色谱

色谱

• 研究论文 • 上一篇    下一篇

蛋白质组衍生肽段文库用于鉴定酪蛋白激酶2的底物模体

张曼玉, 王春丽, 边阳阳, 程凯, 卫晓鸾, 叶明亮*, 邹汉法*   

  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
  • 收稿日期:2011-06-09 修回日期:2011-07-15 出版日期:2011-08-28 发布日期:2011-09-28
  • 通讯作者: 邹汉法,博士,研究员,博士生导师. Tel: (0411)84379610, 叶明亮,博士,研究员,博士生导师. Tel: (0411)84379620
  • 基金资助:

    国家自然科学基金创新研究群体科学基金项目(No. 21021004)

Proteomic peptide library for determination of substrate motif of casein kinase 2

ZHANG Manyu, WANG Chunli, BIAN Yangyang, CHENG Kai, WEI Xiaoluan, YE Mingliang*, ZOU Hanfa*   

  1. CAS Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
  • Received:2011-06-09 Revised:2011-07-15 Online:2011-08-28 Published:2011-09-28

PDF

563

被引次数

6

摘要： 模体是蛋白质二级结构上的一种特征序列,激酶底物通常具有一类特征性的模体,识别底物模体对鉴定激酶底物具有重要意义。为了快速鉴定激酶底物模体,将全细胞蛋白质酶解液作为肽段文库的来源,采用碱性磷酸酶去除肽段的固有磷酸化后构建了用于筛选激酶底物模体的肽段文库。该肽段文库是大量非磷酸化肽段的混合物,将此混合肽段与酪蛋白激酶2和三磷酸腺苷作用30 min后,通过固定化金属离子亲和色谱法富集磷酸化肽段,采用反相液相色谱-串联质谱分析,成功地鉴定到472条非冗余底物肽段,包含451个非冗余磷酸化位点,并由此得到底物模体S/T-D/E-x-D/E。该法能够快速准确地筛选出激酶底物模体,对研究激酶-底物识别以及信号转导过程具有重要意义。

关键词: 蛋白质组学, 反相液相色谱-串联质谱, 固定化金属离子亲和色谱, 激酶, 模体, 肽段文库

Abstract: Substrate motif is of great importance in kinase-substrate recognition and contributes a lot to the understanding of signal transduction. In this study, a proteomic peptide library was generated to determine substrate motif of casein kinase 2. Firstly, whole cell lysate was digested by trypsin to generate a large number of candidate peptides, which were then incubated with alkaline phosphotase to dephosphorylate intrinsic phosphopeptides. Then, the unphosphorylated peptide mixture was incubated with casein kinase 2 (CK2) and adenosine-triphosphate (ATP) for 30 min, and the resulting phosphopeptides were enriched by immobilized metal affinity chromatography (IMAC) followed by reversed-phase liquid chromatographic separation coupled with tandem mass spectrometry detection. Finally, the 472 unique phosphopeptides and 451 unique phosphorylation sites were identified, resulting in the determination of a motif of S/T-D/E-x-D/E for CK2. This method enables accurate determination of substrate motif in a short time and can be readily applied to other kinases.

Key words: kinase, motif, peptide library, proteomics, reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS), immobilized metal affinity chromatography (IMAC)