| ObjectiveThe study combined Laser Capture Microdissection(LCM) with Label-free quantitative technology based on liquid chromatography tandem mass spectrometry (Label-free LC MS/MS) to investigate quantitative proteomes of gastric poorly differential adenocarcinoma and matched normal gastric tissue. Screening and identifying the differential expressed protein between Zymolysing protein of two groups above.These results may provide theoretical basis for the carcinogenic mechanism , early diagnosis and prognostic of gastric carcinoma.Methods1.Six pairs of human gastric adenocarcinoma and their adjacent nontumourous tissues, which were diagnosised for poorly differentiated adenocarcinoma ,were obtained after a surgical resection at the affiliated hospital of Taishan medical college. Malignant and nonmalignant gastric epithelial cells were isolated by laser capture microdissection(LCM).After protein extracted and mixed together,two group samples were analysed by Nano 2DLC-MS/MS for selecting differentially expressed protein . Bioinformatics method (GO and KEGG) were applied to analyse significantly differentially expressed proteins.2.Inorder to validate the reliability of the quantitative proteome result, we use Western blot and immunohistochemiscal method to detect the expression level of four proteins (Integrinα5,Annexin A2,Stat3 andβ-catenin)which were selected from the quantitative proteomes results.Results1.We successfully performed LCM to purify malignant and nonmalignant gastric epithelial cells.2.1114 and 1155 proteins were identified by Label-free LC MS/MS method in gastric adenocarcinoma and their adjacent nontumourous tissues, respectively. In which 75 proteins as endemic in gastric adenocarcinoma while 116 proteins in adjacent nontumourous tissue. We identified 264 significant differential expressed proteins(137 up regulate and 127 down regulate) ammong 1039 proteins in both two groups,.The GO enrichment analysis shows that most of them are located in the intracellular part ,take part in the cell metabolism process .These proteins associate with various molecular functions,include protein binding,nucleotide binding and structural constituent of cytoskeleton. KEGG result indicate that these protein involved in 24 kinds of signal transduction pathway. The highest match rate among these pathway is Oxidative phosphorylation .3.We use western blot and immunohistochemical method to detect the expression level of three proteins such as Integrinα5,β-catenin,Annexin A2.Western blot results indicate that the expression of Integrinα5 and Annexin A2 were overexpressed in gastric adenocarcinoma whileβ-catenin was downexpressed in gastric adenocarcinoma.The results of IHC showed that in gastric cancer , Integrinα5 and Annexin A2 were positively expressed ,their positive expressed rate were and respectively. Meanwhileβ-catenin was positively expressed in nomal gastric tissues.Conclusions1.We analysis gastric cancer relevant differencially expressed proteins by LCM and Lable-free quantitative proteomics technical.Which identified 137 up-regulated proteins and 127 down-regulated proteins,75 proteins only in cancerous tissue and 116 only in normal tissue.The result show that this method may become effective means of gastric cancer difference proteomic study.2.Bioinformatics method was used to do the annotation of protein binding, cellular metabolism, extracellular composition and oxidative phosphorylation of the differential proteins. The results lays foundation for gastric cancer mechanism and tumor markers.3.The expression level of Integrin alpha 5, Annexin A2, Stat3 and beta catenin in 6 pairs of poorly differentiated gastric adenocarcinoma and matched normal gastric mucosal tissue are basically consistent in the results of Western blot,immunohistochemical and Lable-free quantitative proteomics. |
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