Chemical Approaches for Activity Based Proteomics

描述（由申请人提供）：在后基因组时代，化学和生物学研究人员承担着为数千种预测基因产物分配分子和细胞功能的任务。为了解决这个问题，蛋白质组学领域寻求开发和应用蛋白质表达和功能的全局分析方法。传统的蛋白质组学方法测量蛋白质丰度的变化，因此只能提供蛋白质活性的间接估计。基于活性分析蛋白质种类的能力将大大加快蛋白质功能的分配，并为诊断和治疗人类疾病识别新的标记和目标。考虑到这一目标，我们引入了一种称为基于活性的蛋白质分析（ABPP）的化学蛋白质组学策略，该策略利用活性位点定向探针直接测量复杂蛋白质组中酶活性的变化。迄今为止，我们已经合成并应用了针对许多生物医学上重要酶类的ABPP探针，包括蛋白酶、脂肪酶、脱氢酶和谷胱甘肽s -转移酶。本提案的目的是为ABPP开发新的探针和方法，并应用这些试剂和技术来鉴定人类癌症中差异表达的酶活性。

DESCRIPTION (provided by applicant): In the post-genomic era, chemical and biological researchers are charged with the task of assigning molecular and cellular functions to thousands of predicted gene products. To address this problem, the field of proteomics seeks to develop and apply methods for the global analysis of protein expression and function. Conventional proteomic approaches measure variations in protein abundance and therefore provide only an indirect estimate of protein activity. The ability to profile classes of proteins based on activity would greatly accelerate both the assignment of protein function and the identification of new markers and targets for the diagnosis and treatment of human disease. With this goal in mind, we have introduced a chemical proteomic strategy termed activity-based protein profiling (ABPP) that utilizes active site-directed probes to measure changes in enzyme activity directly in complex proteomes. To date, we have synthesized and applied ABPP probes that target a number of biomedically important enzyme classes, including proteases, lipases, dehydrogenases, and glutathione S-transferases. The objective of this proposal is to develop new probes and methodologies for ABPP and apply these reagents and techniques to identify enzyme activities that are differentially expressed in human cancer. In Aim #1, we will apply ABPP probes to identify enzyme activities in cell models of human cancer and primary tumors. In Aim #2, we will optimize procedures for tag-free ABPP to identify enzyme activities in living cancer cells. In Aim #3, we will combine "gel-free" ABPP with isotope labeling to create a general platform for the quantitative analysis of enzyme activities. In Aim #4, we will develop ABPP probes for several new enzyme classes. We anticipate that these studies will: 1) identify enzyme activities up regulated in cancer that may represent new biomarkers and/or therapeutic targets for the diagnosis and treatment of this disease, and 2) produce key methodological advances that enable the general application of ABPP to numerous enzyme classes both in vitro and in vivo.