Quantitative Substrate Kinetics Screening: One-Pot LC/MS Based Approach to Map Protease Specificity

Proteases play critical roles in many biological processes and diseases. Proteases tend to have limited and overlapping specificities, which hinders our understanding of their roles in physiology. We designed a naïve protease substrate library with the aim of fully utilizing modern mass spectrometry proteomics tools to quantitatively map protease substrate specificity. A library of approximately 200,000 peptides was efficiently produced in E. coli with sufficient library diversity to identify cleavage efficiency of thousands of substrates for most proteases in a single assay. Each experiment results in ample substrate kinetics data to build a positional specificity matrix that quantitatively maps protease specificity. We apply this method to two different proteases, GluC commonly used as a proteomics tool, and Fibroblast Activation Protein Alpha that is overexpressed in activated fibroblasts of epithelial cancers and fibrotic diseases. This research quantitatively maps substrate specificity for GluC and FAP in a single assay and can be applied to map substrate specificity for most proteases. Graphical Abstract