Proteomic Characterization of Reversible Thiol Oxidations in Proteomes and Proteins

Figure 4B-to-D summarizes some of the strategies we review in the five sections

Susanna Boronat; Alba Domènech; Elena Hidalgo


Scholarcy highlights

  • The price that organisms have to pay for living in an environment with oxygen is the extent of their energy to cope with the deleterious effects of the oxidation of biomolecules
  • More specific sources of reactive oxygen species and reactive nitrogen species are the enzymatic activities of several enzymes such as nicotinamide adenine dinucleotide phosphate oxidases, mieloperoxidases and nitric oxide synthases, activated in phagocytes in response to microorganisms, or the activities of cytochrome P450 and the enzymatic system of hypoxanthine/xanthine oxidase activated in cases of ischemia/reperfusion, metal-catalyzed reactions, osmotic stress and chemotherapeutic drugs
  • Regarding the proteomic characterization of the protein, following the pipeline described in Figure 4A we obtained acidic cell extracts from cultures untreated or treated with H2O2; reduced thiols were alkylated with NEM; reversibly oxidized thiols were reduced with Tris phosphine hydrochloride and the resulting new thiols were alkylated with IAM
  • The results demonstrated that four Cys residues, Cys278, 285, 501 and 532, were alkylated with IAM only when the protein was purified from H2O2– treated cultures
  • While Cys post-transcriptional modification by oxidants has been reported in many studies of either toxicity or signaling associated to oxidative stress, the development and widespread use of powerful mass spectrometry-based techniques linked to basic redox principles in sample manipulation and preparation is greatly contributing to clarifying the role of ROS fluctuations in health and disease
  • BZIP: basic leucine zipper Cys: cysteine Cys-reacting tandem mass tags: cysteine tandem mass tag DTT: dithiothreitol glutathione peroxidases: glutathione peroxidase Grx: glutaredoxin GSH: glutathione H2O2: hydrogen peroxide IAM: iodoacetamide ICAT: quantitative isotope coded affinity tag LC-MS/MS: liquid chromatography followed by tandem MS MALDI-TOF: matrix-assisted laser desorption/ionization time-of-fly methyl methanethiosulfonate: methyl methanothiosulphonate MS: mass spectrometry NADPH: nicotinamide adenine dinucleotide phosphate NEM: N-ethyl maleimide Prx: peroxiredoxin ROS: reactive oxygen species TMT: tandem mass tag SDS-PAGE: sodium dodecyl sulphate polyacrylamide gel electrophoresis TCA: trichloroacetic acid TCEP: Tris phosphine hydrochloride Trx: thioredoxin

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