Analysis of Ubiquitinated Proteome

描述（由申请人提供）：与磷酸化一样，蛋白质泛素化是一个可逆过程，基本上调节所有细胞事件。泛素化的错误调控在多种疾病的发病机制中起着重要作用，从癌症到神经退行性疾病。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)建立一种串联亲和纯化方法来纯化哺乳动物细胞中的ub偶联物；（ii）开发一种策略，以丰富被特定聚脲b连接修饰的泛素化蛋白亚群；（iii）开发蛋白质组学方法来分析ub偶联物并评估假阳性。这些定量质谱分析策略将用于研究不同条件下泛素化蛋白质组的动态。总之，这些研究将为探索泛素化蛋白质组和更好地理解多聚脲链结构的异质性提供必要的工具，多聚脲链结构在泛素信号传导和疾病发展中起着重要作用。

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.