应用免疫沉淀-质谱法筛选肿瘤血管靶向肽CGNSNPKSC的结合受体  

Screening of receptors of the peptide CGNSNPKSC that bind specifically to tumor vascular endothelium by immunoprecipitation and mass spectrometry

作　　者：贺莉[1] 吴开春[1] 惠晓莉[1] 陈瑜[1] 梁树辉[1] 陈蓓[1] 曹姗姗[1] 闫堃[1] 韩宇[1] 姚芳芳[1] 杨利萍[1] 韩英[1] 樊代明[1] 

机构地区：[1]第四军医大学西京医院消化病研究所,肿瘤生物学国家重点实验室,陕西西安710032

出　　处：《现代肿瘤医学》2008年第8期1259-1263,共5页Journal of Modern Oncology

基　　金：国家自然科学基金面上资助项目(批准号:30400203);全军医药卫生科研基金资助项目(批准号:06G087);863计划资助项目(批准号:2006AA02Z103)

摘　　要：目的:筛选和鉴定肿瘤血管靶向肽GX1(CGNSNPKSC)的结合受体。方法:化学合成生物素标记的GX1,培养内皮细胞并收集细胞膜蛋白;利用免疫沉淀的方法进行免疫磁珠分离,富集能与血管特异性短肽GX1结合的蛋白质;利用生物素-亲和素系统进行western blot免疫检测,筛选出能和GX1结合的目的条带;利用液相色谱-二级质谱(LC-MS/MS)对能与血管特异性短肽GX1结合的蛋白质进行测序鉴定。结果:利用免疫沉淀和western blot的方法,获得了能与GX1结合的蛋白条带,其分子量为13KDa。利用LC-MS/MS及生物信息学分析,获得了8个候选蛋白:cDNA FLJ40018fis,clone STOMA2006398,cytochromeP45019A1,probable histone-lysine N-methyltransferaseASH1L,adenylyl cyclase-associated protein2(CAP2),similar toankyrin repeat domain20A,diacylglycerol kinase iota(DGKI),isoform2of Inositol1,4,5-trisphosphate receptortype1,similar to annexinA2isoform1。结论:利用免疫沉淀-质谱法获得了8个GX1结合受体的候选蛋白,为进一步寻找血管抑制治疗的靶标奠定了一定的实验基础。Objective ：To screen and identify the receptors of GX1 （CGNSNPKSC） peptide binding to tumor vascular endothelium. Methods： We synthesized the biotin -labeled GX1 peptide and collected the membrane proteins of the cultured endothelial cells. By using immunoprecipitation and immune magnetic beads separation, we obtained proteins combined with GX1 peptide. By using biotin - avidin system and western blot, the protein band that specif- ically bound to GX1 peptide was screened. Then, the protein band was identified by tandem mass spectrometry （LC - MS/MS）, and analyzed with bioinformatics. Results： By using immunoprecipitation and western blot, one band in the position of 13KDa was found to bind selectively to the GX1 peptide. Using tandem mass spectrometry analysis and bioinformatics analysis, 8 candidate proteins was obtained, including cDNA FLJ40018fis, clone STOMA2006398, cytochromeP450 19A1, probable histonelysine N- methyltran feraseASH1L, adenylyl cyclaseassociated protein 2 （ CAP2）, similar to ankyrin repeat domain 20A, diacylglycerol kinase iota （ DGKI ）, isoform2 of Inositol 1,4,5 - trisphosphate receptor type 1, and similar to annexinA2 isoforml. Conclusion： By using immunoprecipitation and mass spectrometry, 8 candidate proteins were identified, which may provide some experimental basis for probing into new targets of anti - vascular therapy.

关 键 词：GX1 脐静脉内皮细胞 免疫沉淀 LC-MS/MS 受体 

分 类 号：R730[医药卫生—肿瘤]