Analysis of Ubiquitinated Proteome

描述（由申请人提供）：与磷酸化类似，蛋白质泛素化是一个可逆过程，基本上调节所有细胞事件。泛素化的失调在从癌症到神经退行性疾病的多种疾病的发病机制中起重要作用。虽然已经开发了一些蛋白质组学工具来分析酵母中的泛素化蛋白，但在哺乳动物细胞中进行类似的分析仍然很困难。本申请的目标是开发一系列蛋白质组学工具来分析泛素化蛋白质组，包括：（i）建立串联亲和纯化方法以纯化哺乳动物细胞中的Ub缀合物;（ii）开发富集通过特异性polyUb键修饰的泛素化蛋白质亚群的策略;（iii）开发蛋白质组学方法以分析Ub缀合物并评估假阳性。这些定量质谱策略将被用来研究不同条件下泛素化蛋白质组的动态变化。总之，这些研究将为探索泛素化蛋白质组和更好地理解polyUb链结构的异质性创造必要的工具，polyUb链结构在泛素信号传导和疾病发展中起着重要作用。 公共卫生相关性：像磷酸化一样，蛋白质泛素化是一个可逆的过程，基本上可以调节所有的细胞事件。泛素化的失调在从癌症到神经退行性疾病的多种疾病的发病机制中起重要作用。本申请的目标是开发新的蛋白质组学技术，以分析由特定泛素聚合物修饰的蛋白质底物。一旦建立，这些技术将能够调查泛素信号的特异性及其参与泛素相关疾病。

DESCRIPTION (provided by applicant): Like phosphorylation, protein ubiquitination is a reversible process to regulate essentially all cellular events. Misregulation of ubiquitination plays an important role in the pathogenesis of a variety of diseases, ranging from cancer to neurodegenerative disorders. Although some proteomics tools have been developed to analyze ubiquitinated proteins in yeast, it is still difficult to perform similar analysis in mammalian cells. The goal of this application is to develop a series of proteomics tools to analyze ubiquitinated proteome including: (i) to establish a method of tandem affinity purification to purify Ub-conjugates in mammalian cells; (ii) to develop a strategy to enrich subsets of ubiquitinated proteins that are modified by specific polyUb linkages; (iii) to develop proteomic methods to profile Ub-conjugates and to evaluate false positives. These quantitative mass spectrometry strategies will be implemented to investigate the dynamics of ubiquitinated proteome under different conditions. Taken together, the studies will invent essential tools for exploring ubiquitinated proteome and a better understanding of heterogeneity of polyUb chain structures, which plays an important role in ubiquitin signaling and disease development. PUBLIC HEALTH RELEVANCE: Like phosphorylation, protein ubiquitination is a reversible process to regulate essentially all cellular events. Misregulation of ubiquitination plays an important role in the pathogenesis of a variety of diseases, ranging from cancer to neurodegenerative disorders. The goal of this application is to develop novel proteomics technologies to analyze protein substrates that are modified by specific ubiquitin polymers. Once established, the technologies will enable investigation of the specificity of ubiquitin signaling and its involvement in ubiquitin- related diseases.