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Molecular & Cellular Proteomics 4:626-636, 2005.
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays^*

Dhaval N. Gosalia^,, Cleo M. Salisbury^¶,, Jonathan A. Ellman^¶,|| and Scott L. Diamond^,**,||

From the Department of Bioengineering, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104; ^¶ Center for New Directions in Organic Synthesis, Department of Chemistry, University of California, Berkeley, California 94720; and ^** Department of Chemical and Biomolecular Engineering, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Proteases regulate numerous biological processes with a degree of specificity often dictated by the amino acid sequence of the substrate cleavage site. To map protease/substrate interactions, a 722-member library of fluorogenic protease substrates of the general format Ac-Ala-X-X-(Arg/Lys)-coumarin was synthesized (X = all natural amino acids except cysteine) and microarrayed with fluorescent calibration standards in glycerol nanodroplets on glass slides. Specificities of 13 serine proteases (activated protein C, plasma kallikrein, factor VIIa, factor IXaß, factor XIa and factor XIIa, activated complement C1s, C1r, and D, tryptase, trypsin, subtilisin Carlsberg, and cathepsin G) and 11 papain-like cysteine proteases (cathepsin B, H, K, L, S, and V, rhodesain, papain, chymopapain, ficin, and stem bromelain) were obtained from 103,968 separate microarray fluorogenic reactions (722 substrates x 24 different proteases x 6 replicates). This is the first comprehensive study to report the substrate specificity of rhodesain, a papain-like cysteine protease expressed by Trypanasoma brucei rhodesiense, a parasitic protozoa responsible for causing sleeping sickness. Rhodesain displayed a strong P₂ preference for Leu, Val, Phe, and Tyr in both the P₁ = Lys and Arg libraries. Solution-phase microarrays facilitate protease/substrate specificity profiling in a rapid manner with minimal peptide library or enzyme usage.

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