Modified method for determination of sulfur metabolites in plant tissues by stable isotope dilution-based liquid chromatography–electrospray ionization–tandem mass spectrometry

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Title:	Modified method for determination of sulfur metabolites in plant tissues by stable isotope dilution-based liquid chromatography–electrospray ionization–tandem mass spectrometry 以穩定同位素稀釋液相層析電噴灑游離串聯質譜法定量植物組織之含硫代謝物之方法改進
Authors:	Ya-Lan Chang;Chin-Lin Hsieh;Yao-Moan Huang;Wen-Liang Chiou;Yueh-Hsiung Kuo;Mei-Hwei Tseng;曾梅慧
Keywords:	Sulfur metabolites;LC-S/MS;34S metabolic labeling
Date:	2013-11
Issue Date:	2014-11-27 09:52:44 (UTC+8)
Abstract:	A wide variety of sulfur metabolites play important roles in plant functions. We have developed a precise and sensitive method for the simultaneous measurement of several sulfur metabolites based on liquid chromatography coupled with tandem mass spectrometry (LCâMS/MS) and 34S metabolic labeling of sulfur-containing metabolites in Arabidopsis thaliana seedlings. However, some sulfur metabolites were unstable during the extraction procedure. Our proposed method does not allow for the detection of the important sulfur metabolite homocysteine because of its instability during sample extraction. Stable isotope-labeled sulfur metabolites of A. thaliana shoot were extracted and utilized as internal standards for quantification of sulfur metabolites with LCâMS/MS using S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), methionine (Met), glutathione (GSH), and glutathione disulfide (GSSG) as example metabolites. These metabolites were detected using electrospray ionization in positive mode. Standard curves were linear (r2 > 0.99) over a range of concentrations (SAM 0.01â2.0 Î¼M, SAH 0.002â0.10 Î¼M, Met 0.05â4.0 Î¼M, GSH 0.17â20.0 Î¼M, GSSG 0.07â20.0 Î¼M), with limits of detection for SAM, SAH, Met, GSH, and GSSG of 0.83, 0.67, 10, 0.56, and 1.1 nM, respectively; and the within-run and between-run coefficients of variation based on quality control samples were less than 8%. 各種各樣的含硫代謝物在植物生命系統中扮演重要角色。利用液相層析串聯質譜技術與34S同位素在阿拉伯芥植物體內進行標定含硫代謝物的方法，我們已建立一個靈敏和精確的方法並且同時可以測定幾種含硫代謝物含量。然而，由於一些含硫代謝物於萃取過程中化性不穩定，我們先前所建議的方法無法測定重要含硫代謝物同半胱胺酸(homocysteine)，因其在萃取過程中不穩定。經34S穩定同位素標定的含硫代謝物自阿拉伯芥莖部萃取出並作為內標準品，在液相層析串連質譜技術中進行同步測定S-腺苷甲硫胺酸(S-adenosylmethionine)，S-腺苷高半胱胺酸(S-adenosylhomocysteine)，甲硫酸銨(methionine)，榖胱甘肽(glutathione)，及榖胱甘太(氧化型)( glutathione disulfide)，作為研究例子。使用電灑法正離子模式檢測這些代謝物，可得到可測定範圍的檢量線(r²>0.99)，SAM0.01-2.0μM，SAH0.002-0.10μM，Met0.05-4.0μM，GSH0.17-20.0μM，偵側極限各在SAM(0.83nM)，SAH(0.67nM)，Met(10nM)，GSH(0.56nM)，及GSSG(1.1nM)，於品管檢測下得到回內分析與回間分析的變異係數小於8%。
Relation:	Analytical Biochemistry，v.442n.1，p.24-33
Appears in Collections:	[Department of Applied Physics and Chemistry] Periodical Articles

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