Proteomics Approaches to Protein Turnover

 项目描述（申请人提供）：本项目的目标是开发用于完整测量细胞中蛋白质周转的蛋白质组学技术，重点分析蛋白质丰度、泛素化、合成速率和降解速率。根据分子生物学的中心法则，遗传信息从基因组流向转录组，再流向蛋白质组。理论上，细胞系统中的蛋白质动力学产生一种稳定状态，其中蛋白质的水平取决于其先前存在的浓度，合成速率和降解速率。真核细胞的蛋白质降解主要由泛素-蛋白酶体系统和自噬-溶酶体途径介导。泛素是一种由76个氨基酸组成的小分子蛋白质，作为蛋白酶体降解和其他下游事件的多功能信号修饰数千种蛋白质。泛素以单体和聚合物的形式与底物缀合。在这个应用中，我们建议开发新的质谱（MS）为基础的方法来配置文件蛋白质营业额，分析泛素化蛋白质组和泛素链结构，并研究蛋白质营业额是如何在病理生理条件下的影响。我们的四个具体目标是：（i）开发一个20重集成MS方法测量蛋白质组营业额;（ii）开发中间向下MS方法分析多聚泛素链结构;（iii）研究酵母和哺乳动物细胞中不同的多聚泛素链的功能;（iv）研究人类疾病小鼠模型中蛋白质营业额的变化。蛋白质周转和泛素化是基本的调节事件，有助于人类疾病的发病机制。这项研究将导致开发新的MS技术，用于研究蛋白质周转和对多聚泛素链功能的新理解，以及其与人类疾病的关系。

 DESCRIPTION (provided by applicant): The goal of this project is to develop proteomics technologies for the complete measurement of protein turnover in cells, focusing on the analysis of protein abundance, ubiquitination, synthetic rate, and degradation rate. According to the central dogma of molecular biology, genetic information flows from the genome to the transcriptome to the proteome. Theoretically, protein dynamics in a cellular system yields a steady state in which the level of a protein depends on its preexisting concentration, synthetic rate, and degradation rate. Protein degradation in eukaryotic cells is mainly mediated by the ubiquitin (Ub)-proteasome system and the autophagy-lysosome pathway. Ubiquitin, a small protein of 76 amino acids, modifies thousands of proteins as multifunctional signals for proteasomal degradation and other downstream events. Ubiquitin is conjugated to substrates in the form of monomers and polymers. In this application, we propose to develop novel mass spectrometry (MS)-based methods to profile protein turnover, analyze ubiquitinated proteome and ubiquitin chain structures, and study how protein turnover is affected under pathophysiological conditions. Our four specific aims are to (i) develop a 20-plex integrated MS approach for measuring proteome turnover; (ii) develop middle-down MS methods for analyzing polyubiquitin chain structures; (iii) study the function of diverse polyubiquitin chains in yeast ad mammalian cells; and (iv) investigate protein turnover alterations in mouse models of human disease. Protein turnover and ubiquitination are fundamental regulatory events, contributing to the pathogenesis of human disease. The research will lead to the development of novel MS technologies for studying protein turnover and new understanding of polyubiquitin chain function, as well as its involvement in human disease.