Monitoring in vivo reversible cysteine oxidation in proteins using ICAT and mass spectrometry

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• dc.contributor.author García Santamarina, Sarela, 1978-ca
• dc.contributor.author Boronat i Llop, Susanna, 1965-ca
• dc.contributor.author Domènech, Albaca
• dc.contributor.author Ayté del Olmo, Joséca
• dc.contributor.author Molina, Henrikca
• dc.contributor.author Hidalgo Hernando, Elenaca
• dc.date.accessioned 2016-01-13T14:45:07Z
• dc.date.available 2016-01-13T14:45:07Z
• dc.date.issued 2014
• dc.description.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 labelingca
• dc.description.sponsorship This work was supported by the Spanish Ministry of Science and Innovation (nos.BFU2009-06933 and BFU2012-32045), by PLAN E and Fondo Europeo de Desarrollo Regional (FEDER), by the Spanish program Consolider-Ingenio 2010 (grant no. CSD 2007-0020) and by grant no. SGR2009-195 from Generalitat de Catalunya (Spain) to E.H. E.H. and J.A. are recipients of Institució Catalana de Recerca i Estudis Avançats (ICREA) /nAcademia Awards (Generalitat de Catalunya)
• dc.format.mimetype application/pdfca
• dc.identifier.citation García-Santamarina S, Boronat S, Domènech A, Ayté J, Molina H, Hidalgo E. Monitoring in vivo reversible cysteine oxidation in proteins using ICAT and mass spectrometry. Nature protocols. 2014; 9(5):1131-45. DOI: 10.1038/nprot.2014.065ca
• dc.identifier.doi http://dx.doi.org/10.1038/nprot.2014.065
• dc.identifier.issn 1750-2799
• dc.identifier.uri http://hdl.handle.net/10230/25565
• dc.language.iso engca
• dc.publisher Nature Publishing Groupca
• dc.relation.ispartof Nature protocols. 2014; 9(5):1131-45
• dc.relation.projectID info:eu-repo/grantAgreement/ES/3PN/BFU2009-06933
• dc.relation.projectID info:eu-repo/grantAgreement/ES/3PN/BFU2012-32045
• dc.rights © Nature Publishing Group. http://dx.doi.org/10.1038/nprot.2014.065ca
• dc.rights.accessRights info:eu-repo/semantics/openAccessca
• dc.subject.keyword Thiol oxidation
• dc.subject.keyword Disulfide proteome
• dc.subject.keyword ICAT
• dc.subject.keyword Dimethyl labelling
• dc.subject.keyword Thioredoxin
• dc.subject.keyword H2O2
• dc.subject.other Proteïnes -- Metabolismeca
• dc.subject.other Cromatografia de líquidsca
• dc.title Monitoring in vivo reversible cysteine oxidation in proteins using ICAT and mass spectrometryca
• dc.type info:eu-repo/semantics/articleca
• dc.type.version info:eu-repo/semantics/acceptedVersionca