色谱

色谱

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人神经母细胞瘤的磷酸化膜蛋白质组分析

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   

  1. 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
  • 收稿日期:2011-08-01 修回日期:2011-08-02 出版日期:2011-09-28 发布日期:2011-10-28
  • 通讯作者: 张丽华,研究员,主要从事生物分子的高效分离与表征等研究工作. Tel: (0411)84379720
  • 基金资助:

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

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

HOU Chunyan1,2, MA Junfeng1,2, TAO Dingyin1,2, ZHANG Lihua1*, LIANG Zhen1, ZHANG Yukui1   

  1. 1. CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, National Chromatographic R. & A. Center, Dalian 116023, China; 2. Graduate University of Chinese Academy of Sciences, Beijing 100039, China
  • Received:2011-08-01 Revised:2011-08-02 Online:2011-09-28 Published:2011-10-28

PDF

447

被引次数

5

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(Ti4+-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/L NaCl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Ti4+-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。

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

Abstract: 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.

Key words: immobilized Ti4+affinity chromatography, membrane protein, multi-enzyme digestion, organic-inorganic hybrid silica monolith, human neuroblastoma, phosphopeptide enrichment