hepatocellular carcinoma cell lines LC-MS/MS

Hepatocellular carcinoma (HCC) ranks sixth among the most common malignancies and fourth in mortality among all kinds of cancers in the world. Recent advances in early diagnosis and therapeutics have led to improved survival of HCC patients, but the recurrence and metastasis are still main reasons for the poor prognosis and high mortality of HCC. Many researches on the mechanism of HCC deterioration have uncovered the epithelial-mesenchymal transition (EMT) as a major trigger for HCC metastasis and invasion.In this study, in order to systematically investigate the dynamic site-specific glycosylation alterations associated with the EMT process of HCC, two hepatoma cell lines SMMC-7721 and HepG2 were treated using HGF and the cell proteins were harvested at six treatment time points. Using TMT-labeling, solid phase extraction and mass spectrometry, we not only detailly profiled N-glycoproteome of both cell lines at site-specific glycosylation level, but also dynamically quantified those intact glycopeptides among six increasing HGF-treated time points. By dynamically quantifying enriched glycopeptides and proteins among six HGF-treated time points in two cell lines, we identified 20 up-regulated intact glycopeptides during the process of EMT, with the majority changed at the glycosylation level instead of protein expression level. The site-specific glycosylation change of those glycoproteins was related to the process of EMT, which was further verified by a series of molecular biology methods, mass spectrometry, and migration and invasion assay in vitro.