人神经母细胞瘤的磷酸化膜蛋白质组分析  

Phosphorylated membrane proteome analysis of human neuroblastoma SH-SY5Y cell line

作　　者：侯春彦[1,2] 马俊锋[1,2] 陶定银[1,2] 张丽华[1] 梁振[1] 张玉奎[1] 

机构地区：[1]中国科学院大连化学物理研究所,中国科学院分离分析化学重点实验室,国家色谱研究分析中心,辽宁大连116023 [2]中国科学院研究生院,北京100039

出　　处：《色谱》2011年第9期851-856,共6页Chinese Journal of Chromatography

基　　金：国家重大科学研究计划项目(2007CB914100);国家自然科学基金项目(20935004);科技部创新方法工作专项项目(2009IM031800)

摘　　要：针对人神经母细胞瘤SH-SY5Y细胞系的磷酸化膜蛋白质组,发展了基于多酶酶解法结合杂化硅胶基质固定化钛离子亲和色谱(Ti 4+-IMAC)整体柱富集的分析策略。该方法通过对细胞裂解液进行超速离心,以及1 mol/LNaCl和0.1 mol/L Na2CO3顺序清洗,获得膜蛋白质组分。所提取的蛋白质分别经胰蛋白酶、胰凝乳蛋白酶和胃蛋白酶平行酶解,产生的肽段经Ti 4+-IMAC整体柱选择性富集磷酸肽后,采用纳升级反相液相色谱分离和质谱鉴定,成功鉴定到43个磷酸化蛋白质,其中有14个定位于膜上。研究结果表明,采用该策略开展SH-SY5Y细胞系磷酸化膜蛋白质组学分析有望加速对该肿瘤的研究和相关潜在标记物的筛选。A strategy with the combination of multiprotease digestion and the selective enrichment of phosphopeptides by silica hybrid monolith based immobilized Ti4＋affinity chromatography（Ti4＋-IMAC） was proposed,and applied in the global profiling of phosphorylated membrane proteome of neuroblastoma SH-SY5Y cells.The fraction of membrane proteins was extracted by ultra speed centrifuge,followed by washing with 1 mol/L sodium chloride and 0.1 mol/L sodium carbonate.For digestion,chymotrypsin and pepsin with broader specificity were used as complementary enzymes to trypsin.The phosphopeptides were then selectively enriched by monolithic Ti4＋-IMAC column,and analyzed by nanoflow high performance liquid chromatography and mass spectrometry.A total of 43 phosphoproteins were identified,among which 14 proteins were located on the membrane.All these results demonstrated that the proposed strategy might be promising to promote the in-depth study of neuroblastoma and discover the candidate biomarkers.

关 键 词：人神经母细胞瘤 磷酸化肽富集 膜蛋白质 多酶酶解 固定化钛离子亲和色谱 有机-无机杂化硅胶整体材料 

分 类 号：O658[理学—分析化学]