Simultaneous determination of sulfur metabolites in Arabidopsis thaliana via LC-ESI-MS/MS and 34S-metabolic labeling

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Title:	Simultaneous determination of sulfur metabolites in Arabidopsis thaliana via LC-ESI-MS/MS and 34S-metabolic labeling
Authors:	Chin-Lin Hsieh Kai-Wun Yeh Luit J. De Kok Ryh-Nan Pan Yueh-Hsiung Kuo Mei-Hwei Tseng 曾梅慧
Contributors:	臺北市立教育大學應用物理暨化學系
Date:	2011-08
Issue Date:	2011-12-01 11:03:52 (UTC+8)
Abstract:	Introduction – Sulphur-containing metabolites play an important role in metabolism and homeostasis. Determination of these metabolites is challenging owing to their low concentrations and the interference in mass spectrometry analysis. Objective – To develop a sensitive and accurate method based on liquid chromatography, electrospray ionisation, tandem mass spectrometry (LC-ESI-MS/MS) and 34S-metabolic labelling for quantification of methionine, reduced glutathione, oxidised glutathione in Arabidopsis thaliana. Methodology – A hydroponic set-up was used for the in vivo 34S-metabolic labelling of A. thaliana. The 34S-labelled metabolites biosynthesised in plant were extracted and used as internal standards. Tissue was extracted with perchloric acid (PCA) or PCA containing a known amount of the analytes for recovery analysis. Tissue extract mixed with extract of 34S-labelled A. thaliana in an appropriate ratio was subjected to a LC system and electrospray ionisation-mass spectrometric (ESI-MS) analysis. Quantification of metabolites was measured by comparing the 32S/34S ratios obtained for samples with the calibration curves. Results – Calibration curves showed linearity with regression coefficients in the range of 0.9994–0.9999. Analyte recoveries were approximately 100%. The coefficients of variation of intra-assay and inter-assay were less than 4.2% and 5%, respectively. The ranges for the limits of detection determined for Met, GSSG and GSH were 10 fmol, <10 fmol and 1.12 fmol and the limits of quantification determined for Met, GSSG and GSH were 0.44 pmol, 0.16 pmol and 34 fmol, respectively. Conclusion – The validated method for determination of methionine, reduced glutathione and oxidised glutathione was effectively applied to measure metabolite dynamics of sulphur-containing metabolites at the whole-plant level.
Relation:	Phytochemical Analysis
Appears in Collections:	[Department of Applied Physics and Chemistry] Periodical Articles

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