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Nitric Oxide pp 161-169 | Cite as

Identification of Tyrosine and Nitrotyrosine with a Mixed-Mode Solid-Phase Extraction Cleanup Followed by Liquid Chromatography–Electrospray Time-of-Flight Mass Spectrometry in Plants

  • Mounira Chaki
  • Beatriz Sánchez-Calvo
  • Alfonso Carreras
  • Raquel Valderrama
  • Juan C. Begara-Morales
  • Francisco J. Corpas
  • Juan B. BarrosoEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1747)

Abstract

In higher plants, there is a growing interest in the study of protein tyrosine nitration (NO2Tyr) as well as the identification of in vivo nitrated proteins. Different methods have been developed for identifying nitrotyrosine in biological samples. However, these analyses are difficult because tyrosine nitration is a very low-abundance posttranslational protein modification (PTM) and the lack of efficient enrichment methods for detection. The identification and quantification of NO2Tyr in proteins has represented a challenge for researchers.

In this chapter a new method for determining NO2Tyr and tyrosine (Tyr) in Arabidopsis thaliana cell-suspension culture extracts is proposed. The quantification was performed using a simple, sensitive, and specific sample preparation assay based on mixed-mode solid-phase extraction (SPE) which was developed for the quantification of trace NO2Tyr in Arabidopsis extracts by liquid chromatography–electrospray time-of-flight mass spectrometry (LC-TOFMS).

Key words

Nitrotyrosine Tyrosine Nitrosative stress Arabidopsis thaliana Liquid chromatography Solid-phase extraction Mass spectrometry 

Notes

Acknowledgments

This study was supported by the ERDF grants cofinanced by the Ministry of Economy and Competitiveness (projects BIO2015-66390-P and AGL2015-65104-P) and the Junta de Andalucía (groups BIO286 and BIO192) in Spain.

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Copyright information

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

Authors and Affiliations

  • Mounira Chaki
    • 1
  • Beatriz Sánchez-Calvo
    • 1
  • Alfonso Carreras
    • 1
  • Raquel Valderrama
    • 1
  • Juan C. Begara-Morales
    • 1
  • Francisco J. Corpas
    • 2
  • Juan B. Barroso
    • 1
    Email author
  1. 1.Group of Biochemistry and Cell Signaling in Nitric Oxide, Department of Experimental Biology, Center for Advanced Studies in Olive Grove and Olive Oils, Faculty of Experimental SciencesUniversity of JaénJaénSpain
  2. 2.Group of Antioxidants, Free Radicals, and Nitric Oxide in Biotechnology, Food, and Agriculture, Department of Biochemistry, Cellular and Molecular Biology of Plants, Estación Experimental del ZaidínConsejo Superior de Investigaciones CientíficasGranadaSpain

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