title: 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.
abstract: In this work, a method for the determination of trace nitrotyrosine (NO(2)Tyr) 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 NO(2)Tyr 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 NO(2)Tyr 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.
authors: Analytical Chemistry Research Group, Department of Physical and Analytical Chemistry, University of Jaén, Campus Las Lagunillas, Jaén, Spain.
authors: Paula
authors: P
authors: Berton
authors: Juan C
authors: JC
authors: Domínguez-Romero
authors: Rodolfo G
authors: RG
authors: Wuilloud
authors: Beatriz
authors: B
authors: Sánchez-Calvo
authors: Mounira
authors: M
authors: Chaki
authors: Alfonso
authors: A
authors: Carreras
authors: Raquel
authors: R
authors: Valderrama
authors: Juan C
authors: JC
authors: Begara-Morales
authors: Francisco J
authors: FJ
authors: Corpas
authors: Juan B
authors: JB
authors: Barroso
authors: Bienvenida
authors: B
authors: Gilbert-López
authors: Juan F
authors: JF
authors: García-Reyes
authors: Antonio
authors: A
authors: Molina-Díaz
mesh_terms: D017360:Arabidopsis / Q000737:chemistry*; D002853:Chromatography, Liquid / Q000379:methods; D057230:Limit of Detection; D052616:Solid Phase Extraction / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
chemical_list: C002744:3-nitrotyrosine; D014443:Tyrosine