Isotope ratio-based profiling of microbial folates
Isotope ratio-based profiling of microbial folates
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DOI：
10.1016/j.jasms.2007.01.017
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发表时间：
2007-05-01
影响因子：
3.2
通讯作者：
Rabinowitz, Joshua D.
中科院分区：
文献类型：
--
作者：
Lu, Wenyun;Kwon, Yun Kyung;Rabinowitz, Joshua D.
叶酸代谢是抗生素和抗癌药物最重要的靶点之一，它负责包括胸苷生物合成在内的关键细胞过程中的一碳转移反应。细胞内叶酸的分析因三种不同类型的叶酸修饰而变得复杂：氧化/还原、甲基化和聚谷氨酰化。在这里，我们提出了一种通过液相色谱-串联质谱（LC-MS/MS）定量细胞内叶酸的全部多样性的方法。该方法开始于使用-75 ° C甲醇：水提取叶酸，加入抗坏血酸和乙酸铵以防止叶酸相互转化。然后使用亲水相互作用色谱法和氨基柱分离提取物，通过正离子模式电喷雾电离，并使用多反应监测在三重四极杆仪器上进行分析。该方法已被用于分析大肠杆菌和酿酒酵母中的叶酸库，并在大肠杆菌中获得了所选叶酸的绝对水平。通过用纯化的未标记的叶酸标准品加标均匀饲喂C-13-葡萄糖的细胞提取物来测量。一种基于同位素比例的方法已被应用于研究甲氧苄啶的作用，甲氧苄啶是一种临床上重要的抗生素，可阻断细菌二氢叶酸还原酶。除了引起氧化叶酸的预期增加和还原叶酸的减少外，甲氧苄啶。引发了一个戏剧性的和以前未被认识到的转变，朝着更短的聚谷氨酸链长度。这一发现突出了通过MS/MS分析细胞叶酸全谱的潜力，以揭示新的生物学现象。
Folate metabolism, which is responsible for one-carbon transfer reactions in critical cellular processes including thymidine biosynthesis, is among the most important targets of antibiotic and anticancer drugs. Analysis of intracellular folates is complicated by three different types of folate modification: oxidation/reduction, methylation, and polyglutamylation. Here we present a method for quantifying the full diversity of intracellular folates by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method begins with folate extraction using -75 degrees C methanol:water, with ascorbic acid and ammonium acetate added to prevent folate interconversion. The extract is then separated using hydrophilic interaction chromatography with an amino column, ionized by positive mode electrospray, and analyzed on a triple quadrupole instrument using multiple reaction monitoring. The method has been used to profile the folate pools in Escherichia coli and Saccharomyces cerevisiae, with absolute levels of selected folates in E. coli measured by spiking extracts of cells fed uniformly C-13-glucose with purified, unlabeled folate standards. An isotope-ratio-based approach has been applied to study the effects of trimethoprim, a clinically important antibiotic that blocks bacterial dihydrofolate reductase. In addition to causing the expected increase in oxidized and decrease in reduced folates, trimethoprim. triggered a dramatic and previously unrecognized shift towards shorter polyglutamate chain lengths. This finding highlights the potential for analysis of the full spectrum of cellular folates by MS/MS to unveil novel biological phenomena.