Simultaneous Determination of Sulphur metabolites in Arabidopsis thaliana via LC-ESI-MS/MS and 34S-metabolic Labelling
Resource
PHYTOCHEMICAL ANALYSIS, 23(4), 324-331
Journal
PHYTOCHEMICAL ANALYSIS
Journal Volume
23
Journal Issue
4
Pages
324-331
Date Issued
2012
Date
2012
Author(s)
Hsieh, Chin-Lin
Yeh, Kai-Wun
De Kok, Luit J.
Pan, Ryh-Nan
Kuo, Yueh-Hsiung
Tseng, Mei-Hwei
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 ³⁴S/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.
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