Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Along with a rising number of diagnostic biomarkers were discovered, more and more patients can be diagnosed early. Additionally, patients with early-stage HCC are able to be treated curatively. Unfortunately, 40-70% HCC patients still suffer from recurrence within five years. Accordingly, various reports tried to develop prognosis signature in HCC to find potential prognostic predictors. However, most of the previous studies focused on mRNA, miRNA, and proteins. Phosphorylation catalyzed different vital signaling cascades which may contribute to poor prognosis of HCC patients. Therefore, phosphorylation-related events have generally been considered biomarkers and/or viable therapeutic targets for HCC. In the present study, we aim to discover potential phosphorylation-related biomarkers for predicting prognosis and/or potential therapeutic targets for developing new treatment option for HCC patients.
First, we established a pre-fractionation workflow using strong anion-exchange chromatography under continuous pH gradient, which can efficiently increase the number of identified proteins and phosphopeptides. Next, we applied this pre-fraction workflow and iTRAQ-based labeling strategy to quantitatively analyze proteome and phosphoproteome of paired tissue samples (cancerous and adjacent non-cancerous tissues) from HCC patients with good (no recurrence in 3 years after operation) or poor prognosis (recurrence in one year after operation). In the four pooled tissue samples, we obtained relatively quantitative results of 20,693 phosphopeptides and 9,311 proteins. We found that phosphoprotein signature outperformed protein profile to distinguish poor prognosis from good prognosis group. Moreover, we introduced super-SILAC mix as internal standard into the pooled tissue samples from discovery phase to preliminarily verify dozens of prognosis-related proteins/phosphoproteins by PRM assay. We’ll further validate the significant candidates in hundreds of HCC tissue specimens to determine practical prognostic predictors and investigate the feasibility of treatment targets.