Analysis of Protein-Ligand Binding on the Proteomic Scale

蛋白质组规模上的蛋白质-配体结合分析

• 批准号: 7903143
• 负责人: Michael C Fitzgerald
• 金额: $ 32.43万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7903143
• 关键词: Antineoplastic Agents; Bacteriophage T7; Behavior; Binding; Binding Proteins; Biological Assay; Cells; Cyclosporine; DNA; Deuterium; Development; Digestion; Dissociation; Drug effect disorder; Escherichia coli; Escherichia coli Proteins; Free Energy; Hydrogen; Immunosuppressive Agents; Laboratories; Ligand Binding; Ligands; Mass Spectrum Analysis; Mediating; Modeling; Pathway interactions; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Property; Protein Analysis; Protein Binding; Proteins; Proteome; Proteomics; Protocols documentation; Research; Saccharomyces cerevisiae; Set protein; Signal Pathway; Signal Transduction; Signaling Protein; Tamoxifen; Techniques; Thermodynamics; Validation; Work; Yeasts; base; interest; malignant breast neoplasm; overexpression; oxidation; protein complex; protein function; protein protein interaction; protein quantitation/detection; public health relevance; research study; small molecule; yeast protein

DESCRIPTION (provided by applicant): The detection and quantitation of protein-ligand binding interactions is critical for understanding protein function and drug action. Current assays for the analysis of protein-ligand binding interactions do not have the combination of throughput, generality, and quantitative capabilities necessary for characterizing protein-ligand binding interactions on the proteomic scale. This ultimately precludes a complete understanding of protein function and drug action. Proposed here is the development of a protein-ligand binding platform that is amenable to quantitative analyses on the proteomic scale and that is useful with a wide range of ligand types including small molecules, DNA, peptides, and proteins. The platform exploits an H/D exchange- and mass spectrometry-based technique, termed SUPREX, for the high-throughput and quantitative thermodynamic analysis of protein-ligand binding interactions. The specific aims of this work are focused on the platform's development, validation and initial application to analysis of the protein network in the 55-protein Escherichia coli (E. coli) bacteriophage T7 proteome, to analysis of protein-protein interactions in a subset of 11 proteins involved in a yeast cell-signaling pathway, and to drug mode-of-action studies using selected proteins in the yeast proteome. In the proposed work we will (1) characterize the thermodynamic properties of each model protein in this study using three different protocols; (2) optimize and evaluate the different protocols in (1) in terms of their throughput and multiplex cababilities; (3) use the optimized protocols to detect and quantify the protein-protein interactions between the 55 proteins T7 proteome and between 11 yeast proteins in involved in a Camediated cell-signaling pathway; (4) demonstrate the platform's ability to identify the on-target and off-target interactions of the immunosuppressive drug, cyclosporin A (CsA), with the 11 cell-signaling proteins in yeast; and (5) utilize the platform in a drug mode-of-action study in which tamoxifen will be screened for binding to a set of 1427 yeast proteins in order to better understand the pleiotropic activities of this breast cancer drug. PUBLIC HEALTH RELEVANCE Current assays for protein-ligand binding do not have the combination of throughput, generality, and quantitative capabilities necessary for characterizing protein function and drug action on the proteomic scale. Proposed here is the development of a protein-ligand binding platform that is amenable to quantitative analyses on the proteomic scale. The work will focus on the platform's optimization, validation, and initial application to analysis of the protein network in the 55-protein E. coli bacteriophage T7 proteome, to analysis of protein-protein interactions in a subset of 11 proteins involved in a yeast cell-signaling pathway, and to drug mode-of-action studies using proteins in the yeast proteome; but, the platform will be scaleable and directly applicable to the quantitative analysis of protein-ligand binding in other proteomes.

描述（由申请人提供）：蛋白质 - 配体结合相互作用的检测和定量对于理解蛋白质功能和药物作用至关重要。当前用于分析蛋白质结合相互作用的测定法没有在蛋白质组学量表上表征蛋白质 - 凸结合相互作用所需的吞吐量，一般性和定量能力的组合。这最终排除了对蛋白质功能和药物作用的完全理解。此处提出的是蛋白质结合平台的开发，该平台可与蛋白质组学量表进行定量分析，并且可用于多种配体类型，包括小分子，DNA，肽和蛋白质。该平台利用了H/D交换和质谱技术，称为Suprex，用于对蛋白质 - 配体结合相互作用的高通量和定量热力学分析。这项工作的具体目的集中在平台的开发，验证和初步应用中，用于分析55个蛋白质网络中的蛋白质网络大肠杆菌（E. coli）噬菌体T7蛋白质组，用于分析蛋白质蛋白质相互作用在11个蛋白质中涉及的酵母菌细胞型蛋白质和酵母菌模式蛋白的子集中的蛋白质 - 蛋白相互作用，并使用Y型蛋白质进行了YEATS蛋白质的研究。在拟议的工作中，（1）将使用三种不同方案在本研究中表征每种模型蛋白的热力学特性； （2）根据（1）在其吞吐量和多重障碍方面优化和评估不同的协议； （3）使用优化的方案来检测和量化55蛋白T7蛋白质组之间的蛋白质 - 蛋白质相互作用，以及参与相机细胞信号途径的11种酵母蛋白之间的蛋白质； （4）证明了该平台可以鉴定免疫抑制药物Cyclosporin A（CSA）与酵母11个细胞信号蛋白的靶向和脱靶相互作用的能力； （5）在一项药物行动模式研究中利用该平台，在该研究中，将筛选他莫昔芬与一组1427种酵母蛋白结合，以更好地了解该乳腺癌药物的多效活性。公共卫生相关性的当前蛋白质结合结合的测定不具有表征蛋白质功能和药物作用在蛋白质组学量表上所必需的吞吐量，一般性和定量能力的组合。此处提出的是蛋白质 - 配体结合平台的开发，该平台可以在蛋白质组学量表上进行定量分析。这项工作将集中于该平台的优化，验证和初步应用，以分析55个蛋白质大肠杆菌噬菌体T7蛋白质组中蛋白质网络的分析，以分析涉及11种蛋白质的蛋白质 - 蛋白质相互作用的蛋白质相互作用，该子集参与酵母菌细胞信号途径的11个蛋白质，以及使用蛋白质的蛋白质研究中的酵母菌蛋白质相互作用。但是，该平台将是可扩展的，并且直接适用于对其他蛋白质组中蛋白质 - 配体结合的定量分析。