A strategy to profile prime and non-prime proteolytic substrate specificity

A strategy was developed to determine the prime and non-prime substrate specificity of serine, threonine and cysteine proteases. ACC positional scanning technology was employed to determine the P4–P1 non-prime site substrate specificity. The data was used to synthesize biased donor–quencher positional scanning libraries to profile the P1′–P4′ prime site substrate specificity. Directed sorting using the Irori Nanokan system allowed for the archiving of multiple P1′–P4′ positional scanning libraries. From these libraries focused donor–quencher libraries incorporating P4–P1 data for each protease under study could be rapidly prepared. The profiling of thrombin and caspase-3 P4–P4′ substrate specificity, comparison of the library specificity data to single substrates, and the analysis of physiological cleavage sites are described.