Identification of S-Nitrosylated and Reversibly Oxidized Proteins by Fluorescence Switch and Complementary Techniques

  • Alicia Izquierdo-Álvarez
  • Daniel Tello
  • J. Daniel Cabrera-García
  • Antonio Martínez-Ruiz
Part of the Methods in Molecular Biology book series (MIMB, volume 1747)


S-nitrosylation and other reversible oxidative posttranslational modifications of proteins are part of the nonclassical mechanisms of nitric oxide signaling. The biotin switch technique for specifically labeling S-nitrosylated proteins opened the way to proteomic identification of these modifications. Since then, several variations and adaptations of the original method have been applied.

We describe here the protocols of several techniques that can be used for the proteomic identification of these modifications, as well as for the detailed characterization of the modification of individual proteins. The fluorescence switch technique allows the proteomic identification of S-nitrosylated proteins based on their fluorescent labeling coupled to electrophoretic separation, as well as the comparison of the overall modification in different samples. The redox fluorescence switch is an adaptation to detect all the proteins reversibly oxidized in cysteine residues. We also describe the protocols of complementary techniques that allow comparing the extent of modification of individual proteins in several conditions by biotin switch, and the identification of modified residues by differential labeling adapted for mass spectrometry identification.

Key words

Nitric oxide S-nitrosylation S-glutathionylation Oxidative posttranslational modification Reversible cysteine oxidation 



Research in our lab is supported by grants PI15/00107 and SAF2015-71521-REDC (Consolredox network) from the Spanish Government (partially funded by European Union ERDF), and by a grant from the Fundación Domingo Martínez.


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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Alicia Izquierdo-Álvarez
    • 1
    • 2
  • Daniel Tello
    • 3
    • 4
  • J. Daniel Cabrera-García
    • 1
  • Antonio Martínez-Ruiz
    • 1
    • 4
  1. 1.Servicio de InmunologíaHospital Universitario de La Princesa, Instituto de Investigación Sanitaria Princesa (IIS-IP)MadridSpain
  2. 2.Biomechanics SectionKU LeuvenLeuvenBelgium
  3. 3.Unidad de InvestigaciónHospital Santa Cristina, Instituto de Investigación Sanitaria Princesa (IIS-IP)MadridSpain
  4. 4.Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV)MadridSpain

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