A “fluorescence switch” technique increases the sensitivity of proteomic detection and identification of S‐nitrosylated proteins

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dc.contributor.authorTello, Daniel
dc.contributor.authorTarín, Carlos
dc.contributor.authorAhicart, Patricia
dc.contributor.authorBretón‐Romero, Rosa
dc.contributor.authorLamas, Santiago
dc.contributor.authorMartínez Ruiz, Antonio
dc.date.accessioned2024-05-22T12:45:36Z
dc.date.available2024-05-22T12:45:36Z
dc.date.issued2009-12
dc.description.abstractProtein S-nitrosylation is a reversible post-translational modification of protein cysteines that is increasingly being considered as a signal transduction mechanism. The ‘‘biotin switch’’ technique marked the beginning of the study of the S-nitrosoproteome, based on the specific replacement of the labile S-nitrosylation by a more stable biotinylation that allowed further detection and purification. However, its application for proteomic studies is limited by its relatively low sensitivity. Thus, typical proteomic experiments require high quantities of protein extracts, which precludes the use of this method in a number of biological settings. We have developed a ‘‘fluorescence switch’’ technique that, when coupled to 2-DE proteomic methodologies, allows the detection and identification of S-nitrosylated proteins by using limited amounts of starting material, thus significantly improving the sensitivity. We have applied this methodology to detect proteins that become S-nitrosylated in endothelial cells when exposed to S-nitroso-L-cysteine, a physiological S-nitrosothiol, identifying already known S-nitrosylation targets, as well as proteins that are novel targets. This ‘‘fluorescence switch’’ approach also allowed us to identify several proteins that are denitrosylated by thioredoxin in cytokine-activated RAW264.7 (murine macrophage) cells. We believe that this method represents an improvement in order to approach the identification of S-nitrosylated proteins in physiological conditions.
dc.description.departmentDepto. de Bioquímica y Biología Molecular
dc.description.refereedTRUE
dc.description.sponsorshipGobierno de España
dc.description.statuspub
dc.identifier.doi10.1002/pmic.200900070
dc.identifier.issn1615-9853
dc.identifier.issn1615-9861
dc.identifier.officialurlhttps://doi.org/10.1002/pmic.200900070
dc.identifier.urihttps://hdl.handle.net/20.500.14352/104317
dc.issue.number9
dc.journal.titleProteomics
dc.language.isoeng
dc.page.final5370
dc.page.initial5359
dc.relation.projectIDinfo:eu-repo/grantAgreement/MSC//CP03%00025/ES/CP03%00025/
dc.relation.projectIDinfo:eu-repo/grantAgreement/MSC//CP07%2F00143/ES/CP07%2F00143/
dc.relation.projectIDinfo:eu-repo/grantAgreement/MEC//SAF2006-02410/ES/MODELOS EXPERIMENTALES DE FISIOPATOLOGIA VASCULAR : DE LA MODIFICACION POSTRADUCCIONAL AL ANIMAL TRANSGENICO/
dc.relation.projectIDinfo:eu-repo/grantAgreement/MEC//CSD2007-00020/ES/Papel funcional del estrés oxidativo y nitrosativo en grandes sistemas biológicos/
dc.rights.accessRightsmetadata only access
dc.subject.cdu577.1
dc.subject.keywordCell biology
dc.subject.keywordEndothelium
dc.subject.keywordMacrophage activation
dc.subject.keywordPost-translational modification
dc.subject.keywordProtein oxidation
dc.subject.keywordS-nitrosylation
dc.subject.ucmBioquímica (Farmacia)
dc.subject.unesco2302 Bioquímica
dc.titleA “fluorescence switch” technique increases the sensitivity of proteomic detection and identification of S‐nitrosylated proteins
dc.typejournal article
dc.type.hasVersionVoR
dspace.entity.typePublication
relation.isAuthorOfPublicationeec0b303-34c9-47dd-9ec6-704b6c6c7acd
relation.isAuthorOfPublication.latestForDiscoveryeec0b303-34c9-47dd-9ec6-704b6c6c7acd
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