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

Federico Uliana, Matej Vizovišek, Laura Acquasaliente, Rodolfo Ciuffa, Andrea Fossati, Fabian Frommelt, Sandra Goetze, Bernd Wollscheid, Matthias Gstaiger, Vincenzo Filippis, Ulrich auf dem Keller and Ruedi Aebersold ()
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Federico Uliana: Institute of Molecular Systems Biology, ETH Zürich
Matej Vizovišek: Institute of Molecular Systems Biology, ETH Zürich
Laura Acquasaliente: University of Padua
Rodolfo Ciuffa: Institute of Molecular Systems Biology, ETH Zürich
Andrea Fossati: Institute of Molecular Systems Biology, ETH Zürich
Fabian Frommelt: Institute of Molecular Systems Biology, ETH Zürich
Sandra Goetze: Institute of Translational Medicine, ETH Zürich
Bernd Wollscheid: Institute of Translational Medicine, ETH Zürich
Matthias Gstaiger: Institute of Molecular Systems Biology, ETH Zürich
Vincenzo Filippis: University of Padua
Ulrich auf dem Keller: Technical University of Denmark
Ruedi Aebersold: Institute of Molecular Systems Biology, ETH Zürich

Nature Communications, 2021, vol. 12, issue 1, 1-18

Abstract: Abstract 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.

Date: 2021
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DOI: 10.1038/s41467-021-21754-8

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