Hepatocellular carcinoma (HCC) is one of the most common fatal cancers worldwide. The most clearly established risk factor for HCC is chronic infection with hepatitis B or C virus (HBV or HCV). The characteristic of HCC in Japan is that they originate from the liver tissues of hepatitis HCV or liver cirrhosis. Therefore, it is important to elucidate the factors related to the products from tumor tissues of HCV-infected patients, and to identify some proteins with which we can speculate the mechanism of carcinogenesis of HCC. Liver tissue samples of HCC cancerous tissues or non-cancerous tissues from patients infected with HCV were prepared and separated by means of two-dimensional gel electrophoresis. From proteomic differential display analysis, 11 spots whose expression in cancerous tissues increased, and 11 spots whose expression in tumor tissues decreased, were detected. Eight proteins out of 11 decreasing spots were identified as liver type aldolase, tropomyosin b-chain, ketohexokinase, enoyl-CoA hydratase, albumin, smoothelin, ferritin light chain, and arginase 1. Nine proteins out of 11 increasing spots were identified as GRP78, HSC70, GRP75, HSP70.1, HSP60, ATP synthase b-chain, glutamine synthetase(GS), phosphoglycerate mutase 1, and triosephosphate isomerase. The three protein spots of the same molecular weight (42kDa), whose expression increased in well-differentiated cancerous tissues, were identified as GS. The tryptic peptides of the most acidic GS isoform lost the signal of 899.5 Da corresponding a peptide of SASIRIPR and gained the signal of 1059.5 Da which was submitted to post source decay (PSD) analysis. The PSD analysis suggested the neutral loss by elimination of two phosphate groups which were supposed to be on serine residues of 899.5 Da peptide from serine 320 to arginine 327 in GS. The PMF followed by PSD analysis is thought to be useful for the determination of phosphorylation site of proteins showing molecular heterogeneity.
