Analysis of Protein-Ligand Binding on the Proteomic Scale

摘要 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。蛋白质-配体相互作用分析的最新方法 不具备以下方面所需的吞吐量、通用性和量化能力 在蛋白质组学水平上表征蛋白质-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。在此建议开发一种 蛋白质-配基结合平台，能够在蛋白质组尺度上和 这对包括小分子、DNA、多肽和蛋白质在内的各种配体类型都很有用。 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-配体相互作用的热力学分析。 这项工作的具体目标是平台的开发、验证和初步 55蛋白大肠杆菌噬菌体蛋白质网络分析的应用 T7蛋白质组，用于分析酵母中涉及的11种蛋白质的蛋白质-蛋白质相互作用 细胞信号途径，以及利用酵母中选定的蛋白质进行药物作用模式研究 蛋白质组。在拟议的工作中，我们将(1)描述每个模型的热力学性质 在本研究中使用了三种不同的方案；(2)对不同的方案进行了优化和评估 (1)在吞吐量和多路传输容量方面；(3)使用优化的协议来检测和 量化55个蛋白质T7蛋白质组之间和11个酵母之间的蛋白质-蛋白质相互作用 参与Ca+2介导的细胞信号通路的蛋白质；(4)展示平台的能力 确定免疫抑制药物环孢素A(CsA)的靶上和靶外相互作用， 在酵母中使用11种细胞信号蛋白；以及(5)将该平台用于 为了更好地与一组1427种酵母蛋白结合，将对哪种他莫昔芬进行筛选 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.