title: Monitoring in vivo reversible cysteine oxidation in proteins using ICAT and mass spectrometry.
abstract: Reversible thiol oxidation of cysteine residues occurs in many intracellular catalytic and signaling processes. Here we describe an optimized protocol, which can be completed in ∼5 d, to unambiguously identify specific cysteine residues that are transiently and reversibly oxidized by comparing two complex biological samples obtained from yeast cell cultures at the proteome level. After 'freezing' the in vivo thiol stage of cysteine residues by medium acidification, we first block reduced thiols in extracts with iodoacetamide (IAM), and then we sequentially reduce and label reversible oxidized thiols with the biotin-based heavy or light IAM derivatives, which are known as isotope-coded affinity tag (ICAT) reagents, so that the two samples can be compared at once after combination of the labeled extracts, trypsin digestion, streptavidin-affinity purification of peptides containing oxidized cysteines, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. For the same protein extracts, before cysteine-containing peptide enrichment, individual relative protein concentrations are obtained by stable-isotope dimethyl labeling.
authors.affiliation: Oxidative Stress and Cell Cycle Group, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, Spain.
authors.forename: Sarela
authors.initials: S
authors.lastname: García-Santamarina
authors.affiliation: Oxidative Stress and Cell Cycle Group, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, Spain.
authors.forename: Susanna
authors.initials: S
authors.lastname: Boronat
authors.affiliation: Oxidative Stress and Cell Cycle Group, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, Spain.
authors.forename: Alba
authors.initials: A
authors.lastname: Domènech
authors.affiliation: Oxidative Stress and Cell Cycle Group, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, Spain.
authors.forename: José
authors.initials: J
authors.lastname: Ayté
authors.affiliation: Proteomics Resource Center, The Rockefeller University, New York, New York, USA.
authors.forename: Henrik
authors.initials: H
authors.lastname: Molina
authors.affiliation: Oxidative Stress and Cell Cycle Group, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, Spain.
authors.forename: Elena
authors.initials: E
authors.lastname: Hidalgo
mesh_terms: D002853:Chromatography, Liquid; D003545:Cysteine / Q000378:metabolism*; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods*; D010084:Oxidation-Reduction; D011506:Proteins / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D015003:Yeasts
chemical_list: D011506:Proteins; D013438:Sulfhydryl Compounds; D003545:Cysteine
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