Redox proteomics of thiol proteins in mouse heart during ischemia/reperfusion using ICAT reagents and mass spectrometry

Vikas Kumar, Torsten Kleffmann, Mark B Hampton, Mark B Cannell, Christine C Winterbourn

Research output: Contribution to journalArticle (Academic Journal)

43 Citations (Scopus)

Abstract

There is strong evidence for the involvement of reactive oxygen species in ischemia/reperfusion injury. Although oxidation of individual thiol proteins has been reported, more extensive redox proteomics of hearts subjected to ischemia/reperfusion has not been performed. We have carried out an exploratory study using mass spectrometry with isotope-coded affinity tags (ICAT) aimed at identifying reversible oxidative changes to protein thiols in Langendorff perfused isolated mouse hearts subjected to 20min ischemia with or without aerobic reperfusion for 5 or 30min. Reduced thiols were blocked by adding N-ethylmaleimide during protein extraction, then reversibly oxidized thiols in extracts of control perfused and treated hearts were reduced and labeled with the light and heavy ICAT reagents, respectively. Protein extracts were mixed in equal amounts and relative proportions of the isotope-labeled peaks were used to quantify oxidative changes between the control and the treated groups. Approximately 300 peptides with ICAT signatures were reliably identified in each sample, with 181 peptides from 118 proteins common to all treatments. A proportion showed elevated ICAT ratios, consistent with reversible thiol oxidation. This was most evident after early reperfusion, with apparent reversal after longer reperfusion. In comparison, there was gradual accumulation of protein carbonyls and loss of GSH with longer reperfusion. Many of the thiol changes were in mitochondrial proteins, including components of electron transport complexes and enzymes involved in lipid metabolism. The results are consistent with mitochondria being a major site of oxidant generation during early cardiac reperfusion and mitochondrial thiol proteins being targets for oxidation.
Original languageEnglish
Pages (from-to)109-117
Number of pages9
JournalFree Radical Biology and Medicine
Volume58
Early online date29 Jan 2013
DOIs
Publication statusPublished - 1 May 2013

Keywords

  • Cardiac ischemia/reperfusion
  • Thiol proteomics
  • Mass spectrometry
  • Mitochondria
  • Thiol oxidation
  • Free radicals

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