title: Inhibitor fingerprinting of metalloproteases using microplate and microarray platforms: an enabling technology in Catalomics.
abstract: One of the most fundamental properties of an enzyme is its selectivity, a property that has proved highly challenging to understand. Recent developments offer methodologies to rapidly establish activity-dependent profiles of enzymes. In this protocol, we describe methods to elucidate inhibitor fingerprints of enzymes. By taking advantage of well-defined small-molecule inhibitor libraries and the screening throughput offered from microplate and microarray platforms, we provide step-by-step application of the methodology toward the global characterization of metalloproteases, an important class of enzymes involved in numerous diseases and cellular processes. The same strategy is nonetheless applicable to virtually any given enzyme class, provided suitable experimental design and chemical inhibitor libraries are carefully implemented. We are able to routinely fingerprint as many as 2,000 independent enzyme interactions on the microplate platform within a span of approximately 7 h; however, the same throughput is attained within 5 h on the microarray platform.
authors.affiliation: Department of Biological Science, National University of Singapore, Singapore, Republic of Singapore.
authors.forename: Wei L
authors.initials: WL
authors.lastname: Lee
authors.forename: Junqi
authors.initials: J
authors.lastname: Li
authors.forename: Mahesh
authors.initials: M
authors.lastname: Uttamchandani
authors.forename: Hongyan
authors.initials: H
authors.lastname: Sun
authors.forename: Shao Q
authors.initials: SQ
authors.lastname: Yao
mesh_terms: D002384:Catalysis; D020783:Matrix Metalloproteinase 7 / Q000737:chemistry* / Q000378:metabolism; D045726:Metalloproteases / Q000737:chemistry / Q000378:metabolism; D015336:Molecular Probe Techniques; D019151:Peptide Library; D011480:Protease Inhibitors / Q000737:chemistry*; D040081:Protein Array Analysis / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D019151:Peptide Library; D011480:Protease Inhibitors; D045726:Metalloproteases; D020783:Matrix Metalloproteinase 7