Protease specificity profiling in a pipette tip using “charge-synchronized” proteome-derived peptide libraries

About 2% of the genome of human and other organisms codes for proteases. An important step toward deciphering the biological function of a protease and designing inhibitors is the profiling of protease specificity. In this work we present a novel, label-free, proteomics-based protease specificity profiling method that only requires simple sample preparation steps. It uses proteome-derived peptide libraries and enriches the cleaved sequences using strong cation exchange chromatography (SCX) material in a pipette tip. As a demonstration of the method’s versatility, we successfully determined the specificity of GluC, caspase-3, chymotrypsin and cathepsin G from several hundreds to almost 2000 cleavage events per proteases. Interestingly, we also found and confirmed a novel intrinsic preference of cathepsin G for Asn at the P1 subsite, explaining the reported cleavage position on the P. aeruginosa aeruginosa flagellin by human cathepsin G . Overall, this method is straightforward and requires so far the lowest investment in material and equipment for protease specificity profiling. Therefore, we think it will be applicable in any biochemistry laboratory and promote an increased understanding of protease specificity.