Proteomics Application For Screening Serum Biomarkers Of Hepatocellular Carcinoma Early Detection

Part 1 Serum proteome analysis of primary hepatocellular carcinoma bytwo-dimensioanal electrophoresis and MALDI-TOF MSPurposeAnalyzing the serum proteome of the patients suffering from primaryhepatocellular carcinoma (HCC), liver cirrhosis and healthy donors, to search tumormarkers of hepatocellular carcinoma for early diagnosis.MethodsThe serum proteome of the patients from primary hepatocellular carcinoma,liver cirrhosis and healthy donors were separated and identified by immobilized pHgradient (IPG). Isoelectric focusing (IEF) electrophoresis was run as the firstdimensional electrophoresis, and then horizontal SDS-PAGE as the secondelectrophoresis. After silver staining or coomassie blue staining, images werecaptured by scanner, and then edited and matched by Imagemaster2DE analysissoftware. The differentially expressed proteins were analyzed by peptide massfingerprint based on matrix-assisted laser desorption-ionization time of flight massspectrometry (MALDI-TOF-MS) and SWISS-PROT or BLAST nr databasesearching. RT-PCR and Western blotting analysis were used to comfirmZinc-alpha-2-glycoprotein highly expressed in HCC.Results1. By removing albumin and IgG in serum, trying different loding quantity,procedure of isoelectric focusing electrophoresis, time of equilibrium, density ofSDS-PAGE gel and other important procedures of two-dimensionalelectrophoresis, the images were analyzed successfully and good reproducibilitywere obtained.2. Analyzing by Imagemaster2DE software, thirty-three protein spots were founddifferentially expressed in sera from hepatocellular carcinoma patients, liver cirrhosis patients and healthy donors. After analyzing by MALDI-TOF-MS, fivedifferentially expressed proteins were identified. Albumin, Serotransferrin, CD5antigen-like precursor (IgM-associated peptide) were down-regulated in HCC,and Zinc-alpha-2-glycoprotein and Ig gamma-1 chain C region wereup-regulated in HCC.3. Zinc-alpha-2-glycoprotein (ZAG), a lipid mobilizing factor, is a member of themajor histocompatibility complex (MHC) class I family of protein.Conclusion1. Using immobilized pH gradient two-dimensional polyacrylamide gelelectrophoresis (2-DE), good reproducibility and images could be obtained toseparate and identify the proteome in serum.2. Five proteins which were found differentially expressed in HCC provided usefulinformation for screening diagnostic tunaor markers of human HCC.3. The machenism of Zinc-alpha-2-glycoprotein, which was highly expressed inHCC should be studied in future work.Part 2 Detection and evaluations of serum proteomic patterns for hepatocellularcarcinoma by SELDI-TOF MSPurposeScreening serum biomarkers of hepatocellular carcinoma for early detection bySELDI-TOF mass spectrometry proteinchip in the sera from hepatocellularcarcinoma, liver cirrhosis and healthy people.Methods25 cases of the patients with hepatocellular carcinoma, 25 cases of the patientswith liver cirrhosis and 25 cases of the healthy people were tested by Weak cationicexchang(WCX2) and Hydrophobic (H4) protein chip and surface enhanced laserdesorption ionization-time of flight-mass-spectrometry of Ciphergen Inc. Thedifferentially expressed proteins were analyzed by BioMarker Wizard Software. Results1. At the different M/Z value range, nine proteins were obviously different betweenthe group of the patients with hepatocellular carcinoma, liver cirrhosis and thehealthy people.2. Analyzing by BioMarker Wizard software, five peptides including 6489Da,6662Da, 8582Da, 8720Da, 8960Da were down-regulated in HCC, four including5552Da, 7748Da, 7777Da, 9250Da were up-regulated in HCC.3. We have established a four-peptide profile (5552Da, 7748Da, 7777Da, 9250Da)that can separate patients with hepatocellular carcinoma from liver cirrhosis andnormal controls by use of Biomarker Pattern software. It gives the much-improvedsensitivity of 92.3ï¼…and the specificity of 95.5ï¼….4. Through searching databases, these nine peptides are Galanin-like peptide (P1),Neuregulin-4(P2), small inducible cytokine A15 precursor (P3), CSL-type zincfinger-containing protein(P4), ATP synthase coupling factor 6 mitochondrialprecursor (PS, P10), Ubiquinol-cytochrome C reductase complex 11kD proteinmitochondrial precursor(P6), Transforming growth factor alpha precursor(P7),Mature T-cell proliferation-1 type A(PS), Apolipoprotein A-â…¡precursor(P9).Conclusion1. Using SELDI-TOF MS, the method of sieving the tumor markers from HCCbecomes quick and valid.2. These differentially expressed proteins/peptides could bebiomarkers ofhepatocellular carcinoma in the serum and drug targets for treating hepatocellularcarcinoma.