The development and application of methods for activity-based protein profiling

Considering that proteins mediate nearly all biochemical events underlying cell and organismal physiology and pathophysiology, the need to develop general methods to measure levels and activities of these biomolecules directly in cell, and tissue proteomes is apparent. The ability to profile classes of proteins based on activity would greatly accelerate assignment of protein function and identification of new biomarkers and therapeutic targets for the diagnosis and treatment of human disease. These issues have engendered a new breed of comparative proteomic strategies, in which the complex array of proteins expressed by a given biological sample are parceled into manageable fractions prior to analysis.; Described here is a chemical proteomics method, "activity-based protein profiling" (ABPP), that was developed to compliment conventional genomic and proteomic methods that focus on measuring abundance rather than activity. ABPP employs chemical probes to covalently label active sites of enzyme superfamilies in a manner that provides a direct readout of changes in catalytic activity. By providing a covalent link between labeled proteins and a chemical tag, ABPP permits the consolidated detection, isolation, and identification of active enzymes directly from samples of high biological complexity.; This work represents the application of ABPP for the functional analysis of human cancer. First, we demonstrate the utility of ABPP in classifying phenotypically distinct classes of human cancer lines based on enzyme activity profiles of their secreted and membrane proteomes. These studies are then extended to the profiling of primary human tumors as well as an established mouse model of human cancer, demonstrating the dramatic functional differences that exist between cancer cells grown in culture or in the context of in vivo tumor growth, both of which serve as important, widely used, research models of human cancer. Also shown, is the use of ABPP to assign novel constituents to otherwise well characterized enzyme families. Finally, the development and application of activity-based probes directed at the metalloprotease family of enzymes is described.; Collectively, these studies establish ABPP as a powerful strategy for the functional analysis of large panels of cells/tissues, or to obtain in-depth information on a few samples, or even individual enzymes.