Surface glycoproteomic analysis of hepatocellular carcinoma cells by affinity enrichment and mass spectrometric identification.

Cell surface glycoproteins are one of the most frequently observed phenomena correlated with malignant growth. Hepatocellular carcinoma (HCC) is one of the most malignant tumors in the world. The majority of hepatocellular carcinoma cell surface proteins are modified by glycosylation in the process of tumor invasion and metastasis. Therefore, characterization of cell surface glycoproteins can provide important information for diagnosis and treatment of liver cancer, and also represent a promising source of potential diagnostic biomarkers and therapeutic targets for hepatocellular carcinoma. However, cell surface glycoproteins of HCC have been seldom identified by proteomics approaches because of their hydrophobic nature, poor solubility, and low abundance. The recently developed cell surface-capturing (CSC) technique was an approach specifically targeted at membrane glycoproteins involving the affinity capture of membrane glycoproteins using glycan biotinylation labeling on intact cell surfaces. To characterize the cell surface glycoproteome and probe the mechanism of tumor invasion and metastasis of HCC, we have modified and evaluated the cell surface-capturing strategy, and applied it for surface glycoproteomic analysis of hepatocellular carcinoma cells. In total, 119 glycosylation sites on 116 unique glycopeptides were identified, corresponding to 79 different protein species. Of these, 65 (54.6%) new predicted glycosylation sites were identified that had not previously been determined experimentally. Among the identified glycoproteins, 82% were classified as membrane proteins by a database search, 68% had transmembrane domains (TMDs), and 24% were predicted to contain 2-13 TMDs. Moreover, a total of 26 CD antigens with 50 glycopeptides were detected in the membrane glycoproteins of hepatocellular carcinoma cells, comprising 43% of the total glycopeptides identified. Many of these identified glycoproteins are associated with cancer such as CD44, CD147 and EGFR. This is a systematic characterization of cell surface glycoproteins of HCC. The membrane glycoproteins identified in this study provide very useful information for probing the mechanism of liver cancer invasion and metastasis.