Reducing time and increasing sensitivity in sample preparation for adherent mammalian cell metabolomics.
Reducing time and increasing sensitivity in sample preparation for adherent mammalian cell metabolomics.
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DOI：
10.1021/ac103313x
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发表时间：
2011-05-01
影响因子：
7.4
通讯作者：
Kennedy, Robert T.
中科院分区：
文献类型：
--
作者：
Lorenz, Matthew A.;Burant, Charles F.;Kennedy, Robert T.
使用克隆 β 细胞系 INS-1 作为模型样品，开发了一种简单、快速且可重复的样品制备程序，用于贴壁哺乳动物细胞中代谢物的相对定量。该方法是通过评估不同样品制备程序对直接基础上参与糖酵解和三羧酸循环的 27 种代谢物的高效液相色谱-质谱定量以及非定向基础上所有可检测色谱特征的影响而开发的。我们证明，在代谢淬灭之前的快速水冲洗步骤可减少抑制电喷雾电离的成分，从而增加 27 种目标代谢物中 26 种的信号，并将检测到的特征总数从 237 增加到 452，而代谢物含量没有可检测到的变化。采用一种新颖的淬灭技术，该技术涉及将液氮直接添加到培养皿中，并允许样品在提取前在-80°C下保存至少7天。淬灭和萃取步骤的分离提供了增加实验便利性和样品稳定性的优点，同时保持代谢物含量，类似于采用冷有机溶剂同时淬灭和萃取的技术。研究发现，在代谢物回收率和提取物稳定性方面，提取溶剂 9:1 甲醇:氯仿比乙腈、乙醇和甲醇具有更优异的性能。使用单个快速（约 1 分钟）提取步骤即可实现最大回收率。这种快速制备方法（约 5 分钟）的实用性通过精确的代谢物测量（无内标的平均相对标准偏差为 11%）与葡萄糖浓度的阶跃变化（在克隆 β 细胞系 INS-1 中引起胰岛素分泌）相关来证明。
A simple, fast, and reproducible sample preparation procedure was developed for relative quantification of metabolites in adherent mammalian cells using the clonal β-cell line INS-1 as a model sample. The method was developed by evaluating the effect of different sample preparation procedures on high performance liquid chromatography- mass spectrometry quantification of 27 metabolites involved in glycolysis and the tricarboxylic acid cycle on a directed basis as well as for all detectable chromatographic features on an undirected basis. We demonstrate that a rapid water rinse step prior to quenching of metabolism reduces components that suppress electrospray ionization thereby increasing signal for 26 of 27 targeted metabolites and increasing total number of detected features from 237 to 452 with no detectable change of metabolite content. A novel quenching technique is employed which involves addition of liquid nitrogen directly to the culture dish and allows for samples to be stored at −80 °C for at least 7 d before extraction. Separation of quenching and extraction steps provides the benefit of increased experimental convenience and sample stability while maintaining metabolite content similar to techniques that employ simultaneous quenching and extraction with cold organic solvent. The extraction solvent 9:1 methanol: chloroform was found to provide superior performance over acetonitrile, ethanol, and methanol with respect to metabolite recovery and extract stability. Maximal recovery was achieved using a single rapid (~1 min) extraction step. The utility of this rapid preparation method (~5 min) was demonstrated through precise metabolite measurements (11% average relative standard deviation without internal standards) associated with step changes in glucose concentration that evoke insulin secretion in the clonal β-cell line INS-1.
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影响因子：
14.8
作者：
Bennett, Bryson D.;Yuan, Jie;Rabinowitz, Joshua D.
通讯作者：
Rabinowitz, Joshua D.
影响因子：
7.4
作者：
Canelas, Andre B.;ten Pierick, Angela;Heijnen, Joseph J.
通讯作者：
Heijnen, Joseph J.
影响因子：
7.4
作者：
Myint, Khin Than;Uehara, Taisuke;Oda, Yoshiya
通讯作者：
Oda, Yoshiya
影响因子：
4.1
作者：
Bajad, Sunil U.;Lu, Wenyun;Rabinowitz, Joshua D.
通讯作者：
Rabinowitz, Joshua D.
DOI：
10.1016/j.jchromb.2007.03.029
发表时间：
2007-06-15
影响因子：
3
作者：
Gao, Lan;Chiou, William;Burns, David J.
通讯作者：
Burns, David J.