Profiling enzyme activities in vivo using click chemistry methods.
Methods for profiling the activity of enzymes in vivo are needed to understand the role that these proteins and their endogenous regulators play in physiological and pathological processes. Recently, we introduced a tag-free strategy for activity-based protein profiling (ABPP) that utilizes the copper(I)-catalyzed azide-alkyne cycloaddition reaction ("click chemistry") to analyze the functional state of enzymes in living cells and organisms. Here, we report a detailed characterization of the reaction parameters that affect click chemistry-based ABPP and identify conditions that maximize the speed, sensitivity, and bioorthogonality of this approach. Using these optimized conditions, we compare the enzyme activity profiles of living and homogenized breast cancer cells, resulting in the identification of several enzymes that are labeled by activity-based probes in situ but not in vitro.
Speers
Anna E
AE
The Skaggs Institute for Chemical Biology, Departments of Chemistry and Cell Biology, Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Cravatt
Benjamin F
BF
D000480:Alkynes / Q000737:chemistry; D000818:Animals; D001386:Azides / Q000737:chemistry; D001943:Breast Neoplasms / Q000201:enzymology*; D002384:Catalysis; D045744:Cell Line, Tumor; D003300:Copper / Q000737:chemistry; D004798:Enzymes / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D006801:Humans; D008099:Liver / Q000201:enzymology; D051379:Mice; D008954:Models, Biological; D015335:Molecular Probes / Q000737:chemistry* / Q000378:metabolism*; D015394:Molecular Structure; D040901:Proteomics / Q000379:methods*; D013329:Structure-Activity Relationship
D000480:Alkynes; D001386:Azides; D004798:Enzymes; D015335:Molecular Probes; D003300:Copper