Screening And Identification Of Gastric Cancer Associated Proteins By Proteomic Analysis

ObjectiveIdentification of the specific biomarkers of cancers has become one of the reliable approaches in the early-stage diagnosis.Nowadays,Proteomic technologies are providing the tools needed to discover and identify disease-associating biomarkers. In this study,protemic technologies were used to establish 2-DE profiles including gastric cancer tissues and paired tumor-adjacent tissues,the protein spots were screened out and indentified by MS,then detected by Western blot analysis to search the specific tumor markers of gastric cancer.Materials and Methods1.The soluble proteins from human gastric cancer tissue and paired tumoradjacent tissue were purified with lysis buffer of 2-DE.And Bradford's method was used to determine the total protein concentration of the samples.2.The soluble proteins of human gastric cancer tissue and paired tumor-adjacent tissue were separated by 2-DE and stained by silver,then got the imanges of two-dimensional gels with PowerLook 1120,and analyzed with ImageMaster 2D platinum6.0 software to acquire accordant ratio of protein spots in gels.3.The two-dimensional gels for the soluble proteins of gastric cancer tissue and paired tumor-adjacent tissue were obtained with protein standard quantity for preparation gel,then scanning gels images with PowerLook 1120,and analyzed the date by ImageMaster 2D platinum6.0 software to acquire the differential protein spots and identified their corresponding spots in gel.4.The different protein spots were cut out from preparation gels and then digested with typsin.Through matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS),searched on the protein database and identified the protein as the candidate proteins of tumor-associated proteins of gastric cancer.5.In order to validate the reliability of the identified results,S100A9 was detected by Western-blot analysis.Results1.Well-resolved,highly reproducible 2-DE profiles of human gastric cancer tissue and tumor-adjacent tissue were established.For tumor tissue,average spots of 3 gels were 737±33,average matching rate was 81.1%.For control,average spots of 3 gels were 713±29,and average matching rate was 87.0%.The average position deviation of matched spots was(1.23±0.27)mm in IEF direction,and(1.49±0.35)mm in SDS-PAGE direction.2.Forty-two distinct protein spots were found between the electrophoretic images of human gastric cancer tissue and paired tumor-adjacent tissue.3.Ten protein spots were cut out from the preparation gel and were detected by MALDI-TOF-MS.After searching the protein database,there were ten proteins identified out:S100 calcium-binding protein A8,Tropomyosinl,Transgelin,F-actin capping protein alpha-1,S100 calcium-binding protein A9,Heat Shock Protein 27,: response regulator,Profilin,Heat Shock Protein 60 and Cytokeratin10.4.Western blot analysis was able to confirm S100A9 was up-regulated in gastric cancer tissue,down-regulated in tumor-adjacent tissue,which is consistent with our proteome analysis results.Conclusions1.2-DE profiles of human gastric cancer tissue and tumor-associated proteins was successfully established and ten protein spots were detected,which can be effectively applied in proteomics of the gastric cancer.2.S100A9 was up-regulated in gastric cancer tissue,down-regulated in tumor-adjacent tissue,which show S100A9 is a marker of gastric cancer probaboly.