激酶底物筛选新方法的研究

项目名称： 激酶底物筛选新方法的研究

项目编号： No.21275142

项目类型： 面上项目

立项/批准年度： 2013

项目学科： 数理科学和化学

项目作者： 叶明亮

作者单位： 中国科学院大连化学物理研究所

项目金额： 80万元

中文摘要： 蛋白质可逆的磷酸化是细胞信号转导的基础，激酶与底物蛋白质之间的特异性磷酸化相互作用构成了复杂的磷酸化调控网络。由于缺少有效的激酶底物筛选技术，人们对激酶与底物的对应关系知之甚少。一个蛋白质可以被多个激酶磷酸化，因此是多个激酶的底物，但是它们的作用位点即磷酸化位点往往是不同的，因此在确定激酶与底物对应关系的同时应确定磷酸化位点。基于质谱分析的磷酸化蛋白质组学的发展为通量化鉴定磷酸化位点创造了条件。本项目提出了一种将细胞提取物中的总蛋白质固载于固相微球上进行体外磷酸化，然后利用磷酸化蛋白质组学分析技术鉴定潜在的激酶底物蛋白及其磷酸化位点的新方法。在细胞提取物中的干扰激酶由于被固载在微球上而使其难以对其它蛋白质进行磷酸化，因此可以在一定程度上抑制背景磷酸化的影响。通过本项目的研究希望实现通量化鉴定激酶底物蛋白质及其作用磷酸化位点的目的，同时希望所建立的固相方法可以适用于其它修饰性酶底物的筛选。

中文关键词： 蛋白质组；翻译后修饰；底物筛选；；

英文摘要： Reversible protein phosphorylation play important role in signal transduction. The specific interactions between kinases and their substrates form the complex phosphorylation network. Due to lacking of effective kinase substrate screening methods, the knowledge of the kinase substrate relationship is still superficial. One protein could be phosphorylated by different kinases at different residues. Therefore, it is crucial to determine the phosphorylation site for the kinase substrate relationship. The development of mass spectrometry-based proteomics paves the way to determine phosphorylation site in a high throughput way. We proposed a new proteomics approach to screen the kinase substrate relationship. In the approach, proteins in total cell lysate are first immobilized onto beads. The immobilized proteins are then incubated with kinase for in-vitro phosphorylation. The putative substrates, i.e. the phosphorylated proteins, and their phosphorylation sites are finally identified by proteomics approach. Because all the proteins are immobilized, the background phosphorylation resulted from the intrinsic kinases presented in the lysate is expected to be reduced significantly. With the accomplish of this project, an new proteomics approach is expected to be developed to screen the kinase substrate relationship and

英文关键词： Proteomics；post-translational modifications；substrate screening；；