Analysis of Protein-Ligand Binding on the Proteomic Scale

摘要 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。用于分析蛋白质-配体结合相互作用的当前测定法 不具备必要的吞吐量、通用性和定量能力， 在蛋白质组学规模上表征蛋白质-配体结合相互作用。这最终排除了 对蛋白质功能和药物作用有全面的了解。这里提出的是发展一个 蛋白质-配体结合平台，其适于蛋白质组学规模的定量分析， 其可用于广泛的配体类型，包括小分子、DNA、肽和蛋白质。 该平台利用了一种基于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是侧重于平台的开发、验证和初步实施。 应用于55蛋白大肠杆菌（Escherichia coli，E.大肠杆菌噬菌体 T7蛋白质组，用于分析酵母中涉及的11种蛋白质的子集中的蛋白质-蛋白质相互作用 细胞信号传导途径，以及使用酵母中选定的蛋白质进行药物作用模式研究 蛋白质组在所提出的工作中，我们将（1）表征每个模型的热力学性质 蛋白质在这项研究中使用三种不同的协议;（2）优化和评估不同的协议， (1)在它们的吞吐量和多路复用能力方面;（3）使用优化的协议来检测和 量化55种蛋白质T7蛋白质组之间和11种酵母之间的蛋白质-蛋白质相互作用 参与Ca +2介导的细胞信号传导途径的蛋白质;（4）证明该平台能够 确定免疫抑制药物环孢菌素A（CsA）的靶向和脱靶相互作用， 与酵母中的11种细胞信号蛋白;以及（5）在药物作用模式研究中利用该平台， 将筛选哪种他莫昔芬与一组1427种酵母蛋白的结合， 了解这种乳腺癌药物的多效性。

ABSTRACT 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 Ca+2-mediated 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.