修饰性酶与底物相互作用的蛋白质组学分析方法研究

Analysis of post-translational modifications (PTMs) by MS based proteomics approaches is able to identify thousands of modified sites which indicates that numerous PTM enzyme catalyzed reactions occurred in the cells. However, these modified sites do not directly reveal the complex interactions mediated by the PTM enzymes. In this propose, we are going to develop new proteomics approaches to reveal the interaction of PTM enzymes with their substrates. Firstly, we plan to develop methods to decipher the PTM enzyme-substrate relationship. To facilitate the screening of enzyme substrates, we proposed a solid phase based approach using immobilized total cell lysate as the protein library. To enable the determination of the kinetics constants by proteomics approaches, we plan to study the enzyme kinetics theory in complex system where an enzyme coexists with huge number of substrates with different abundance. Secondly, we plan to develop methods to reveal the interaction of domain with PTM proteins. To enable the identification of PTM proteins with interaction with specific protein domains, we aim to develop domain based affinity chromatography. To enable the determination of the interaction strength with high throughput, we plan to develop new methods by combining quantitative proteomics with chromatographic techniques. Thirdly, we plan to develop new methods to analyze the protein complex, which should be able to quantitatively reveal the composition and the PTMs on individual proteins. With the completion of this project, we aim to significantly improve our ability to decipher the PTM enzyme mediated biological processes.

蛋白质组修饰谱的分析可以鉴定大量的修饰位点，说明在生物体内发生了大量的修饰酶与底物蛋白质之间的酶促化学反应。但是这些位点并不能直接揭示生物体内修饰性酶介导的复杂相互作用。本项目拟发展揭示修饰性酶与底物相互作用的蛋白质组学分析新方法，包括1）发展揭示修饰性酶与底物蛋白质之间的直接相互作用的方法。拟通过发展底物筛选的通量化鉴定技术，揭示酶与底物之间的特异性相互作用；通过发展复杂体系中的酶促动力学理论，实现酶与底物之间的特异性相互作用的定量化表述。2）建立结构域对修饰蛋白质的特异性相互作用的表征新方法，期望建立复杂体系中蛋白相互作用强度或解离常数的通量化测定方法；3）蛋白质复合物组成、修饰的定性定量分析方法。通过本项目的研究，建立多项修饰性酶与底物蛋白相互作用表征的新技术新方法，期望显著提高修饰性酶与底物蛋白相互作用研究水平，增强解析蛋白质翻译后修饰调控复杂生命活动的能力。

蛋白质组修饰谱的分析可以鉴定大量的修饰位点，说明在生物体内发生了大量的修饰酶与底物蛋白质之间的酶促化学反应。但是这些位点并不能直接揭示生物体内修饰性酶介导的复杂相互作用。针对这一问题，结合申请者在修饰蛋白质组研究中所形成的基础和积累，开展了揭示修饰性酶与底物相互作用的蛋白质组学分析新方法的创新性研究，有效提高了我国在修饰性酶与底物蛋白相互作用表征的新技术新方法方面的研究水平，形成一个蛋白质组修饰谱的研究团队。主要研究成果包括建立了酪氨酸磷酸化蛋白质组的规模化分析平台，发展了复杂酶促体系酶促动力学分析方法，开发了修饰酶与底物相互作用的分析新方法，以及建立了小分子与修饰酶相互作用的筛选方法等。这些技术和方法的突破大大提升了修饰性酶与底物蛋白相互作用研究水平，增强解析蛋白质翻译后修饰调控复杂生命活动的能力。