Quantitative metabolome analysis using liquid chromatography-high-resolution mass spectrometry
Quantitative metabolome analysis using liquid chromatography-high-resolution mass spectrometry
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DOI：
10.1016/j.ab.2008.07.010
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发表时间：
2008-11-15
影响因子：
2.9
通讯作者：
Vorholt, Julia A.
中科院分区：
文献类型：
--
作者：
Kiefer, Patrick;Portais, Jean-Charles;Vorholt, Julia A.
在这份报告中，我们介绍了一种使用LTQ Orbitrap高分辨率质谱仪进行代谢组定量的液相色谱-单质谱方法。用亲水作用液相色谱分离分析物。在工作分辨率为30,000(m/z为400)时，25种代谢物的检出限从50fmol到5pmol不等。就代谢物浓度而言，大多数化合物(R-2和GT；0.99)的线性关系约为2-3个数量级。为了确定该系统在复杂样品基质中的准确性，对同位素稀释法进行了评估，从纯化合物和均匀的C-13标记的细胞提取物的混合物中进行了评估。应用这种方法，即使在单个代谢物的池大小从0.13到55.6微米的范围内，也可以在单次运行中进行定量。作为案例研究，测定了在甲醇和琥珀酸两种不同碳源上生长的甲基洛伯杆菌AMI胞内中心代谢物的浓度。从技术和生物重复获得了可重复性的结果，揭示了细胞内代谢物池大小的显著差异，这取决于碳源。LTQ Obitrap为代谢组定量提供了新的视角和策略。(C)2008 Elsevier Inc.保留所有权利。
in this report, we introduce a liquid chromatography single-mass spectrometry method for metabolome quantification, using the LTQ Orbitrap high-resolution mass spectrometer. Analytes were separated with hydrophilic interaction liquid chromatography. At a working resolution of 30,000 (at m/z 400), the limit of detection varied from 50 fmol to 5 pmol for 25 metabolites tested. In terms of metabolite concentration, the linearity was about 2 to 3 orders of magnitude for most compounds (R-2 > 0.99). To determine the accuracy of the system in complex sample matrices, the isotope dilution method was evaluated from mixtures of pure compounds and uniformly C-13-labeled cell extracts. With the application of this method, quantification was possible within single runs even when the pool sizes of individual metabolites varied from 0.13 to 55.6 mu M. As a case Study, intracellular concentrations of central metabolites were determined for Methylobacterium extorquens AMI during growth on two different carbon sources, methanol and succinate. Reproducible results from technical and biological repetitions were obtained that revealed significant variations of intracellular metabolite pool sizes, depending on the carbon source. The LTQ Obitrap offers new perspectives and strategies for metabolome quantification. (C) 2008 Elsevier Inc. All rights reserved.