Screening Human Gastric Carcinoma Associated Antigens By Serologic Proteome Analysis (SERPA)

OBJECTIVE: To seek the human gastric carcinoma biomarkers or tumor associated antigens, the western-blot imaging films reacted with the patient autologous serum and with control serum were compared and human gastric carcinoma associated antigens were identified by using Serologic Proteome Analysis (SERPA). METHODS: In this study, Serologic Proteome Analysis (SERPA) of ten human gastric carcinoma tissues were performed. Three gels were run simultaneously with 1000μg, 300μg, 300μg amounts of proteins from the same gastric carcinoma by IPG -2D PAGE. After separation, the proteins of the gel (replica gel) which has 1000lμg amounts of proteins was visualized by Coomassie brilliant blue staining technique. The other two gels were used for western blot analysis. Proteins are transferred to PVDF membranes, and then reacted with autologous patient serum and with control normal serum, respectively. The Western blot imaging films were obtained and the differential reacting protein spots were recognized, then the differential reacting protein spots in the replica gel by matching analysis using artificial method and computer analysis. Finally identified by peptide mass fingerprint(PMF) based on matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and database searching.RESULTS: (1) The results showed that well-resolved, reproducible 2-DE maps of human gastric carcinoma tissues were obtained. The number of average spots of 10 gels was827±39. There were 732±32 spots matched. The average match rate was 88.5 %. (2) Well-resolved 2-DE western blot imaging films of human gastric carcinoma reacted with autologous patient sera and with control sera were obtained. (3) Sixteen differentially expressed proteins which only were reactive with gastric carcinoma patient serum were found and fifteen proteins, which are human gastric carcinoma associated antigens, were identified by MS.They are heterogeneous nuclear ribonucleoprotein K(hnRNP K), hnRNP HI, hnRNP H2, cytokeratin 18(CK18), crystal structure of Fkbp52 c-terminal domain complex with the c-terminal peptide meevd of Hsp90, serum vitamin D-binding protein precursor(DBP), selenium binding protein 1(SBP1), eukaryotic translation elongation factor 1 delta(eEF-l), ACTB protein,...