An efficient LC-MS method for isomer separation and detection of sugars, phosphorylated sugars, and organic acids
An efficient LC-MS method for isomer separation and detection of sugars, phosphorylated sugars, and organic acids
复制标题
用于糖、磷酸化糖和有机酸异构体分离和检测的高效 LC-MS 方法
DOI：
10.1093/jxb/erac062
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发表时间：
2021
影响因子：
6.9
通讯作者：
Allen, Doug K
中科院分区：
文献类型：
--
作者：
Koley, Somnath;Chu, Kevin L;Gill, Saba S;Allen, Doug K
由于库大小的动态范围，评估植物中的中心碳代谢可能具有挑战性，相对于更丰富的糖和有机酸，重要的磷酸化糖水平较低。在这里，我们报告了一个敏感的液相色谱-质谱法分析中心代谢物的混合柱，其中阴离子交换和亲水相互作用色谱（HILIC）配体嵌入在固定相。液相色谱法的开发，以提高选择性的27个中心代谢物在一个单一的运行与灵敏度在飞摩尔水平观察到大多数磷酸化糖。该方法分离了磷酸化的己糖、戊糖和丙糖异构体，否则这些异构体具有挑战性。与标准HILIC方法相比，由于生物样品基质中的离子增强或低离子抑制，这些代谢物使用我们的方法具有改善的峰面积。该方法被应用于研究高脂烟草叶片的代谢，表现出增加的乙酰辅酶A水平，油生物合成的前体。通过对13 C标记亚麻荠种子的评价，探讨了该方法在同位素体检测和定量中的应用。该方法为更全面地分析植物组织中心代谢的中间产物提供了一种手段。
Assessing central carbon metabolism in plants can be challenging due to the dynamic range in pool sizes, with low levels of important phosphorylated sugars relative to more abundant sugars and organic acids. Here, we report a sensitive liquid chromatography–mass spectrometry method for analysing central metabolites on a hybrid column, where both anion-exchange and hydrophilic interaction chromatography (HILIC) ligands are embedded in the stationary phase. The liquid chromatography method was developed for enhanced selectivity of 27 central metabolites in a single run with sensitivity at femtomole levels observed for most phosphorylated sugars. The method resolved phosphorylated hexose, pentose, and triose isomers that are otherwise challenging. Compared with a standard HILIC approach, these metabolites had improved peak areas using our approach due to ion enhancement or low ion suppression in the biological sample matrix. The approach was applied to investigate metabolism in high lipid-producing tobacco leaves that exhibited increased levels of acetyl-CoA, a precursor for oil biosynthesis. The application of the method to isotopologue detection and quantification was considered through evaluating13C-labeled seeds fromCamelina sativa. The method provides a means to analyse intermediates more comprehensively in central metabolism of plant tissues.