Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan, 430072, China.
Analyst. 2011 Apr 7;136(7):1515-22. doi: 10.1039/c0an00736f. Epub 2011 Feb 18.
In order to quantitatively study the jasmonate biosynthetic pathway, we chemically synthesized a pair of isotope mass probes and established a labeling protocol. The pair of mass probes used in our work were ω-bromoacetonylpyridinium bromide (BPB) and d(5)-ω-bromoacetonylpyridinium bromide (d(5)-BPB), which contain carboxylic acid reactive groups, isotopically labeled groups and permanent positive charges. High performance liquid chromatography (HPLC) and electrospray ionization quadrupole-time of flight mass spectrometry (ESI-QTOF-MS) were used for the detection of labeled standard mixtures and plant samples. In comparison to negative mode electrospray ionization detection of unlabeled analytes, the ESI signal of reverse charge labeled compounds was shown to improve by 20- to 80-fold. Accurate relative quantification was achieved as no isotopic effects of the different isotope labeled phytohormones during RP/SCX mixed-mode liquid chromatographic separation were observed. A data analysis method was established for analyzing metabolic pathways using our labeling strategy. We then applied our method and examined the jasmonate biosynthetic pathway of rice under salt stress and the premature senescence mutant. Here we found that under salt stress conditions, rice showed up-regulation in (13S)-hydroperoxyoctadecatrienoic acid (HOPT), cis-(+)-12-oxophytodienoic acid (OPDA), 3-oxo-2-(2'-pentenyl)-cyclopentane-1-octanoic acid (OPC-8) and jasmonoyl-valine (JA-Val) levels, while α-linolenic acid (LA) and jasmonic acid (JA) showed down-regulation, and three components (HPOT, OPC-8 and JA-Val) were accumulated. The premature senescence mutant showed up-regulation in all major components of the jasmonate biosynthetic pathway with the exception of LA, and an accumulation of HPOT, OPC-6 and JA-Val. This study demonstrates that our chemical stable isotope labeling strategy can be used as a powerful tool for metabolic pathway analysis of phytohormones in plants.
为了定量研究茉莉酸生物合成途径，我们化学合成了一对同位素质量探针，并建立了标记方案。我们工作中使用的一对质量探针是ω-溴代乙酰基吡啶溴化物（BPB）和 d（5）-ω-溴代乙酰基吡啶溴化物（d（5）-BPB），它们包含羧酸反应基团、同位素标记基团和永久正电荷。高效液相色谱（HPLC）和电喷雾电离四极杆飞行时间质谱（ESI-QTOF-MS）用于标记标准混合物和植物样品的检测。与未标记分析物的负离子电喷雾检测相比，反向电荷标记化合物的 ESI 信号提高了 20 到 80 倍。由于在 RP/SCX 混合模式液相色谱分离过程中不同同位素标记植物激素没有同位素效应，因此实现了准确的相对定量。我们建立了一种数据分析方法，用于使用我们的标记策略分析代谢途径。然后，我们应用我们的方法并检查了盐胁迫下水稻和早衰突变体的茉莉酸生物合成途径。在这里，我们发现，在盐胁迫条件下，水稻中（13S）-过氧氧代十八碳三烯酸（HOPT）、顺式（+）-12-氧代-植二烯酸（OPDA）、3-氧代-2-（2'-戊烯基）-环戊烷-1-辛酸（OPC-8）和茉莉酰-缬氨酸（JA-Val）水平上调，而α-亚麻酸（LA）和茉莉酸（JA）下调，三种成分（HPOT、OPC-8 和 JA-Val）积累。早衰突变体除了 LA 外，茉莉酸生物合成途径的所有主要成分都上调，并且 HPOT、OPC-6 和 JA-Val 积累。这项研究表明，我们的化学稳定同位素标记策略可以作为植物中植物激素代谢途径分析的有力工具。
