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 protein products encoded by the human genome. In order to accomplish this daunting task, methods will be required that facilitate the global analysis of proteins in complex biological systems. Recently, methods have been described for simultaneously monitoring the activity of multiple enzymes in crude proteomes based on their reactivity with tagged chemical probes. These activity based probes (ABPs) have used either radiochemical or biotin/avidin-based detection methods to allow consolidated visualization of numerous enzyme 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, sensitivity, and quantitative accuracy when compared to previously described ABPs, and provide a straight-forward platform for high-throughput proteome analysis.
Patricelli
M P
MP
ActivX Biosciences, La Jolla, CA 92037, USA. mattp@activx.com
Giang
D K
DK
Stamp
L M
LM
Burbaum
J J
JJ
D000818:Animals; D019556:COS Cells; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000378:metabolism*; D006801:Humans; D015336:Molecular Probe Techniques; D015394:Molecular Structure; D020543:Proteome; D051381:Rats; D012694:Serine / Q000031:analogs & derivatives*; D012697:Serine Endopeptidases / Q000378:metabolism*
D005456:Fluorescent Dyes; D020543:Proteome; C028002:serine hydroxamate; D012694:Serine; D012697:Serine Endopeptidases