Proteomic analyses reveal divergent ubiquitylation patterns in hepatocellula carcinoma cell lines with different metastasis potential.

Abstract Hepatocellular carcinoma (HCC) is one of the most common malignant tumours, metastasis and recurrence remain the primary reasons for poor prognosis. Ubiquitination serves as a degradation mechanism of proteins, but it is involved in additional cellular processes including metastasis. Here, by using label-free quantification, double-glycine (di-Gly) antibody affinity purification and high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS), we investigated quantitative proteome, ubiquitylome, and the crosstalk between the two datasets in HCC cell lines with different metastasis potential to identify biomarkers associated with HCC metastasis. In total, 83 ubiquitinated proteins significantly and steadily changed their abundance according to their metastatic potential, and the participated biological processes of these ubiquitinated proteins were tightly associated with tumour metastasis. Further signaling pathway analysis revealed that the ribosome and proteasome were significantly over-activated in the highly metastatic cells. Furthermore, we analyzed the crosstalk between the whole proteome and the ubiquitylome, and further discussed the mechanism that how ubiquitination events affect HCC metastasis. Eventually, the ubiquitination of Ku80 was validated to be significantly down-regulated in the high-metastatic cells comparing with the low-metastatic cells. We believe that these findings will help us better understand the underlying molecular mechanisms of the metastasis of HCC. Significance In this manuscript, we used label free based proteomics combined with diglycine antibody (di-Gly) affinity purification approach to identify biomarkers associated with HCC recurrence/metastasis in in a serial HCC cell lines with increasing invasion and metastasis potential. And then, we analyzed the crosstalk between the whole proteome and the ubiquitylome. Eventually, the ubiquitination of Ku80 was confirm to be closely associated with invasion and migration of HCC cells. As far as we know, this is the first time to use quantitative proteomic approach to study the ubiquitylomics in HCC cell lines with increasing metastasis ability.