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
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 wordsNitrotyrosine Tyrosine Nitrosative stress Arabidopsis thaliana Liquid chromatography Solid-phase extraction Mass spectrometry
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.
- 5.Chaki M, Valderrama R, Fernández-Ocaña AM, Carreras A, López Jaramillo J, Luque F, Palma JM, Pedrajas JR, Begara-Morales JC, Sánchez-Calvo B, Gómez-Rodríguez MV, Corpas FJ, Barroso JB (2009) Protein targets of tyrosine nitration in sunflower (Helianthus annuus L.) hypocotyls. J Exp Bot 60:4221–4234CrossRefPubMedGoogle Scholar
- 6.Chaki M, Fernandez-Ocana AM, Valderrama R, Carreras A, Esteban FJ, Luque F, Gomez-Rodriguez MV, Begara-Morales JC, Corpas FJ, Barroso JB (2008) Involvement of reactive nitrogen and oxygen species (RNS and ROS) in sunflower-mildew interaction. Plant Cell Physiol 50:265–279CrossRefPubMedGoogle Scholar
- 12.Berton P, Domínguez-Romero JC, Wuilloud RG, Sánchez-Calvo B, Chaki M, Carreras A, Valderrama R, Begara-Morales JC, Corpas FJ, Barroso JB, Gilbert-López B, García-Reyes JF, Molina-Díaz A (2012) 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 404:1495–1503CrossRefPubMedGoogle Scholar
- 13.Axelos M, Curie C, Mazzolini L, Bardet C, Lescure B (1992) A protocol for transient gene expression in Arabidopsis thaliana protoplasts isolated from cell suspension cultures. Plant Physiol Biochem 30:123–128Google Scholar
- 15.Ishii Y, Iijima M, Umemura T, Nishikawa A, Iwasaki Y, Ito R, Saito K, Hirose M, Nakazawa H (2006) Determination of nitrotyrosine and tyrosine by high-performance liquid chromatography with tandem mass spectrometry and immunohistochemical analysis in livers of mice administered acetaminophen. J Pharm Biomed Anal 41:1325–1331CrossRefPubMedGoogle Scholar