Cracking the Ubiquitination Code by Top Down Mass Spectrometry

 描述（由申请人提供）：泛素化机制将泛素的单个分子或链连接到底物蛋白，从而靶向它们进行降解，调节细胞定位，调节炎症反应，并介导蛋白质-蛋白质相互作用。由于异常泛素化导致的癌蛋白和肿瘤抑制因子的无效清除可能诱导恶性转化。泛素化系统与癌症之间的关系是一个复杂的难题，有许多未解决的问题，涉及泛素化在介导细胞增殖和凋亡中的作用。泛素链的长度和连接的变化导致了这个问题的复杂性，并使其成为一个重大的分析挑战。有7个赖氨酸残基的泛素作为潜在的多聚泛素连接位点，每一个具有不同的反应性，每个推测调节不同的细胞途径。绘制泛素化图谱的能力是建立对链长度、连接性和编码蛋白质命运的功能作用之间相互作用的全面理解的第一步。该建议解决了这个问题的发展，自上而下的紫外光解离（UVPD）质谱鉴定的目标蛋白质的分布和泛素链的连接。本论文的主要目标包括：（1）建立UVPD-MS、MS 3和中下策略用于泛素链的表征。将通过UVPD-MS和MS 3（主要是HCD，然后是选定大碎片离子的UVPD）方法分析Lys 48和Lys 63连接的二聚体和三聚体以及线性和Lys 48连接的四聚体、五聚体和较大的多聚体，以评价连接位置和分支的测定。NanoLC条件将被优化用于分离和分析聚泛素混合物。(2)生理靶点的方案测试和开发：p21、NF-κ B抑制剂α和p53的泛素化种类的表征。将对蛋白酶体抑制（硼替佐米）和去泛素化酶抑制前后的三种靶蛋白进行高分辨率UVPD质谱分析，以确定泛素链长的分布和连接的化学计量。 自上而下UVPD-MS技术的发展和应用将有助于解读蛋白质泛素化的范围，并支持构建泛素化状态与癌症之间的相关性。

 DESCRIPTION (provided by applicant): The ubiquitination machinery attaches single molecules or chains of ubiquitins to substrate proteins, thus targeting them for degradation, regulating cellular localization, modulating the inflammatory response, and mediating protein-protein interactions. Ineffective removal of oncoproteins and tumor suppressors due to abnormal ubiquitination may induce malignant transformation. The relationship between the ubiquitination system and cancer is a complicated puzzle with numerous unresolved questions related to the role of ubiquitination in mediating cellular proliferation and apoptosis. The variation in lengths and linkages of the ubiquitin chains contribute to the complexity of this problem and make it a significant analytical challenge. There are seven lysine residues of ubiquitin which serve as potential poly-ubiquitin linkage sites, each with varying reactivities and each presumed to modulate different cellular pathways. The ability to map ubiquitination is the first step towards building a comprehensive understanding of the interplay between chain length, connectivity, and functional role in encoding protein fate. This proposal addresses this problem by the development of top down ultraviolet photodissociation (UVPD) mass spectrometry for identification of the distribution and linkages of ubiquitin chains on targeted proteins. The two proposed aims include: (1) Development of UVPD-MS, MS3, and middle down strategies for characterization of ubiquitin chains. Lys48- and Lys63-linked dimers and timers, and linear and Lys48-linked tetramers, pentamers and larger multimers will be analyzed by UVPD-MS and MS3 (primarily HCD followed by UVPD of selected large fragment ions) methods to evaluate the determination of linkage position and branching. NanoLC conditions will be optimized for separation and analysis of polyubiquitin mixtures. (2) Protocol testing and development on physiological targets: characterization of ubiquitinated species of p21, NF-kΒ inhibitor alpha, and p53. High resolution UVPD mass spectrometric analysis of three target proteins before and after proteasome inhibition (bortezomib) and deubiquitinating enzyme inhibition will be undertaken to determine the distribution of ubiquitin chain lengths and stoichiometry of the linkages. The development and application of top-down UVPD-MS will help unscramble the scope of ubiquitination of proteins and support the construction of correlations between the status of ubiquitination and implications for cancer.