应用iTRAQ技术筛选高转移和低转移肺腺癌细胞中差异表达的膜蛋白  

Screening for membrane proteins differentially expressed between the lung adenocarcinoma cell lines with high-and low-metastatic potential using iTRAQ technology

作　　者：林河春[1] 张方琳[1] 耿沁[1] 赵方瑜[1] 孙磊[1] 孔韩卫[1] 李静[1] 姚明[1] 

机构地区：[1]上海交通大学医学院附属仁济医院上海市肿瘤研究所癌基因及相关基因国家重点实验室,上海200032

出　　处：《肿瘤》2013年第2期150-156,共7页Tumor

基　　金：上海市科学技术委员会科研计划项目(编号:12140901500;10140902400)

摘　　要：目的:筛选高转移和低转移肺腺癌细胞中差异表达的膜蛋白,以寻找肺癌的生物学标志物或治疗的潜在靶点。方法:提取具有相同来源、不同转移潜能的肺腺癌SPC-A-1和SPC-A-1sci细胞的膜蛋白,应用核素标记相对和绝对定量(isobaric tags for relative and absolute quantitation,iTRAQ)技术标记膜蛋白,联合纳升液相色谱和串联质谱(nanoliquid chromatography-tandem mass spectrometry,Nano-LC-MS/MS)分离、分析肽段,采用Proteinpilot 4.0软件对蛋白进行鉴定和定量分析,对获得的差异蛋白进行GO(Gene Ontology)分析,应用实时荧光定量-PCR和蛋白质印迹法进行验证。结果:样品进行4次NanoLC-MS/MS,鉴定出符合假阳性率<1%的分别有1413、1374、1297和1351个蛋白,标记率>95%,其中4次都上调的蛋白有27个(膜蛋白20个)、下调的蛋白有32个(膜蛋白25个)。GO分析显示,差异蛋白的分子功能主要集中在细胞骨架蛋白结合、同源蛋白结合和酶结合。生物学进程主要集中在代谢进程和细胞定位。SPC-A-1sci细胞中ITGA3(integrin alpha-3)、MYH9(myosin,heavy chain9)、PLEC1(plectin 1)、HADHA(3-hydroxyacyl-CoA dehydrogenase)、HK1(hexokinase-1)、KTN1(kinectin 1)、ESYT1(extended synaptotagmin-like protein 1)、ALDH18A1(aldehyde dehydrogenase 18 family,member A1)、ATP5A1(ATP synthase alpha-subunit)、LMNB2(lamin-B2)、CAV1(caveolin-1)和CK-19(keratin,typeI cytoskeletal19)mRNA的表达水平以及CLTC(clathrin,heavy chain)、HK1、LMNB2和CK-19蛋白的表达水平均高于SPC-A-1细胞,与质谱定量结果一致。结论:iTRAQ联合NanoLC-MS/MS技术能高通量地筛选与转移相关的膜蛋白,为肺腺癌患者转移的诊断和预后提供可能的生物学标志物或治疗靶点。Objective： To screen for the membrane proteins differentially expressed between the lung adenocarcinoma cell lines with high- and low- metastatic potential, and to explore the potential targets for biomarkers and biological therapy. Methods： The membrane proteins were extracted from lung adenocarcinoma SPC-A-1 and SPC-A-1 sci cells which were generated from the same parental cell type and had low- and high- metastatic potential, respectively. The membrane proteins were labeled and the peptides were separated and analyzed by iTRAQ （isobaric tags for relative and absolute quantitation） technology combined with Nano-LC-MS/MS （nano liquid chromatography-tandem mass spectrometry）. The identification and quantitation of the proteins were analyzed by Proteinpilot 4.0 solfware. The membrane proteins differentially expressed were analyzed and verified by GO （Gene Ontology） terms and real-time fluorescence quantitative-PCR in combination with Western blotting, respectively. Results： The identified numbers of proteins with FDRs （false discovery rates） 〈 1% were 1 413, 1 374, 1 297, and 1 351 in the experiment which were repeated four times by Nano LC-MS/MS, and the rate of labelling was above 95%. Among the 27 proteins up-regulated in total four experiments, 20 proteins were identified as membraneprotein. Among the 32 proteins down-regulated in total four experiments, 25 proteins were identified as membrane protein. The GO analysis demonstrated the major molecular functions of the proteins differentially expressed including cytoskeletal protein binding, identical protein binding and enzyme binding as well as the catabolic process and cellular localization in biological processes. The expression levels of ITGA3 （integrin alpha-3）, MYH9 （myosin, heavy chain 9）, PLECI （plectin 1）, HADHA （3-hydroxyacyl- CoA dehydrogenase）, HKI （hexokinase-1）, KTNI （kinectin 1）, ESYTI （extended synaptotagmin-like protein 1）, ALDH18A1 （aldehyde dehydrogenase 18 family, member AI）, ATP5AI （

关 键 词：肺肿瘤 膜蛋白质类 肿瘤转移 ITRAQ 

分 类 号：R734.2[医药卫生—肿瘤]