Institute for Advanced Biosciences, Keio University, Nipponkoku 403-1, Daihouji, Tsuruoka City, Yamagata, Japan.
PLoS One. 2012;7(8):e44283. doi: 10.1371/journal.pone.0044283. Epub 2012 Aug 29.
There is an increasing call for the absolute quantification of time-resolved metabolite data. However, a number of technical issues exist, such as metabolites being modified/degraded either chemically or enzymatically during the extraction process. Additionally, capillary electrophoresis mass spectrometry (CE-MS) is incompatible with high salt concentrations often used in extraction protocols. In microbial systems, metabolite yield is influenced by the extraction protocol used and the cell disruption rate. Here we present a method that rapidly quenches metabolism using dry-ice ethanol bath and methanol N-ethylmaleimide solution (thus stabilising thiols), disrupts cells efficiently using bead-beating and avoids artefacts created by live-cell pelleting. Rapid sample processing minimised metabolite leaching. Cell weight, number and size distribution was used to calculate metabolites to an attomol/cell level. We apply this method to samples obtained from the respiratory oscillation that occurs when yeast are grown continuously.
人们越来越呼吁对时间分辨代谢物数据进行绝对定量。然而，存在许多技术问题，例如在提取过程中化学或酶促修饰/降解代谢物。此外，毛细管电泳质谱 (CE-MS) 与提取方案中常用的高盐浓度不兼容。在微生物系统中，代谢物的产量受到所用提取方案和细胞破碎率的影响。在这里，我们提出了一种使用干冰乙醇浴和甲醇 N-乙基马来酰亚胺溶液快速淬灭代谢物的方法（从而稳定硫醇），使用珠磨高效地破坏细胞，并避免由活细胞沉淀产生的假象。快速的样品处理最大限度地减少了代谢物的浸出。使用细胞重量、数量和大小分布来计算细胞内代谢物的含量，达到了 attomol/细胞的水平。我们将该方法应用于从酵母连续生长时发生的呼吸振荡中获得的样品。
