Department of Disaster Management, Taiwan Police College, Taipei 11696, Taiwan.
Phytochem Anal. 2012 Jul-Aug;23(4):324-31. doi: 10.1002/pca.1361. Epub 2011 Oct 10.
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.
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.
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.
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.
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.
含硫代谢物在代谢和内稳态中起着重要作用。由于其浓度低，以及在质谱分析中的干扰，这些代谢物的测定具有挑战性。
开发一种基于液相色谱、电喷雾电离、串联质谱（LC-ESI-MS/MS）和 ³⁴S 代谢标记的灵敏、准确的方法，用于定量拟南芥中的蛋氨酸、还原型谷胱甘肽和氧化型谷胱甘肽。
使用水培装置对拟南芥进行体内 ³⁴S 代谢标记。植物中生物合成的 ³⁴S 标记代谢物被提取出来作为内标。用高氯酸（PCA）或含有已知分析物量的 PCA 提取组织进行回收分析。将组织提取物与适当比例的 ³⁴S 标记拟南芥提取物混合，进行液相色谱系统和电喷雾电离-质谱（ESI-MS）分析。通过比较样品中获得的 ³⁴S/³⁴S 比值与校准曲线，来测量代谢物的定量。
校准曲线呈线性，相关系数在 0.9994-0.9999 范围内。分析物回收率约为 100%。日内和日间变异系数均小于 4.2%和 5%。测定 Met、GSSG 和 GSH 的检测限分别为 10 fmol、<10 fmol 和 1.12 fmol，定量限分别为 0.44 pmol、0.16 pmol 和 34 fmol。
该方法有效应用于测定含硫代谢物的代谢动力学，可用于测量植物整体水平的含硫代谢物。
