Sampling for metabolome analysis of microorganisms
Sampling for metabolome analysis of microorganisms
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DOI：
10.1021/ac0623888
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发表时间：
2007-05-15
影响因子：
7.4
通讯作者：
Wittmann, Christoph
中科院分区：
文献类型：
--
作者：
Bolten, Christoph J.;Kiefer, Patrick;Wittmann, Christoph
在目前的工作中，我们调查了代谢组学领域中最常用的微生物采样方法，以揭示以前被忽视但对准确的代谢组分析至关重要的潜在误差源。为了扩大我们的研究意义，我们调查了不同的革兰氏阴性和革兰氏阳性细菌，即枯草芽孢杆菌、谷氨酸棒杆菌、大肠杆菌、氧化葡萄糖杆菌、恶臭假单胞菌和运动发酵单胞菌，并分析了不同分解代谢和合成代谢途径的代谢产物。在细胞分离和提取之前用冷甲醇猝灭细胞会导致所有被测代谢物由于非特异性泄漏而急剧损失(>；60%)。使用快速过滤，当使用不适当的低离子强度洗涤液时，革兰氏阴性菌也显示出显著的损失(>；80%)。调整洗涤液的离子强度以适应培养介质的离子强度几乎可以完全避免这一问题。与洗涤液无关，革兰氏阳性菌株没有明显的渗漏。快速过滤，在提取前采样几秒钟，似乎是一种适合于细胞内水平相对较高和周转较低的代谢物，如氨基酸或TCA循环中间体的方法。对培养上清液和细胞内部代谢物水平的比较表明，整个肉汤猝灭方案将发现的代谢物专门归因于细胞内池的常见假设在许多情况下可能不成立。在这种情况下，需要对含有中间成分的代谢物进行差示校正。
In the present work we investigated the most commonly applied methods used for sampling of microorganisms in the field of metabolomics in order to unravel potential sources of error previously ignored but of utmost importance for accurate metabolome analysis. To broaden the significance of our study, we investigated different Gram-negative and Gram-positive bacteria, i.e., Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli, Gluconobacter oxydans, Pseudomonas putida, and zymomonas mobilis, and analyzed metabolites from different catabolic and anabolic intracellular pathways. Quenching of cells with cold methanol prior to cell separation and extraction led to drastic loss (> 60%) of all metabolites tested due to unspecific leakage. Using fast filtration, Gram-negative bacteria also revealed a significant loss (> 80%) when inappropriate washing solutions with low ionic strength were applied. Adapting the ionic strength of the washing solution to that of the cultivation medium could almost completely avoid this problem. Gram-positive strains did not show significant leakage independent of the washing solution. Fast filtration with sampling times of several seconds prior to extraction appears to be a suitable approach for metabolites with relatively high intracellular level and low turnover such as amino acids or TCA cycle intermediates. Comparison of metabolite levels in the culture supernatant and the cell interior revealed that the common assumption of whole broth quenching protocols attributing the metabolites found exclusively to the intracellular pools may not be valid in many cases. In such cases a differential approach correcting for medium-contained metabolites is required.