Analysis of Protein-Ligand Binding on the Proteomic Scale

描述（由申请人提供）：在过去十年中，基于基因组和蛋白质组的基因表达谱研究已被广泛用于表征生物学状态和药物作用。虽然这种差异基因表达谱研究可以帮助鉴定与生物状态或与治疗剂的生物活性相关的细胞途径和生理过程，但它们通常不能产生对生物状态或药物作用模式的详细分子水平理解，因为基因表达水平不直接与蛋白质功能相关。这限制了有用的蛋白质生物标志物的数量 以及从这些研究中发现的治疗靶点，并在我们对药物作用的理解中留下了空白。这里提出的是使用蛋白质稳定性的热力学测量来描绘与不同生物状态和药物作用相关的蛋白质相互作用网络。预计蛋白质稳定性的这种热力学测量与蛋白质功能的关系比蛋白质表达水平的关系更密切，从而产生更有用的蛋白质生物标志物和疾病的治疗靶点，并产生对药物作用的更好理解。 拟议的工作将使用一种基于化学修饰和质谱的方法，称为SPROX，在几种不同的年龄，疾病和药物相关的生物状态下，在蛋白质组学规模上对蛋白质稳定性进行热力学测量。本研究的具体目标是：（1）简化和改进全蛋白组SPROX实验中的数据分析流程;（2）利用SPROX技术表征来自四种不同乳腺癌细胞培养模型的蛋白质的热力学稳定性，并确定哪些蛋白质在不同细胞培养模型中的稳定性发生了改变;（3）使用SPROX技术表征来源于在存在和不存在他莫昔芬的情况下生长的乳腺癌细胞的蛋白质的热力学稳定性，并确定哪些蛋白质在他莫昔芬的存在下具有改变的稳定性;（4）使用SPROX技术表征源自小鼠衰老模型的小鼠蛋白质的热力学稳定性，并确定哪些蛋白质具有作为年龄的函数的改变的稳定性;和（5）使用（2）-（4）中鉴定的蛋白质来表征与不同年龄，疾病，和药物相关的生物学状态。

DESCRIPTION (provided by applicant): Genome- and proteome-based gene expression profiling studies have been widely used over the past decade to characterize biological states and drug action. While such differential gene expression profiling studies can help identify the cellular pathways and physiological processes associated with a biological state or with the biological activity of a therapeutic agent, they often fail to produce a detailed molecular level understanding of the biological state or drug mode-of-action because gene expression levels are not directly tied to protein function. This has limited the number of useful protein biomarkers and therapeutic targets discovered from such studies, and left gaps in our understanding of drug-action. Proposed here is the use of thermodynamic measurements of protein stability to profile the protein-interaction networks associated with different biological states and drug action. Such thermodynamic measurements of protein stability are expected to be more closely related to protein function than are protein expression levels, and thus produce more useful protein biomarkers and therapeutic targets of disease and generate a better understanding of drug action. The proposed work will use a chemical modification- and mass spectrometry-based method, termed SPROX, to make thermodynamic measurements of protein stability on the proteomic scale in several different age-, disease-, and drug-related biological states. The specific aims of this work are: (1) to streamline and improve the data analysis pipeline in proteome-wide SPROX experiments; (2) to characterize the thermodynamic stability of proteins derived from four different cell culture models of breast cancer using the SPROX technique and determine which proteins have altered stabilities in the different cell culture models; (3) to characterize the thermodynamic stability of proteins derived from breast cancer cells grown in the presence and in the absence of tamoxifen using the SPROX technique and determine which proteins have altered stabilities in the presence of tamoxifen; (4) to characterize the thermodynamic stability of mouse proteins derived from a mouse model of aging using the SPROX technique and determine which proteins have altered stabilities as a function of age; and (5) to use the proteins identified in (2)-(4) to characterize the altered protein interaction networks associated with the different age-, disease-, and drug-related biological states in this study.