Proteome analysis of human liver tumor tissue by two‐dimensional gel electrophoresis and matrixassisted laser desorption/ionization‐mass spectrometry for identification of disease‐related proteins

Abstract

Hepatocellular carcinoma (HCC) is a common malignancy worldwide and is a leading cause of death. To contribute to the development and improvement of molecular markers for diagnostics and prognostics and of therapeutic targets for the disease, we have largely expanded the currently available human liver tissue maps and studied the differential expression of proteins in normal and cancer tissues. Reference two-dimensional electrophoresis (2-DE) maps of human liver tumor tissue include labeled 2-DE images for total homogenate and soluble fraction separated on pH 3-10 gels, and also images for soluble fraction separated on pH 4-7 and pH 6-9 gels for a more detailed map. Proteins were separated in the first dimension by isoelectric focusing on immobilized pH gradient (IPG) strips, and by 7.5-17.5% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels in the second dimension. Protein identification was done by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-time of flight-mass spectrometry (DE-MALDI-TOF-MS). In total, 212 protein spots (117 spots in pH 4-7 map and 95 spots in pH 6-9) corresponding to 127 different polypeptide chains were identified. In the next step, we analyzed the differential protein expression of liver tumor samples, to find out candidates for liver cancer-associated proteins. Matched pairs of tissues from 11 liver cancer patients were analyzed for their 2-DE profiles. Protein expression was comparatively analyzed by use of image analysis software. Proteins whose expression levels were different by more than three-fold in at least 30% (four) of the patients were further analyzed. Numbers of protein spots overexpressed or underexpressed in tumor tissues as compared with nontumorous regions were 9 and 28, respectively. Among these 37 spots, 1 overexpressed and 15 underexpressed spots, corresponding to 11 proteins, were identified. The physiological significance of the differential expressions is discussed.

Enhanced nuclear factor-kappa B-associated Wnt-1 expression in hepatitis B- and C-related hepatocarcinogenesis: identification by functional proteomics

“…These data suggest that overexpression of NF-jB associated Wnt-1 protein might be an important mechanism of hepatocarcinogenesis common to HBV and HCV infection. Several studies have reported proteomic analysis of HCC, either from tumor tissues [29,32,33], or from patients' sera [34]. All these studies attempted to identify specific factors correlated with hepatocarcinogenesis or novel tumor markers for early diagnosis of HCC, but these goals seem far from reach [35,36].…”

“…Tryptic peptides from 2-DE protein spots were subjected to peptide mass fingerprinting (PMF) using a matrix-assisted laser desorption/ionization quadrupole-time of flight (MALDI-Q-TOF) mass spectrometer (M@LDI TM ; Micromass, Manchester, UK) operated in reflectron positive ion mode as described [27][28][29]. Briefly, samples were spotted onto the 96-well format MALDI target plate using a saturated matrix solution of a-cyano-4-hydroxycinnamic acid (CHCA) in 60% ACN/1% TFA.…”

A possible predictive marker of progression for hepatocellular carcinoma

“…Consequently, molecular biomarkers associated with HCC should be identified. Different proteomics have been applied to the study of HCC, resulting in panels of altered proteins that are proposed as markers with key roles in the progression of the disease in certain cases (28)(29)(30)(31)(32)(33)(34)(35). However, the protein repertoires reported in these studies are barely coincident and, consequently, their value as biomarkers for HCC is limited.…”

Serum biomarker screening for the diagnosis of early gastric cancer using SELDI-TOF-MS

“…Over the years, SELDI-TOF-MS has been successfully applied in many types of tumors. A number of biomarkers in this process have been identified and further characterized, associated with cancers such as breast (16)(17)(18), liver (19)(20)(21), lung (22,23), prostate (24) and ovarian cancers (25). These studies suggest that SELDI-TOF-MS ProteinChip technology can distinguish cancer patients from normal subjects with relatively high sensitivity and specificity.…”