Chemical Approaches for Activity Based Proteomics

基于活性的蛋白质组学的化学方法

• 批准号: 7227508
• 负责人: BENJAMIN F CRAVATT
• 金额: $ 32.04万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7227508
• 关键词: Active Sites; Address; Alkynes; Analytical Biochemistry; Analytical Chemistry; Azides; Biological; Biological Markers; Breast; Cancer cell line; Cell model; Cell physiology; Cells; Cellular biology; Charge; Chemicals; Class; Complex; Diagnosis; Diagnostic Neoplasm Staging; Dipeptides; Disease; Drug resistance; Endopeptidases; Enzymes; Exhibits; Family; Gel; Genomics; Glutathione S-Transferase; Goals; Human; Immunohistochemistry; In Situ Hybridization; In Vitro; Isotope Labeling; Label; Libraries; Life; Link; Lipase; Malignant Neoplasms; Malignant neoplasm of ovary; Matrix Metalloproteinases; Measures; Metalloproteases; Methodology; Methods; Mind; Molecular; Numbers; Organism; Ovarian Tissue; Oxidoreductase; Peptide Hydrolases; Primary Neoplasm; Procedures; Property; Proteins; Proteome; Proteomics; Reaction; Reagent; Research Personnel; Resolution; Screening procedure; Staging; Synthesis Chemistry; Techniques; Testing; Variant; base; cancer cell; cycloaddition; enzyme activity; human disease; in vivo; interdisciplinary approach; novel; ovarian neoplasm; protein expression; protein function; therapeutic target

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.

描述(由申请人提供)：在后基因组时代，化学和生物研究人员负责将分子和细胞功能分配给数千种预测的基因产品。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球分析方法。传统的蛋白质组学方法测量蛋白质丰度的变化，因此只能间接估计蛋白质的活性。根据活性分析蛋白质类别的能力将大大加快蛋白质功能的分配以及为人类疾病的诊断和治疗确定新的标志物和靶点。考虑到这一目标，我们引入了一种名为基于活性的蛋白质图谱(ABPP)的化学蛋白质组学策略，它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。到目前为止，我们已经合成并应用了针对一些生物医学上重要的酶类的ABPP探针，包括蛋白酶、脂肪酶、脱氢酶和谷胱甘肽S转移酶。这项建议的目的是为ABPP开发新的探针和方法，并应用这些试剂和技术来识别在人类癌症中差异表达的酶活性。 在目标1中，我们将应用ABPP探针来确定人类癌症和原发肿瘤细胞模型中的酶活性。在目标2中，我们将优化无标签ABPP的程序，以确定活的癌细胞中的酶活性。在目标3中，我们将结合“无凝胶”ABPP和同位素标记来创建一个用于定量分析酶活性的通用平台。在目标4中，我们将为几种新的酶类开发ABPP探针。我们预计这些研究将：1)确定在癌症中上调的酶活性，这些酶活性可能代表诊断和治疗这种疾病的新的生物标记物和/或治疗靶点；2)产生关键的方法学进展，使ABPP能够在体外和体内广泛应用于多种酶类。