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

Methods Mol Biol. 2018;1841:189-206. doi: 10.1007/978-1-4939-8695-8_14.

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.

Keywords: AAA+ protease; FtsH; Label-free proteomics; Mass spectrometry; Regulated proteolysis; Trapping; protease substrate.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP-Dependent Proteases
  • Chromatography, High Pressure Liquid
  • Data Science
  • Enzyme Assays* / methods
  • Escherichia coli / metabolism
  • Mass Spectrometry
  • Peptide Hydrolases* / metabolism
  • Peptides / chemistry
  • Proteolysis
  • Proteome*
  • Proteomics* / methods
  • Substrate Specificity

Substances

  • Peptides
  • Proteome
  • Peptide Hydrolases
  • ATP-Dependent Proteases