凝胶电泳-毛细管液相色谱-质谱联用技术分析大肠癌肿瘤与癌旁组织的差异蛋白质组
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摘要
通过凝胶电泳将大肠癌患者的手术标本组织(肿瘤与癌旁)中的总蛋白按照分子量分成不同的组分,经胶内水解后,利用毛细管液相色谱-质谱联用技术对各个组分的多肽混合物进行蛋白质组学分析,共鉴定出29种差异表达的蛋白质,其中肿瘤组织中表达上调的蛋白质19种,如Fibrinogen beta chain,Vimentin,Fibrinogen gamma chain,Histone H2A type 1-B/E,Isoform 1 of Periostin,Fibrinogen alpha chain,Histone H3.1,Alphaenolase,Protein disulfide-isomerase A3等;在肿瘤组织中表达下调的有10种蛋白质,如Sarcomeric tropomyosin kappa,Transgelin,Heat shock protein beta-1,Talin-1,Isoform 2 of Vinculin,Isoform 1 of Filamin-C等。通过蛋白质印迹实验对其中的踝蛋白1(Talin-1)和烯醇化酶(Alpha-enolase)的蛋白质组学鉴定结果进行了验证。生物信息学分析表明在大肠癌组织中表达上调的蛋白多具有胞外分泌的属性,提示这些蛋白很有可能会渗透到机体内的循环系统中,从而在患者血液或者腹水中能够被检测到;而在大肠癌组织中表达下调的蛋白则多为细胞骨架、胞外基质纤维等的组成成分,这些蛋白的下调常与肿瘤细胞向周围组织的侵袭和转移有密切关系。本研究为大肠癌的早期诊断、大肠癌初筛等方面的研究等提供了基础数据,对进一步深入研究大肠癌发病机制具有重要意义。
The whole protein extracts from tissue specimens( tumor and noncancerous) from patients with colorectal cancer were divided into several fractions with different molecular weight by gel electrophoresis,which were then subject to in-gel digestion and proteomics analysis by capillary liquid chromatography-mass spectrometry. A total of 29 differentially expressed proteins were identified,among which 19 were up-regulated proteins in tumor tissues,such as Fibrinogen beta chain,Vimentin,Fibrinogen gamma chain,Histone H2 A type 1-B / E,Isoform 1 of Periostin,Fibrinogen alpha chain,Histone H3.1,Alpha-enolase,Protein disulfideisomerase A3,and so on. Ten proteins were found to be down-regulated,including Sarcomeric tropomyosin kappa,Transgelin,Heat shock protein beta-1,Talin-1,Isoform 2 of Vinculin,Isoform 1 of Filamin-C,and so on. The differential expressions of Talin-1 and Alpha-enolase were selected to be verified by Western blot analysis. Bioinformatics analysis results indicated that most of the up-regulated proteins in colorectal cancer tissues had the feature of protein exocytosis,suggesting that these proteins were likely to penetrate into the circulatory system in the body,which could be detected in blood or ascites. On the other hand,most of the down-regulated proteins belonged to the cytoskeleton and extracellular matrix fibers,which were closely related to the invasion and metastasis of tumor cells into their surrounding tissues. This study provided basic data for the early diagnosis of colorectal cancer,colorectal cancer screening and other aspects,which had important significance to further study the pathogenesis of colorectal cancer.
The whole protein extracts from tissue specimens( tumor and noncancerous) from patients with colorectal cancer were divided into several fractions with different molecular weight by gel electrophoresis,which were then subject to in-gel digestion and proteomics analysis by capillary liquid chromatography-mass spectrometry. A total of 29 differentially expressed proteins were identified,among which 19 were up-regulated proteins in tumor tissues,such as Fibrinogen beta chain,Vimentin,Fibrinogen gamma chain,Histone H2 A type 1-B / E,Isoform 1 of Periostin,Fibrinogen alpha chain,Histone H3.1,Alpha-enolase,Protein disulfideisomerase A3,and so on. Ten proteins were found to be down-regulated,including Sarcomeric tropomyosin kappa,Transgelin,Heat shock protein beta-1,Talin-1,Isoform 2 of Vinculin,Isoform 1 of Filamin-C,and so on. The differential expressions of Talin-1 and Alpha-enolase were selected to be verified by Western blot analysis. Bioinformatics analysis results indicated that most of the up-regulated proteins in colorectal cancer tissues had the feature of protein exocytosis,suggesting that these proteins were likely to penetrate into the circulatory system in the body,which could be detected in blood or ascites. On the other hand,most of the down-regulated proteins belonged to the cytoskeleton and extracellular matrix fibers,which were closely related to the invasion and metastasis of tumor cells into their surrounding tissues. This study provided basic data for the early diagnosis of colorectal cancer,colorectal cancer screening and other aspects,which had important significance to further study the pathogenesis of colorectal cancer.
