TY - JOUR
T1 - Mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen
AU - Uliana, Federico
AU - Vizovišek, Matej
AU - Acquasaliente, Laura
AU - Ciuffa, Rodolfo
AU - Fossati, Andrea
AU - Frommelt, Fabian
AU - Goetze, Sandra
AU - Wollscheid, Bernd
AU - Gstaiger, Matthias
AU - De Filippis, Vincenzo
AU - auf dem Keller, Ulrich
AU - Aebersold, Ruedi
PY - 2021
Y1 - 2021
N2 - Proteases are among the largest protein families and critical regulators of biochemical processes like apoptosis and blood coagulation. Knowledge of proteases has been expanded by the development of proteomic approaches, however, technology for multiplexed screening of proteases within native environments is currently lacking behind. Here we introduce a simple method to profile protease activity based on isolation of protease products from native lysates using a 96FASP filter, their analysis in a mass spectrometer and a custom data analysis pipeline. The method is significantly faster, cheaper, technically less demanding, easy to multiplex and produces accurate protease fingerprints. Using the blood cascade proteases as a case study, we obtain protease substrate profiles that can be used to map specificity, cleavage entropy and allosteric effects and to design protease probes. The data further show that protease substrate predictions enable the selection of potential physiological substrates for targeted validation in biochemical assays.
AB - Proteases are among the largest protein families and critical regulators of biochemical processes like apoptosis and blood coagulation. Knowledge of proteases has been expanded by the development of proteomic approaches, however, technology for multiplexed screening of proteases within native environments is currently lacking behind. Here we introduce a simple method to profile protease activity based on isolation of protease products from native lysates using a 96FASP filter, their analysis in a mass spectrometer and a custom data analysis pipeline. The method is significantly faster, cheaper, technically less demanding, easy to multiplex and produces accurate protease fingerprints. Using the blood cascade proteases as a case study, we obtain protease substrate profiles that can be used to map specificity, cleavage entropy and allosteric effects and to design protease probes. The data further show that protease substrate predictions enable the selection of potential physiological substrates for targeted validation in biochemical assays.
U2 - 10.1038/s41467-021-21754-8
DO - 10.1038/s41467-021-21754-8
M3 - Journal article
C2 - 33727531
SN - 2041-1723
VL - 12
JO - Nature Communications
JF - Nature Communications
M1 - 1693
ER -