The first step of many metabolomics studies is quenching, a technique vital for rapidly halting metabolism and ensuring that the metabolite profile remains unchanging during sample processing. The most widely used approach is to plunge the sample into prechilled cold methanol; however, this led to significant metabolite loss in Synecheococcus sp. PCC 7002. Here we describe our analysis of the impacts of cold methanol quenching on the model marine cyanobacterium Synechococcus sp. PCC 7002, as well as our brief investigation of alternative quenching methods. We tested several methods including cold methanol, cold saline, and two filtration approaches. Targeted central metabolites were extracted and metabolomic profiles were generated using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). The results indicate that cold methanol quenching induces dramatic metabolite leakage in Synechococcus, resulting in a majority of central metabolites being lost prior to extraction. Alternatively, usage of a chilled saline quenching solution mitigates metabolite leakage and improves sample recovery without sacrificing rapid quenching of cellular metabolism. Finally, we illustrate that metabolite leakage can be assessed, and subsequently accounted for, in order to determine absolute metabolite pool sizes; however, our results show that metabolite leakage is inconsistent across various metabolite pools and therefore must be determined for each individually measured metabolite.

中文翻译：

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光自养聚球藻代谢物回收淬灭方法的评价。PCC 7002。

许多代谢组学研究的第一步是淬灭，这项技术对于快速停止代谢和确保代谢物在样品处理过程中保持不变至关重要。最广泛使用的方法是将样品放入预冷的冷甲醇中；然而，这导致了聚球藻属的显着代谢物损失。PCC 7002。在这里，我们描述了我们对冷甲醇淬火对模型海洋蓝藻聚球藻的影响的分析sp. PCC 7002，以及我们对替代淬火方法的简要调查。我们测试了几种方法，包括冷甲醇、冷盐水和两种过滤方法。提取目标中心代谢物，并使用液相色谱-串联质谱 (LC-MS/MS) 生成代谢组学特征。结果表明，冷甲醇淬灭导致聚球藻中的代谢物显着泄漏，导致大部分中心代谢物在提取前丢失。或者，使用冷冻盐水淬灭溶液可减轻代谢物泄漏并提高样品回收率，而不会牺牲细胞代谢的快速淬灭。最后，我们说明可以评估代谢物泄漏，并随后解释，以确定绝对代谢物池大小；然而，我们的结果表明代谢物泄漏在各种代谢物库中是不一致的，因此必须针对每个单独测量的代谢物进行确定。