Mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen

Abstract Proteases are among the largest protein families and critical regulators of biochemical processes like apoptosis blood coagulation. Knowledge proteases has been expanded by development proteomic approaches, however, technology for multiplexed screening within native environments is currently lacking behind. Here we introduce a simple method to profile protease activity based on isolation products from lysates using 96FASP filter, their analysis in mass spectrometer custom data pipeline. The significantly faster, cheaper, technically less demanding, easy multiplex produces accurate fingerprints. Using cascade as case study, obtain substrate profiles that can be used map specificity, cleavage entropy allosteric effects design probes. further show predictions enable selection potential physiological substrates targeted validation assays.