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Determination of nitrotyrosine in Arabidopsis thaliana cell cultures with a mixed-mode solid-phase extraction cleanup followed by liquid chromatography time-of-flight mass spectrometry

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Abstract

In this work, a method for the determination of trace nitrotyrosine (NO2Tyr) and tyrosine (Tyr) in Arabidopsis thaliana cell cultures is proposed. Due to the complexity of the resulting extracts after protein precipitation and enzymatic digestion and the strong electrospray signal suppression displayed in the detection of both Tyr and NO2Tyr from raw A. thaliana cell culture extracts, a straightforward sample cleanup step was proposed. It was based on the use of mixed-mode solid-phase extraction (SPE) using MCX-type cartridges (Strata™-X-C), prior to identification and quantitation using fast liquid chromatography–electrospray time-of-flight mass spectrometry. Unambiguous confirmation of both amino acids was accomplished with accurate mass measurements (with errors lower than 2 ppm) of each protonated molecule along with a characteristic fragment ion for each species. Recovery studies were accomplished to evaluate the performance of the SPE sample preparation step obtaining average recoveries in the range 92–101 %. Limit of quantitation obtained for NO2Tyr in A. thaliana extracts was 3 nmol L−1. Finally, the proposed method was applied to evaluate stress conditions of the plant upon different concentrations of peroxynitrite, a protein-nitrating compound, which induces the nitration of Tyr at the nanomolar range. Detection and confirmation of the compounds demonstrated the usefulness of the proposed approach.

Determination of trace nitrotyrosine and tyrosine in Arabidopsis thaliana cell cultures by liquid chromatography time-of-flight mass spectrometry is achieved

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Acknowledgments

The authors acknowledge funding support from the Spanish “Ministerio de Asuntos Exteriores y de Cooperación” (Program PCI-AECID Ref. A/026661/09), Junta de Andalucía (Research Groups FQM323, BIO-286, BIO-192), and the Spanish “Ministerio de Ciencia e Innovación” (BIO2009-12003-C02-01 and BIO2009-12003-C02-02).

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Authors and Affiliations

  1. Analytical Chemistry Research Group, Department of Physical and Analytical Chemistry, University of Jaén, Campus Las Lagunillas, Edif. B-3, 23071, Jaén, Spain

    Paula Berton, Juan C. Domínguez-Romero, Bienvenida Gilbert-López, Juan F. García-Reyes & Antonio Molina-Díaz

  2. Analytical Chemistry Research and Development Group (QUIANID), Instituto de Ciencias Básicas, Universidad Nacional de Cuyo, Padre Jorge Contreras 1300, Parque Gral., San Martín, M5502JMA, Mendoza, Argentina

    Paula Berton & Rodolfo G. Wuilloud

  3. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), C1033AAJ, Caba, Argentina

    Paula Berton & Rodolfo G. Wuilloud

  4. Grupo de Señalización Molecular y Sistemas Antioxidantes en Plantas, Unidad asociada al CSIC (EEZ), Departamento de Bioquímica y Biología Molecular, Universidad de Jaén, 23071, Jaén, Spain

    Beatriz Sánchez-Calvo, Mounira Chaki, Alfonso Carreras, Raquel Valderrama, Juan C. Begara-Morales, Francisco J. Corpas & Juan B. Barroso

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  1. Paula Berton
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  2. Juan C. Domínguez-Romero
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  3. Rodolfo G. Wuilloud
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  4. Beatriz Sánchez-Calvo
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  8. Juan C. Begara-Morales
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  9. Francisco J. Corpas
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  10. Juan B. Barroso
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  11. Bienvenida Gilbert-López
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  12. Juan F. García-Reyes
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  13. Antonio Molina-Díaz
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Correspondence to Antonio Molina-Díaz.

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Berton, P., Domínguez-Romero, J.C., Wuilloud, R.G. et al. Determination of nitrotyrosine in Arabidopsis thaliana cell cultures with a mixed-mode solid-phase extraction cleanup followed by liquid chromatography time-of-flight mass spectrometry. Anal Bioanal Chem 404, 1495–1503 (2012). https://doi.org/10.1007/s00216-012-6220-3

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  • DOI: https://doi.org/10.1007/s00216-012-6220-3

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