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Duplexing complexome profiling with SILAC to study human respiratory chain assembly defects.

Petra Páleníková1, Michael E Harbour1, Federica Prodi2

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|February 18, 2021
PubMed
Summary
This summary is machine-generated.

Complexome Profiling (CP) with Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) analyzes mitochondrial respiratory chain complexes. This method reveals assembly defects in cells with pathological variants, offering insights into complex assembly.

Keywords:
Complex IIIComplex IVMitochondrial DNA mutationsMitochondrial diseaseQuantitative proteomicsSILAC

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Area of Science:

  • Biochemistry
  • Proteomics
  • Cell Biology

Background:

  • Complexome Profiling (CP) integrates size separation with mass spectrometry (MS) for analyzing native protein complexes.
  • Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) enables quantitative comparisons of protein abundance between samples.
  • Combining CP and SILAC allows for direct comparison of protein migration and abundance within mitochondrial respiratory chain complexes.

Purpose of the Study:

  • To review the advantages and challenges of SILAC-CP for characterizing cell lines with pathological variants in MT-CO3 and MT-CYB.
  • To present an unpublished example of SILAC-CP in a cell line with an MT-CYB deletion.
  • To investigate mitochondrial respiratory chain complex assembly defects.

Main Methods:

  • Complexome Profiling (CP) using size separation and mass spectrometry (MS).
  • Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) for quantitative proteomics.
  • Bioinformatic tool development for normalized protein abundance profile generation.

Main Results:

  • SILAC-CP successfully compared mitochondrial respiratory chain complexes in two different samples.
  • Analysis of a cell line with an MT-CYB deletion showed partial Complex III assembly associated with Complex I.
  • Profuse accumulation of Complex III assembly intermediates and a Complex IV assembly defect were observed.

Conclusions:

  • SILAC-CP is a powerful technique for studying mitochondrial respiratory chain complex assembly.
  • The method revealed specific assembly defects in cells with pathological mitochondrial DNA variants.
  • This approach provides valuable insights into the functional consequences of genetic mutations on protein complex formation.