Identification of Putative Mitochondrial Protease Substrates

Methods Mol Biol. 2021;2192:313-329. doi: 10.1007/978-1-0716-0834-0_21.

Abstract

Mitochondrial proteases constitute a fundamental part of the organellar protein quality control system to ensure the timely removal of damaged or obsolete proteins. The analysis of proteases is often limited to the identification of bona fide substrates that are degraded in the presence and become more abundant in the absence of the respective protease. However, proteases in numerous organisms from bacteria to humans can process specific substrates to release shortened proteins with potentially altered activities. Here, we describe an adaptation of the substrate-trapping approach, as well as the N-terminal profiling protocol Terminal Amine Isotope Labeling of Substrates (TAILS) for the identification of bona fide substrates and mitochondrial proteins that undergo complete or partial proteolysis.

Keywords: Immunoprecipitations; Mitochondria; N Termini profiling; Proteases; Proteolysis; Substrate-trapping; Substrates; TAILS.

MeSH terms

  • ATP-Dependent Proteases / chemistry*
  • ATP-Dependent Proteases / genetics
  • ATP-Dependent Proteases / metabolism*
  • Animals
  • Cells, Cultured
  • Chromatography, Liquid / methods
  • Fibroblasts / cytology
  • Immunoprecipitation / methods
  • Isotope Labeling / methods
  • Mice
  • Mice, Knockout
  • Mitochondria / enzymology*
  • Mitochondrial Proteins / metabolism*
  • Myocardium / cytology
  • Protein Processing, Post-Translational
  • Proteolysis
  • Substrate Specificity
  • Tandem Mass Spectrometry / methods
  • Transfection / methods

Substances

  • Mitochondrial Proteins
  • ATP-Dependent Proteases