Quantification and identification of mitochondrial proteins containing vicinal dithiols

Vicinal dithiols may play a role in mitochondrial antioxidant defences and in redox signalling. We quantified protein vicinal dithiols within mammalian mitochondria using the vicinal dithiol-specific reagent phenylarsine oxide (PAO). We found 5–15% of thiols exposed on mitochondrial proteins were vicinal dithiols and that these thiols were particularly sensitive to oxidation by hydrogen peroxide. To visualise these proteins we used PAO to block vicinal dithiols, followed by alkylation of other thiols with N-ethylmaleimide (NEM). The PAO was then removed with 2,3-dimercapto-1-propanesulfonic acid (DMPS) and the exposed vicinal dithiols were labelled with iodoacetamide-biotin. To identify these proteins, we developed a selective proteomic methodology, based on Redox difference in gel electrophoresis (Redox-DIGE). Vicinal dithiol proteins were selectively labelled with a red fluorescent thiol-reactive Cy5 maleimide and mixed with Cy3 maleimide labelled protein in which vicinal dithiols remained untagged. Individual proteins were resolved by 2D gel electrophoresis and fluorescent scanning revealed vicinal dithiol proteins by the increase in Cy5 red fluorescence. These proteins were identified by peptide mass fingerprinting and mass spectrometry. These findings are consistent with roles for mitochondrial vicinal dithiol proteins in antioxidant defence and redox signalling and these methodologies will enable these roles to be explored.