title: The oxidized thiol proteome in fission yeast--optimization of an ICAT-based method to identify H2O2-oxidized proteins.
abstract: Major intracellular disulfide formation is prevented in the cytosol by potent reducing systems. However, protein thiols can be oxidized as a consequence of redox-mediated physiological reactions or due to the unwanted toxicity of reactive oxygen species. In addition, the reactivity of cysteine residues towards peroxides is used by H(2)O(2) sensors in signal transduction pathways in a gain-of-function process to induce transcriptional antioxidant responses. Thus, the Schizosaccharomyces pombe peroxiredoxin Tpx1 and the transcription factor Pap1 are sensors of H(2)O(2) meant to promote cell survival. In an attempt to compare signaling events versus global thiol oxidation, we have optimized thiol-labeling approaches to characterize the disulfide proteome of fission yeast in response to added H(2)O(2). We propose a method based on (i) freezing the redox state of thiols with strong acids prior to cell lysis; (ii) blocking thiol groups with iodoacetamide, and reversibly oxidized thiols with heavy and light isotope-coded affinity tags (ICAT) reagents; and (iii) quantifying individual relative protein concentrations with stable-isotope dimethyl labeling. We have applied this highly sensitive strategy to provide a map of H(2)O(2)-dependent oxidized thiols in fission yeast, and found Tpx1 and Pap1 as some of the major targets.
authors.affiliation: Oxidative Stress and Cell Cycle Group, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, C/ Dr. Aiguader 88, E-08003 Barcelona, Spain.
authors.forename: Sarela
authors.initials: S
authors.lastname: García-Santamarina
authors.forename: Susanna
authors.initials: S
authors.lastname: Boronat
authors.forename: Guadalupe
authors.initials: G
authors.lastname: Espadas
authors.forename: José
authors.initials: J
authors.lastname: Ayté
authors.forename: Henrik
authors.initials: H
authors.lastname: Molina
authors.forename: Elena
authors.initials: E
authors.lastname: Hidalgo
mesh_terms: D000603:Amino Acids, Sulfur / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism; D002138:Calibration; D002853:Chromatography, Liquid; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology*; D007553:Isotope Labeling / Q000379:methods / Q000592:standards; D013058:Mass Spectrometry; D008954:Models, Biological; D010084:Oxidation-Reduction / Q000187:drug effects; D000073718:Pancreatitis-Associated Proteins; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D020543:Proteome / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism; D040901:Proteomics / Q000379:methods / Q000592:standards; D017382:Reactive Oxygen Species / Q000378:metabolism; D012568:Schizosaccharomyces / Q000737:chemistry / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000032:analysis* / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism*
chemical_list: D000603:Amino Acids, Sulfur; D000073718:Pancreatitis-Associated Proteins; D020543:Proteome; C000612141:REG3A protein, human; D017382:Reactive Oxygen Species; D029702:Schizosaccharomyces pombe Proteins; D013438:Sulfhydryl Compounds; D006861:Hydrogen Peroxide