INVESTIGADORES
WUILLOUD Rodolfo German
artículos
Título:
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
Autor/es:
BERTON, PAULA; DOMINGUEZ-ROMERO, JUAN C.; WUILLOUD, RODOLFO G.; SANCHEZ-CALVO, BEATRIZ; CHAKI, MOUNIRA; CARRERAS, ALFONSO; VALDERRAMA, RAQUEL; BEGARA-MORALES, JUAN C.; CORPAS, FRANCISCO J.; BARROSO, JUAN B.; GILBERT-LOPEZ, BIENVENIDA; GARCIA-REYES, JUAN F.; MOLINA-DIAZ, ANTONIO
Revista:
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
Editorial:
SPRINGER HEIDELBERG
Referencias:
Lugar: HEIDELBERG; Año: 2012 vol. 404 p. 1495 - 1503
ISSN:
1618-2642
Resumen:
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 Arabidopsis 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 (LC-TOFMS). Unambiguous confirmation of both aminoacids 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 (LOQ) obtained for NO2Tyr in Arabidopsis 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.
