Serum Proteome Analysis Of Primary Hepatocellular Carcinoma By Two-dimensional Gel Electrophoresis And Matrix-assisted Laser Desorption-Ionization Time Of Flight Mass Spectrometry

Analyzing the serum proteome of the patients suffering from primary hepatocellular carcinoma (PHCC ) and healthy donors, to search tumor markers of hepatocellular carcinoma for diagnosis.MethodsThe serum proteome of the patients from primary hepatocellular carcinoma and healthy donors were separated and identified using immobilized pH gradient (IPG). Isoelectric focusing (IEF) electrophoresis was run as the first dimensional electrophoresis, and then horizontal SDS-PAGE as the second electrophoresis. After silver staining or coomassie blue staining, images were captured by scanner, and then the images were edited and matched using Imagemaster2DE analysis software. The differentially expressed proteins were analyzed by peptide mass fingerprint based on matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF-MS) and SWISS-PROT or BLAST nr database searching.Results1. By removing albumin and IgG in serum, trying different loding quantity, procedure of isoelectric focusing electrophoresis, time of equilibrium, density of SDS-PAGE gel and other important procedures of two-dimensional electrophoresis, the images were analyzed successfully and good reproducibility were obtained.2. Analyzing by Imagemaster2DE software, thirty-three proteins were found differentially expressed in sera from hepatocellular carcinoma patients and healthy donors. Six proteins were down-regulated in HCC, five proteins were up-regulated in HCC, seventeen proteins were found especially in healthy controls and five proteins were found especially in HCC.3. After analyzing by MALDI-TOF-MS, five differentially expressed proteins were identified. Albumin , Serotransferrin, CD5 antigen-like precursor ( IgM -associated peptide) were down-regulated in HCC, and Zinc-alpha-2-glycoprotein and Ig gamma-1 chain C region were up-regulated in HCC.Conclusion1. Using immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis (2-DE), good reproducibility and images could be obtained to separate and identify the proteome in serum.2. Thirty-three proteins which were found differentially expressed in HCC and healthy donors provided useful information for screening diagnostic tumor markers of human HCC.