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Next-Generation Trapping of Protease Substrates by Label-Free Proteomics

  • Claudia Lindemann
  • Nikolas Thomanek
  • Katja Kuhlmann
  • Helmut E. Meyer
  • Katrin Marcus
  • Franz Narberhaus
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1841)

Abstract

AAA+ proteases (ATPases associated with various cellular activities) shape the cellular protein pool in response to environmental conditions. A prerequisite for understanding the underlying recognition and degradation principles is the identification of as many protease substrates as possible. Most previous studies made use of inactive protease variants to trap substrates, which were identified by 2D-gel based proteomics. Since this method is known for limitations in the identification of low-abundant proteins or proteins with many transmembrane domains, we established a trapping approach that overcomes these limitations. We used a proteolytically inactive FtsH variant (FtsHtrap) of Escherichia coli (E. coli) that is still able to bind and translocate substrates into the proteolytic chamber but no longer able to degrade proteins. Proteins associated with FtsHtrap or FtsHwt (proteolytically active FtsH) were purified, concentrated by an 1D-short gel, and identified by LC-coupled mass spectrometry (LC-MS) followed by label-free quantification. The identification of four known FtsH substrates validated this approach and suggests that it is generally applicable to AAA+ proteases.

Key words

Label-free proteomics Mass spectrometry Trapping protease substrate FtsH Regulated proteolysis AAA+ protease 

Notes

Acknowledgments

The authors would like to thank Alexandra Müller, Sina Langklotz, and Thilo Lerari for carefully reading the manuscript and for many helpful comments. The work was supported by grants from the German Research Foundation (DFG, SFB642: GTP- and ATP-dependent membrane processes) and P.U.R.E. (Protein Unit for Research in Europe, funded by the German federal state North Rhine-Westphalia).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Claudia Lindemann
    • 1
  • Nikolas Thomanek
    • 1
  • Katja Kuhlmann
    • 1
  • Helmut E. Meyer
    • 1
  • Katrin Marcus
    • 1
  • Franz Narberhaus
    • 2
  1. 1.Medizinisches Proteom-CenterRuhr-Universität BochumBochumGermany
  2. 2.Lehrstuhl für Biologie der MikroorganismenRuhr-Universität BochumBochumGermany

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