| Proteomics research requires methods to characterize the expression and function of proteins in complex mixtures. Toward this end, chemical probes that incorporate known affinity labeling agents have facilitated the activity-based profiling of certain enzyme families. This thesis describes the development of a non-directed, or combinatorial, strategy to accelerate the discovery of proteomics probes for enzyme classes lacking cognate affinity labels. Members of a probe library bearing a sulfonate ester chemotype were screened against complex proteomes for activity-dependent protein reactivities. Most proteins showed preferential labeling with specific members of the library, indicating that the varied binding group element was at least in part specifying the proteome reactivity of the probes. Biotin-tagged or trifunctional probes were applied to the affinity isolation of these reactivities which were subsequently identified by mass spectrometry methods as sulfonate targets from nine mechanistically distinct enzyme classes. Surprisingly, none of these enzymes represented targets of previously described proteomics probes. The identification of the amino acid residue(s) labeled in several of these enzyme targets confirmed that sulfonate esters function as active site-directed probes and are capable of profiling enzymes by modification of a variety of active site residues. The sulfonate library was also used to identify several targets, including glutathione S-transferase GSTO 1-1, tissue transglutaminase, and platelet-type phosphofructokinase, that were found to be upregulated in invasive human breast cancer cell lines. These results indicate that activity-based probes compatible with whole-proteome analysis can be developed for numerous enzyme classes and applied to identify novel protein markers of discrete pathological states. |
