Microarray platform for profiling enzyme activities in complex proteomes.
Activity-based protein profiling (ABPP) is a chemical method that utilizes active-site-directed probes to determine the functional state of enzymes in complex proteomes. Probe-labeled enzymes are typically detected by in-gel fluorescence scanning, a robust technique that nonetheless exhibits some key deficiencies, including limited sensitivity and resolution, as well as ambiguity regarding the molecular identity of the enzymes under investigation. Here, we report a microarray platform for ABPP that addresses these limitations. In this platform, proteomes are treated with ABPP probes in solution, after which labeled enzymes are captured and visualized on glass slides displaying an array of anti-enzyme antibodies. We show that ABPP microarrays exhibit superior sensitivity and resolution compared to gel-based methods, permitting the parallel analysis of several enzyme activities in proteomes, including cancer-associated proteases such as urokinase, matrix metalloproteinase-9, and prostate-specific antigen.
Sieber
Stephan A
SA
The Skaggs Institute for Chemical Biology, Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Mondala
Tony S
TS
Head
Steven R
SR
Cravatt
Benjamin F
BF
D000906:Antibodies / Q000737:chemistry; D001577:Benzophenones / Q000737:chemistry; D001665:Binding Sites; D020780:Matrix Metalloproteinase 9 / Q000276:immunology / Q000378:metabolism*; D040081:Protein Array Analysis / Q000379:methods*; D020543:Proteome / Q000032:analysis / Q000378:metabolism*; D012235:Rhodamines / Q000737:chemistry; D012680:Sensitivity and Specificity; D012697:Serine Endopeptidases / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D001577:Benzophenones; D020543:Proteome; D012235:Rhodamines; D012697:Serine Endopeptidases; D020780:Matrix Metalloproteinase 9