Two-dimensional electrophoresis for comparative proteomic analysis of human bile.

BACKGROUND: Proteomic analysis of bile fluid holds promise as a method to identify biomarkers of bile tract diseases, especially for tumors. Two-dimensional electrophoresis (2-DE) is a popular and proven separation technique for proteome analysis, but using this strategy for bile fluid analysis is still not fully developed. This study was undertaken to (a) establish a reliable method for general clean-up to make bile fluid samples suitable for 2-DE; (b) obtain 2-D biliary maps with high reproducibility and resolution; and (c) identify protein patterns present in 2-D biliary maps for potential tumor biomarker discovery, with the intention of distinguishing malignant from benign causes of bile duct obstruction. METHODS: Bile fluid samples were obtained from two patients suffering from malignant and benign bile tract obstruction (one patient with cholangiocarcinoma as the experimental case, the other with cholelithiasis as control). A variety of sample preparation options, including delipidation, desalination and nucleic acid removal, were adopted to remove contaminants that affect 2-DE results. After that, each 350 microg purified sample was loaded onto nonlinear IPG strips (18 cm, pH 3-10 and pH 4-7) for first-dimension isoelectric focusing, and 12.5% SDS-PAGE electrophoresis for second dimension separation. Then 2-D maps were visualized after silver staining and analyzed with the Image Master 2-D software. RESULTS: A large number of protein spots were separated in 2-D maps from the experimental and control groups, with means of 250 and 216 spots on pH 3-10 IPG strips, and 182 and 176 spots on pH 4-7 strips, respectively. Approximately 16 and 23 spots were differentially expressed in matched pairs from the experimental and control cases using pH 3-10 and pH 4-7 strips. CONCLUSIONS: This study established a reliable sample preparation process suitable for 2-DE of bile fluid. By this method, 2-D biliary maps with high reproducibility and resolution were obtained. The differentially displayed proteomes in the 2-D biliary maps from the experimental and control groups indicated the potential application for bile fluid analysis to identify disease-associated biomarkers, especially for biliary tract tumors.