A proteomics approach to ubiquitination

描述(由申请人提供)： 本研究的目的是建立一种基于高分辨质谱学的蛋白质组学方法，从肿瘤组织中分离和定量泛素化蛋白质组。泛素修饰通过将泛素共价连接到靶底物来调节真核细胞中的各种细胞事件。泛素途径的失调参与了多种癌症的发病机制。然而，目前还没有可靠的方法来全面分析癌症样本中的泛素化蛋白。最近，我们在开发基于质谱学的分析酵母泛素化蛋白质组的新技术方面取得了进展。在这里，我们建议开发一种通用的方法来捕获和量化哺乳动物细胞中泛素化的蛋白质组。该方法将利用由泛素结合域组成的亲和层析。将检测一系列具有不同泛素结合域的融合蛋白，以优化结合亲和力，以改进泛素底物的产量和纯度方面的方法。分离的泛素化蛋白将通过质谱学进一步分析，以确定蛋白质的同一性和泛素化位点。定量质谱学策略将被用来研究泛素化蛋白质组的动力学。一旦建立，该方法将对包括临床肿瘤组织在内的哺乳动物样本中泛素化蛋白质组的图谱非常有用。

DESCRIPTION (provided by applicant): The goal of this research is to develop a proteomics approach to isolate and quantify ubiquitinated proteome from tumor tissues based on high resolution mass spectrometry. Ubiquitin modification regulates a variety of cellular events in eukaryotic cells by covalently attaching ubiquitin to target substrates. The dysregulation of ubiquitin pathways has been involved in the pathogenesis of many types of cancers. However, there is no reliable method to globally analyze ubiquitinated proteins in cancer samples. Recently, we have made progress in developing novel mass spectrometry-based technologies to analyze ubiquitinated proteome in yeast. Here we propose to develop a generic method to capture and quantify ubiquitinated proteome from mammalian cells. The approach will utilize affinity chromatography composed of ubiquitin-binding domains. An array of fusion proteins with various ubiquitin-binding domains will be examined to optimize the binding affinity in order to improve the method with respect to the yield and purity of ubiquitin substrates. The isolated ubiquitinated proteins will be further analyzed by mass spectrometry for the determination of protein identity and ubiquitination sites. Quantitative mass spectrometry strategies will be implemented to investigate the dynamics of ubiquitinated proteome. Once established, the method will be highly useful for profiling ubiquitinated proteome in mammalian samples including clinical tumor tissues.