Albrecht-von-Haller-Institute for Plant Sciences, Department of Plant Biochemistry, Georg-August-University, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany.
Plant Methods. 2013 Jul 6;9(1):24. doi: 10.1186/1746-4811-9-24.
Wax esters are highly hydrophobic neutral lipids that are major constituents of the cutin and suberin layer. Moreover they have favorable properties as a commodity for industrial applications. Through transgenic expression of wax ester biosynthetic genes in oilseed crops, it is possible to achieve high level accumulation of defined wax ester compositions within the seed oil to provide a sustainable source for such high value lipids. The fatty alcohol moiety of the wax esters is formed from plant-endogenous acyl-CoAs by the action of fatty acyl reductases (FAR). In a second step the fatty alcohol is condensed with acyl-CoA by a wax synthase (WS) to form a wax ester. In order to evaluate the specificity of wax ester biosynthesis, analytical methods are needed that provide detailed wax ester profiles from complex lipid extracts.
We present a direct infusion ESI-tandem MS method that allows the semi-quantitative determination of wax ester compositions from complex lipid mixtures covering 784 even chain molecular species. The definition of calibration prototype groups that combine wax esters according to their fragmentation behavior enables fast quantitative analysis by applying multiple reaction monitoring. This provides a tool to analyze wax layer composition or determine whether seeds accumulate a desired wax ester profile. Besides the profiling method, we provide general information on wax ester analysis by the systematic definition of wax ester prototypes according to their collision-induced dissociation spectra. We applied the developed method for wax ester profiling of the well characterized jojoba seed oil and compared the profile with wax ester-accumulating Arabidopsis thaliana expressing the wax ester biosynthetic genes MaFAR and ScWS.
We developed a fast profiling method for wax ester analysis on the molecular species level. This method is suitable to screen large numbers of transgenic plants as well as other wax ester samples like cuticular lipid extracts to gain an overview on the molecular species composition. We confirm previous results from APCI-MS and GC-MS analysis, which showed that fragmentation patterns are highly dependent on the double bond distribution between the fatty alcohol and the fatty acid part of the wax ester.
蜡酯是高度疏水的中性脂质，是角质层和栓质层的主要成分。此外，它们具有作为工业应用商品的有利特性。通过在油料作物中转基因表达蜡酯生物合成基因，可以在种子油中实现高水平积累特定的蜡酯组成，为这种高价值脂质提供可持续的来源。蜡酯的脂肪醇部分是由植物内源性酰基辅酶 A 通过脂肪酰基辅酶 A 还原酶 (FAR) 的作用形成的。在第二步中，脂肪醇与酰基辅酶 A 通过蜡合酶 (WS) 缩合形成蜡酯。为了评估蜡酯生物合成的特异性，需要提供从复杂脂质提取物中提供详细蜡酯谱的分析方法。
我们提出了一种直接注入 ESI 串联 MS 方法，该方法允许从复杂的脂质混合物中半定量测定蜡酯组成，涵盖 784 种偶数链分子物种。根据其碎裂行为定义校准原型组，可将结合的蜡酯组合在一起，从而通过应用多重反应监测进行快速定量分析。这为分析蜡层组成或确定种子是否积累所需的蜡酯谱提供了一种工具。除了分析方法外，我们还根据其碰撞诱导解离光谱系统地定义蜡酯原型，提供了有关蜡酯分析的一般信息。我们将开发的方法应用于特征明确的霍霍巴种子油的蜡酯分析，并将其与表达蜡酯生物合成基因 MaFAR 和 ScWS 的蜡酯积累拟南芥进行了比较。
我们开发了一种快速的蜡酯分析方法，用于在分子物种水平上进行分析。该方法适用于筛选大量转基因植物以及其他蜡酯样品，如角质层脂质提取物，以获得分子物种组成的概述。我们证实了 APCI-MS 和 GC-MS 分析的先前结果，这些结果表明，碎裂模式高度依赖于蜡酯中脂肪醇和脂肪酸部分之间的双键分布。
