Profiling of protease specificity is crucial for characterization of these important enzymes that play numerous roles in health and disease. In the past, several proteomic methods have been developed that enable profiling of protease specificities. Although able to identify thousands of protease cleavage events, these degradomics approaches are often time consuming and methodologically challenging, which limits their application to specialized proteomic groups or the laboratories that initially introduced them. The FPPS approach described here (i.e., fast proteomic profiling of protease specificity) is simple and straightforward and produces reliable results comparable to other more elaborate procedures. It employs labeling the novel N-termini generated by the protease under investigation and subsequent peptide fractionation on SAX-C18 Stage Tips. The procedure can be performed in 2 days, does not require peptide enrichment steps, and can thus be implemented in any proteomic laboratory.
Keywords: In-solution labeling; Intact protein-based cleavage site discovery; N-terminomics; Protease specificity profiling.