Analysis of Protein-Ligand Binding on the Proteomic Scale

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。目前用于分析蛋白质-配体结合相互作用的分析方法不具备在蛋白质组学尺度上表征蛋白质-配体结合相互作用所需的吞吐量、通用性和定量能力。这最终阻碍了对蛋白质功能和药物作用的完整理解。本文提出了一种蛋白质-配体结合平台的开发，该平台可用于蛋白质组学规模的定量分析，并可用于广泛的配体类型，包括小分子，DNA，肽和蛋白质。该平台利用基于H/D交换和质谱的技术，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集中在该平台的开发、验证和初步应用，以分析55个蛋白质的大肠杆菌（E. coli）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个蛋白质子集中的蛋白质-蛋白质相互作用，以及使用酵母蛋白质组中选定的蛋白质进行药物作用模式研究。在提出的工作中，我们将(1)使用三种不同的协议表征本研究中每个模型蛋白的热力学性质；(2)优化和评估(1)中不同协议的吞吐量和多路复用能力；(3)利用优化后的方案，检测并定量了T7蛋白组中55个蛋白与11个酵母蛋白之间介导细胞信号通路的蛋白-蛋白相互作用；(4)证明该平台能够识别酵母中免疫抑制药物环孢素A （cyclosporin A, CsA）与11种细胞信号蛋白的靶向和非靶向相互作用；(5)利用该平台进行药物作用模式研究，筛选他莫昔芬与1427组酵母蛋白的结合，以便更好地了解这种乳腺癌药物的多效性活性。目前的蛋白质-配体结合检测方法不具备在蛋白质组学尺度上表征蛋白质功能和药物作用所需的吞吐量、通用性和定量能力。这里提出的是一种蛋白质-配体结合平台的发展，适用于蛋白质组学规模的定量分析。这项工作将侧重于平台的优化、验证和初始应用，以分析55个蛋白质的大肠杆菌噬菌体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个蛋白质子集中的蛋白质-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，该平台将具有可扩展性，并可直接应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.