Analysis of Protein-Ligand Binding on the Proteomic Scale

描述(由申请人提供)：蛋白质-配体结合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。目前用于分析蛋白质-配体结合作用的分析方法不具备在蛋白质组学规模上表征蛋白质-配体结合作用所必需的吞吐量、通用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了解。本文提出了一种蛋白质-配体结合平台的开发，该平台能够在蛋白质组尺度上进行定量分析，并且适用于包括小分子、DNA、多肽和蛋白质在内的多种配体类型。该平台利用一种基于H/D交换和质谱学的技术，称为SUPREX，用于高通量和定量的蛋白质-配体结合作用的热力学分析。这项工作的具体目标集中在该平台的开发、验证和初步应用，以分析55蛋白的大肠杆菌噬菌体T7蛋白质组中的蛋白质网络，分析酵母细胞信号通路中涉及的11种蛋白质的子集中的蛋白质-蛋白质相互作用，以及使用酵母蛋白质组中的选定蛋白质进行药物作用模式研究。在拟议的工作中，我们将(1)使用三种不同的方案来表征本研究中每个模型蛋白质的热力学性质；(2)优化和评估不同的方案(1)根据它们的吞吐量和多路传输能力；(3)使用优化的方案来检测和量化55个蛋白质T7蛋白质组之间以及11个酵母蛋白之间的蛋白质-蛋白质相互作用；(4)展示该平台能够识别免疫抑制剂环孢菌素A(CsA)与酵母中的11个细胞信号蛋白的靶上和靶外相互作用；以及(5)在药物作用模式研究中利用该平台，其中将筛选他莫昔芬与一组1427种酵母蛋白的结合，以便更好地了解这种乳腺癌药物的多效性。目前用于蛋白质-配体结合的公共卫生相关性分析不具备在蛋白质组尺度上表征蛋白质功能和药物作用所必需的吞吐量、通用性和定量能力的组合。本文提出了一种可在蛋白质组水平上进行定量分析的蛋白质-配体结合平台的开发。这项工作将侧重于该平台的优化、验证和初步应用，以分析55蛋白大肠杆菌噬菌体T7蛋白质组中的蛋白质网络，分析酵母细胞信号通路中涉及的11种蛋白质的子集中的蛋白质-蛋白质相互作用，以及使用酵母蛋白质组中的蛋白质进行药物作用模式研究；但是，该平台将是可扩展的，并直接适用于其他蛋白质组中蛋白质-配体结合的定量分析。

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.