Core B: Proteomics and Protein Analysis Core

核心 B：蛋白质组学和蛋白质分析核心

• 批准号: 9121496
• 负责人: Mamuka Kvaratskhelia
• 金额: $ 18.71万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9121496
• 关键词: Affinity; Amino Acids; Antibodies; Antibody Formation; Binding; Biochemical; Biological; Cancer Model; Collaborations; Column Chromatography; Complex; Comprehensive Cancer Center; Energy Transfer; Escherichia coli; Fluorescence; Funding; Goals; Human Resources; Human T-lymphotropic virus 1; Individual; Mammalian Cell; Maps; Mass Spectrum Analysis; Measures; Monitor; Nucleic Acids; Nucleoproteins; Oligonucleotides; Peptides; Pharmacy facility; Play; Post-Translational Protein Processing; Preparation; Protein Analysis; Protein Chemistry; Protein Footprinting; Proteins; Proteomics; Reader; Recombinant Proteins; Research; Resources; Retroviridae; Role; Sampling; Services; Site; T-Cell Leukemia; Technology; Testing; Time; Validation; Veterinary Medicine; base; biophysical properties; college; cost; cost effective; design; fast protein liquid chromatography; instrumentation; meetings; milligram; mutant; novel; protein E; protein complex; protein protein interaction; research study; skills; virology

PROJECT SUMMARY Core B will provide proteomics and protein analysis services for PPG projects. Throughout the last funding cycle, the protein analysis component of Core B has significantly expanded to meet the needs of the individual projects in the PPG. In addition to mass spectrometry (MS)-based footprinting technology for the mapping of protein-protein and protein-nucleic acid interactions proposed in the previous application, we have added the following new services to the Core: 1) the AKTA-fast protein liquid chromatography (FPLC), which has played a critical role in obtaining purified recombinant proteins for biochemical/biophysical characterization for former Project 3 and antibody production for former Project 2; and 2) the EnSpire multimode plate reader to provide a new service for highly sensitive, rapid and robust monitoring of protein-protein and protein-nucleic acid interactions. MS-based proteomic approaches enabled us to identify new protein-protein interactions and posttranslational modifications, and have thus been instrumental for advancing the research of former Projects 1 and 2, respectively. The above technologies will continue to play an important role in accomplishing the research objectives of the PPG projects proposed in this competitive renewal. In particular, Specific Aims of the Proteomics and Protein Analysis Core are: Aim 1: to perform MS-based protein footprinting experiments to identify contact amino acids in the context of large biologically relevant nucleoprotein and protein-protein complexes; Aim 2: to develop novel applications of the EnSpire multimode plate reader for biochemical characterization of protein-nucleic acid and protein-protein interactions using homogeneous time-resolved fluorescence energy transfer (HTRF), AlphaLISA, and AlphaScreen (ALPHA) based technologies; Aim 3: to provide a column chromatography service using AKTA-FPLC to assist individual PPG projects to isolate low abundant native protein-protein complexes from mammalian cells and purify mg quantities of poorly soluble recombinant proteins from E. coli for their subsequent biochemical and biophysical characterization; Aim 4: to conduct MS-based proteomics experiments to identify interacting partners and protein posttranslational modifications. Taken together, Core B will provide the wide range of protein analysis services. Importantly, these services will be utilized by all four PPG projects.

项目摘要 核心B将为PPG项目提供蛋白质组学和蛋白质分析服务。在最后一个资金中 循环，核心B的蛋白质分析成分已显着扩展以满足个人的需求 PPG中的项目。除了质谱法（MS）的足迹技术，用于映射 蛋白质 - 蛋白质蛋白和蛋白质核酸相互作用在上一个应用中提出，我们添加了 遵循核心的新服务：1）播放的Akta-fast蛋白液相色谱（FPLC） 以前 前项目2的项目3和抗体生产； 2）Enspire多模板读取器提供 用于高度敏感，快速且可靠的蛋白质 - 蛋白质和蛋白质核酸的新服务 互动。基于MS的蛋白质组学方法使我们能够确定新的蛋白质 - 蛋白质相互作用和 翻译后修改，因此有助于推进以前项目的研究 分别为1和2。上述技术将继续在完成 PPG项目的研究目标是在此竞争性更新中提出的。特别是，特定目标 蛋白质组学和蛋白质分析核心是：目标1：进行基于MS的蛋白质足迹实验 在大型生物学相关的核蛋白和蛋白质蛋白质的背景下，鉴定接触氨基酸 复合物；目的2：开发Enspire多模板读取器的新颖应用 使用均匀时间分辨的蛋白质核酸和蛋白质 - 蛋白质相互作用的表征 基于荧光能量转移（HTRF），α和α-基于α的技术；目标3：到 使用AKTA-FPLC提供列色谱服务，以帮助各个PPG项目隔离低 来自哺乳动物细胞的丰富天然蛋白质 - 蛋白质复合物，并净化毫克可溶量的Mg量 大肠杆菌的重组蛋白随后的生化和生物物理表征；目标4：到 进行基于MS的蛋白质组学实验，以鉴定相互作用的伴侣和翻译后蛋白质 修改。综上所述，核心将提供广泛的蛋白质分析服务。重要的是， 这些服务将由所有四个PPG项目使用。