Improved rapid sampling for in vivo kinetics of intracellular metabolites in Saccharomyces cerevisiae
Improved rapid sampling for in vivo kinetics of intracellular metabolites in Saccharomyces cerevisiae
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DOI：
10.1002/bit.10048
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发表时间：
2001-11-20
影响因子：
3.8
通讯作者：
Heijnen, JJ
中科院分区：
文献类型：
--
作者：
Lange, HC;Eman, M;Heijnen, JJ
一个集成的方法是用来开发一个快速的采样策略，在体内动力学行为的基础上测得的浓度在酿酒酵母细胞内代谢产物的定量分析。重点放在抽样和分析过程中的小样本量。亚秒停留时间是通过最小化无菌取样系统的死体积和通过除了发酵罐中的过压之外对取样管施加真空来最大化流速来实现的。一个专门设计的样品管适配器有助于为各种试管类型提供4至5秒的采样间隔。代谢物浓度的酶促和液相色谱质谱（LC-MSMS）分析结果的统计分析用于优化采样方案。最显著的改进是通过引入细胞提取物的真空干燥来实现的。所提出的系统能够可靠地处理小至1克的发酵罐样品，变化小于3%，因此非常适合于小型实验室规模发酵罐的细胞内测量。(C)John Wiley & Sons，Inc.
An integrated approach is used to develop a rapid sampling strategy for the quantitative analysis of in vivo kinetic behavior based on measured concentrations of intracellular metabolites in Saccharomyces cerevisiae. Emphasis is laid on small sample sizes during sampling and analysis. Subsecond residence times are accomplished by minimizing the dead volume of the sterile sampling system and by maximizing flow rates through application of vacuum to the sampling tubes in addition to the overpressure in the fermenter. A specially designed sample tube adapter facilitates sampling intervals of 4 to 5 s for various test tube types. Statistical analysis of the results obtained from enzymatic and liquid chromatography mass spectrometry (LC-MSMS) analysis of the metabolite concentrations was used to optimize the sampling protocol. The most notable improvement is reached through the introduction of vacuum drying of the cell extract. The presented system is capable of reliably dealing with fermenter samples as small as 1-g with a variation of less than 3%, and is thus ideally suited for intracellular measurements on small, lab-scale fermenters. (C) 2001 John Wiley & Sons, Inc.