Federico Uliana1, Matej Vizovišek1, Laura Acquasaliente2, Rodolfo Ciuffa1, Andrea Fossati1, Fabian Frommelt1, Sandra Goetze3,4, Bernd Wollscheid3,4, Matthias Gstaiger1, Vincenzo De Filippis2, Ulrich Auf dem Keller5, Ruedi Aebersold6,7. 1. Department of Biology, Institute of Molecular Systems Biology, ETH Zürich, Zürich, Switzerland. 2. Department of Pharmaceutical and Pharmacological Sciences, Laboratory of Protein Chemistry and Molecular Hematology, University of Padua, Padua, Italy. 3. Department of Health Sciences and Technology, Institute of Translational Medicine, ETH Zürich, Zürich, Switzerland. 4. Swiss Institute of Bioinformatics, Lausanne, Switzerland. 5. Department of Biotechnology and Biomedicine, Technical University of Denmark, Lyngby, Denmark. 6. Department of Biology, Institute of Molecular Systems Biology, ETH Zürich, Zürich, Switzerland. aebersold@imsb.biol.ethz.ch. 7. Faculty of Science, University of Zürich, Zürich, Switzerland. aebersold@imsb.biol.ethz.ch.
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.
Proteases are among the ln class="Chemical">argest 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 profileprotease 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.
Authors: Swati Prasad; Angelene M Cantwell; Leslie A Bush; Peter Shih; Hong Xu; Enrico Di Cera Journal: J Biol Chem Date: 2003-12-16 Impact factor: 5.157
Authors: Peter A Bell; Sophia Scheuermann; Florian Renner; Christina L Pan; Henry Y Lu; Stuart E Turvey; Frédéric Bornancin; Catherine H Régnier; Christopher M Overall Journal: Comput Struct Biotechnol J Date: 2022-08-19 Impact factor: 6.155