Quenching for Microalgal Metabolomics: A Case Study on the Unicellular Eukaryotic Green Alga Chlamydomonas reinhardtii.
Quenching for Microalgal Metabolomics: A Case Study on the Unicellular Eukaryotic Green Alga Chlamydomonas reinhardtii.
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DOI：
10.3390/metabo8040072
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发表时间：
2018-10-31
期刊：
影响因子：
4.1
通讯作者：
Vaidyanathan S
中科院分区：
文献类型：
--
作者：
Kapoore RV;Vaidyanathan S
捕捉有效的代谢组快照需要迅速猝灭酶的活性。这是阻止新陈代谢持续流动和代谢产物高周转率的关键一步。到目前为止，冷水甲醇猝灭被视为金标准，然而，由于与细胞内代谢物泄漏有关的潜在问题，所获得的代谢组学数据的可靠性受到质疑。因此，我们考察了淬灭溶剂、甲醇浓度、缓冲液添加剂包被物、淬灭时间和剂样比等参数对莱茵衣藻细胞内代谢产物渗漏的影响。我们用气相色谱-质谱仪(GC-MS)测量了所有可能组分中12种代谢物的回收率，并建立了质量平衡来追踪猝灭处理过程中代谢物的去向。我们的数据表明，使用传统的60%甲醇时，细胞内代谢物的显著损失，增加甲醇浓度或猝灭时间也会导致更高的泄漏。加入各种缓冲添加剂表明，70 mM HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic酸)是合适的。综上所述，我们建议用60%含水的甲醇和70 mM HEPES(−40°C)在1：1的样品与淬灭溶剂比下淬灭，因为这样可以提高细胞内代谢物的回收率，并随后减少所有代谢物类别的代谢物泄漏。
Capturing a valid snapshot of the metabolome requires rapid quenching of enzyme activities. This is a crucial step in order to halt the constant flux of metabolism and high turnover rate of metabolites. Quenching with cold aqueous methanol is treated as a gold standard so far, however, reliability of metabolomics data obtained is in question due to potential problems connected to leakage of intracellular metabolites. Therefore, we investigated the influence of various parameters such as quenching solvents, methanol concentration, inclusion of buffer additives, quenching time and solvent to sample ratio on intracellular metabolite leakage from Chlamydomonas reinhardtii. We measured the recovery of twelve metabolite classes using gas chromatography mass spectrometry (GC-MS) in all possible fractions and established mass balance to trace the fate of metabolites during quenching treatments. Our data demonstrate significant loss of intracellular metabolites with the use of the conventional 60% methanol, and that an increase in methanol concentration or quenching time also resulted in higher leakage. Inclusion of various buffer additives showed 70 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) to be suitable. In summary, we recommend quenching with 60% aqueous methanol supplemented with 70 mM HEPES (−40 °C) at 1:1 sample to quenching solvent ratio, as it resulted in higher recoveries for intracellular metabolites with subsequent reduction in the metabolite leakage for all metabolite classes.
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影响因子：
5
作者：
Schaedel, Friederike;David, Florian;Franco-Lara, Ezequiel
通讯作者：
Franco-Lara, Ezequiel
影响因子：
7.4
作者：
Bolten, Christoph J.;Kiefer, Patrick;Wittmann, Christoph
通讯作者：
Wittmann, Christoph
影响因子：
7.4
作者：
Kim, Sooah;Lee, Do Yup;Kim, Kyoung Heon
通讯作者：
Kim, Kyoung Heon
影响因子：
4.4
作者：
Soga, T;Ohashi, Y;Nishioka, T
通讯作者：
Nishioka, T
影响因子：
7.4
作者：
Bölling, C;Fiehn, O
通讯作者：
Fiehn, O