In this review we describe in detail the available technologies used for investigating the substrate specificity of proteases. Critical comparison of the available detection methods and their choice for certain type of screening is discussed. We present successful strategies along with appropriate examples for the design and synthesis of combinatorial libraries of substrates using both chemical and biological approaches. Proteomic tools for the identification of natural substrates of proteases are also discussed.
Keywords: Protease, substrate, library, fluorogenic, substrate specificity, combinatorial, positional scanning substrate combinatorial library, MEROPS, Activity Based Probes, ABPs, AMC, 7-amino-4-methylcoumarin, AFC, 7-amino-4-trifluoromethylcoumarin, ACC, 7-amino-4-carbamoylmethylcoumarin, 6-amino-1-naphtalenesulfonamide, ANSN, substrate activity screening, SAS, N-acyl-7-amino-4-methylcoumarin acetic acid, AMCA, p-nitroanilide, pNA, 5-fluorosalicylic acid, fsa, Tb(EDTA), rhodesain, Trypanasoma brucei, quencher, Leishmania mexicana, stromelysin, matrilysin, collagenase-3, MMP-2, MT1-MMP, furin, thrombin, granzyme B, PROTOMAP, iTRAQ, iCat, PICS, Proteomic Identification of protease Cleavage Sites, serine proteases, aspartic proteases, cysteine proteases