Analysis of Protein-Ligand Binding on the Proteomic Scale

描述（由申请方提供）：蛋白质-配体结合相互作用的检测和定量对于了解蛋白质功能和药物作用至关重要。目前用于分析蛋白质-配体结合相互作用的测定不具有在蛋白质组学规模上表征蛋白质-配体结合相互作用所必需的通量、一般性和定量能力的组合。这最终妨碍了对蛋白质功能和药物作用的完全理解。这里提出的是一种蛋白质-配体结合平台的开发，该平台适合于蛋白质组学规模的定量分析，并且可用于广泛的配体类型，包括小分子、DNA、肽和蛋白质。该平台利用H/D交换和质谱技术，称为SUPREX，用于蛋白质-配体结合相互作用的高通量和定量热力学分析。这项工作的具体目标是集中在该平台的开发，验证和初步应用于分析的蛋白质网络在55蛋白大肠杆菌（E。coli）噬菌体T7蛋白质组，分析参与酵母细胞信号传导途径的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。本论文的主要工作是：（1）利用三种不同的方法对模型蛋白质的热力学性质进行表征;（2）对（1）中的不同方法进行优化和评价，以提高其通量和多重性;（3）采用优化的方法对蛋白质进行检测和定量-55种蛋白质T7蛋白质组和11种酵母蛋白质之间的蛋白质相互作用参与了一个介导的细胞信号传导途径;（4）证明该平台能够识别免疫抑制药物环孢菌素A（CsA）与酵母中的11种细胞信号蛋白的靶向和脱靶相互作用;和（5）在药物作用模式研究中利用该平台，其中筛选他莫昔芬与一组1427种酵母蛋白的结合，以便更好地理解这种乳腺癌药物的多效性活性。公共卫生相关性目前的蛋白质-配体结合测定不具有在蛋白质组学规模上表征蛋白质功能和药物作用所需的通量、通用性和定量能力的组合。这里提出的是一个蛋白质-配体结合平台的发展，是服从定量分析的蛋白质组学规模。这项工作将集中在该平台的优化，验证和初步应用于分析55蛋白E中的蛋白质网络。大肠杆菌噬菌体T7蛋白质组，蛋白质-蛋白质相互作用的分析在一个子集的11个蛋白质参与酵母细胞信号通路，并使用酵母蛋白质组中的蛋白质药物作用模式的研究，但该平台将是可扩展的，并直接适用于定量分析蛋白质-配体结合在其他蛋白质组。

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.