Cracking the Ubiquitination Code by Top Down Mass Spectrometry

通过自上而下的质谱法破解泛素化密码

• 批准号: 8959461
• 负责人: Jennifer S. Brodbelt
• 金额: $ 18.94万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-09 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8959461
• 关键词: Address; Affinity Chromatography; Amines; Amino Acids; Analytical Biochemistry; Apoptosis; Biochemical; Biological Process; Bortezomib; C-terminal; Cell Cycle Regulation; Cell Line; Cell Proliferation; Cell physiology; Code; Deubiquitinating Enzyme; Development; Disease; Enzyme Inhibition; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Evaluation; Excision; Foundations; Genetic Transcription; Glycine; Immune response; Inflammatory Response; Ions; Length; Light; Link; Lysine; Malignant - descriptor; Malignant Neoplasms; Mammalian Cell; Maps; Mass Spectrum Analysis; Mediating; Methodology; Methods; Molecular Analysis; Monitor; Multiple Myeloma; Oncogene Proteins; Pathway interactions; Pattern; Physiological; Play; Polyubiquitin; Positioning Attribute; Post-Translational Protein Processing; Process; Proteasome Inhibition; Proteasome Inhibitor; Protein Fragment; Proteins; Proteolysis; Protocols documentation; Research; Resolution; Role; Side; Site; Sum; System; Testing; Tumor Suppressor Proteins; Ubiquitin; Ubiquitination; Variant; age related; analytical method; anti-cancer therapeutic; dimer; functional outcomes; inhibitor/antagonist; multicatalytic endopeptidase complex; neoplastic cell; protein degradation; protein misfolding; protein protein interaction; public health relevance; single molecule; stoichiometry; tumorigenesis; ultraviolet

 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.

 描述（由申请人提供）：泛素化机制将单分子或泛素链附着到底物蛋白上，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，有效去除癌蛋白和肿瘤抑制因子。由于泛素化异常可能诱发恶性转化，泛素化系统与癌症之间的关系是一个复杂的谜题，有许多与其作用相关的未解决的问题。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。泛素有七个赖氨酸残基可作为潜在的多泛素连接位点。每个都有不同的反应性，并且每个都被认为调节不同的细胞途径。绘制泛素化图谱的能力是全面了解链长度、连接性和功能作用之间相互作用的第一步。该提案通过开发自上而下的紫外光解离 (UVPD) 质谱来解决这一问题，以鉴定目标蛋白上泛素链的分布和连接。提出的两个目标包括：(1) 开发 UVPD-。用于表征 Lys48 和 Lys63 连接的二聚体和计时器，以及线性和 Lys48 连接的四聚体、五聚体的 MS、MS3 和中下策略。较大的多聚体将通过 UVPD-MS 和 MS3（主要是 HCD，然后是选定的大片段离子的 UVPD）方法进行分析，以评估连接位置和分支的确定，并将优化用于多泛素混合物的分离和分析 (2. ) 生理目标的方案测试和开发：对 p21、NF-kβ 抑制剂 α 和 p53 的泛素化种类进行前后三种目标蛋白的高分辨率 UVPD 质谱分析的表征。将采用蛋白酶体抑制（硼替佐米）和去泛素化酶抑制来确定泛素链长度的分布和化学计量。自上而下的 UVPD-MS 的开发和应用将有助于阐明蛋白质泛素化的范围并支持构建。泛素化状态与癌症影响之间的相关性。