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. ? ?
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