Proteomic profiling of cathepsin specificity by FPPS

Identification of protease substrates and determination of their specificities can provide important insights into their function. In the past decade, several proteomic approaches for identification of protease cleavage sites were developed, each relying on various strategies for peptide labelling and enrichment. Those approaches often rely on several experimental steps, amongst others involving sample labelling and/or several cycles of chromatographic separation. Here we present a simple and straightforward approach for proteomic identification of protease specificities in a complex proteome background. By combining in solution isotopic labelling with Stage Tip fractionation we developed a simple method to profile protease specificities. We tested our approach by profiling the substrate specificities of the cathepsins K, L and S, three lysosomal proteases known to play important roles in numerous molecular processes.