Direct visualization of serine hydrolase activities in complex proteomes using fluorescent active site-directed probes

The field of biochemistry is currently faced with the enormous challenge of assigning functional significance to more than thirty thousand predicted p rotein products encoded by the human genome. In order to accomplish this da unting task, methods will be required that facilitate the global analysis o f proteins in complex biological systems. Recently, methods have been descr ibed for simultaneously monitoring the activity of multiple enzymes in crud e proteomes based on their reactivity with tagged chemical probes. These ac tivity based probes (ABPs) have used either radiochemical or biotin/avidin- based detection methods to allow consolidated visualization of numerous enz yme activities. Here we report the synthesis and evaluation of fluorescent activity based probes for the serine hydrolase super-family of enzymes. The fluorescent methods detailed herein provide superior throughput, sensitivi ty, and quantitative accuracy when compared to previously described ABPs, a nd provide a straightforward platform for high-throughput proteome analysis .