引文
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2 Mrmol I,Snchez-de-Diego C,Pradilla Dieste A,Cerrada E,Rodriguez Yoldi M J.Int.J.Mol.Sci.,2017,18(1):197
3 Guina T,Biasi F,Calfapietra S,Nano M,Poli G.Ann.N.Y.Acad.Sci.,2015,1340:95-103
4 Giusti L,Iacconi P,Da Valle Y,Ciregia F,Ventroni T,Donadio E,Giannaccini G,Chiarugi M,Torregrossa L,Proietti A,Basolo F,Lucacchini A.Mol.Biosyst.,2012,8(4):1088-1099
5 Hammoudi A,Song F,Reed K R,Jenkins R E,Meniel V S,Watson A J,Pritchard D M,Clarke A R,Jenkins J R.Biochem.Biophys.Res.Commun.,2013,440(3):364-370
6 Yamamoto T,Kudo M,Peng W X,Takata H,Takakura H,Teduka K,Fujii T,Mitamura K,Taga A,Uchida E,Naito Z.Tumour Biol.,2016,37(10):13595-13606
7 Yu K H,Snyder M.Mol.Cell.Proteomics,2016,15(8):2525-2536
8 WANG Zi-Jian,GAO Xiang,YANG Jing-Bo,SUN Wan-Chun,WU Dong-Lin,PENG Qi-Sheng,LIU Ning.Chinese J.Anal.Chem.,2016,44(10):1521-1527王子健,高祥,杨静波,孙万春,吴东林,彭其胜,刘宁.分析化学,2016,44(10):1521-1527
9 BAI Hai-Xin,LIU Xiao-Hua,YANG Fan,YANG Xiu-Rong,WANG Er-Kang.Chem.J.Chinese Universities,2010,31(7):1314-1321白海鑫,刘小花,杨帆,杨秀荣,汪尔康.高等学校化学学报,2010,31(7):1314-1321
10 LIN Lin,LUO Shu-Sheng,WANG Lin-Jue,YANG Jie,SHEN Hai-Nan,TIAN Rui-Jun.Chinese J.Anal.Chem.,2015,43(10):1479-1489林琳,罗树生,王灵珏,杨杰,沈海南,田瑞军.分析化学,2015,43(10):1479-1489
11 Broeckx V,Boonen K,Pringels L,Sagaert X,Prenen H,Landuyt B,Schoofs L,Maes E.Mol.Biosyst.,2016,12(2):553-565
12 Cutillas P R,Crnogorac-Jurcevic T.Methods Mol.Biol.,2016,1381:201-209
13 WU Peng,HE Fu-Chu,JIANG Ying.Prog.Biochem.Biophys.,2013,40(3):281-292武鹏,贺福初,姜颖.生物化学与生物物理进展,2013,40(3):281-292
14 Washburn M P,Wolters D,Yates J R 3rd.Nat.Biotechnol.,2001,19(3):242-247
15 Haining A W,Lieberthal T J,Del Río Hernández A.FASEB J.,2016,30(6):2073-2085
16 Fang K P,Zhang J L,Ren Y H,Qian Y B.Asian Pac.J.Cancer Prev.,2014,15(6):2655-2661
17 SUN Li,ZHAO Hai-Feng,GUO Hong-Hua,YANG Guang-Yuan,HE Cheng-Yan,SHI Qing-Hong,WEN Xue,WANG Xiao-Ting,ZHAO Li-Chun.Chinese J.Anal.Chem.,2012,40(10):1521-1527孙莉,赵海丰,郭宏华,杨光远,何成彦,师庆红,文雪,王小婷,赵丽纯.分析化学,2012,10(40):1500-1506
18 Shi Q N,Gao S,Song L N,Zhao Y M,Li X O,Wu J L,He C Y,Li H J,Zhao H F.Proteome Sci.,2015,13(1):1