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 De Filippis, Ulrich auf dem Keller, Ruedi Aebersold*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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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.
Original languageEnglish
Article number1693
JournalNature Communications
Volume12
Number of pages18
ISSN2041-1723
DOIs
Publication statusPublished - 2021

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