WebPICS Interface


Protease specificity relies largely on the active site-mediated recognition of amino acid sequences that encompass the scissile peptide bond. Mapping of active site specificity is essential to characterise substrate preferences as well as for assay development and inhibitor studies. We have developed PICS (Proteomic Identification of protease Cleavage Sites), a technique that employs proteome-derived peptide libraries as proteolytic substrate screens for the characterisation of protease active site specificity (Schilling & Overall 2008). PICS simultaneously profiles prime and non-prime specificity and allows to investigate subsite cooperativity by identifying 10s – 100s of individual cleavage products.

For PICS, proteomes such as cell lysates are endoproteolytically digested into peptides, followed by chemical protection of sulfhydryls and primary amines. This proteome-derived peptide library is employed as a protease substrate screen. Prime-side cleavage products possess free amine groups at the newly formed amino-termini. These neo amino-termini are biotinylated, affinity isolated, and identified by liquid chromatography-tandem mass spectrometry. The corresponding non-prime side sequences are derived bioinformatically by data base searches, hence reconstructing the sequence information of the full-length peptidic substrate. PICS data analysis has now been automated with the present PICS web service.

PICS has been successfully applied to serine-, cysteine-, aspartate-, and metalloproteases, including matrix metalloprotease (MMP)-2, HIV protease 1, caspases 3 and 7, cathepsins K and G, elastase and thrombin. In all cases, PICS corroborates and refines previous specificity studies. For MMP-2 alone, >1,200 peptidic cleavage sites were identified. For HIV protease 1, positive cooperativity between leucine in P3 and alanine in P1 was detected. The PICS web service offers a basic screening functionality to study potential subsite cooperativity.

In conclusion, proteome-derived, database-searchable peptide libraries are a powerful degradomic tool for the peptidic specificity determination and rapid functional profiling of proteolytic enzymes.