Quantification of intracellular metabolites in Escherichia coli K12 using liquid chromatographic-electrospray ionization tandem mass spectrometric techniques
Quantification of intracellular metabolites in Escherichia coli K12 using liquid chromatographic-electrospray ionization tandem mass spectrometric techniques
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DOI：
10.1006/abio.2001.5183
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发表时间：
2001-08-15
影响因子：
2.9
通讯作者：
Wandrey, C
中科院分区：
文献类型：
--
作者：
Buchholz, A;Takors, R;Wandrey, C
微生物代谢网络的定量理解是有效的合理菌株改良（“代谢工程”）的先决条件。因此有必要精确地确定大量反应物的浓度（即，代谢物、核苷酸、辅因子），以便理解“体内”反应动力学。采用高氯酸萃取和LC-ESI-MS方法定量测定了大肠杆菌K12细胞内糖酵解中间产物和核苷酸的浓度。细胞内代谢物（例如，葡萄糖6-磷酸、果糖1，6-二磷酸、6-磷酸葡萄糖酸、乙酰辅酶A、腺嘌呤核苷酸）在确定的（葡萄糖限制的稳态）生长条件下定量。通过比较通过LC-ESI-MS测量的细胞内代谢物浓度与酶测定来验证该方法。因此，可以用最小量的样品体积平行鉴定和定量超过15种细胞内代谢物。(C)北京：科学出版社.
The quantitative comprehension of microbial metabolic networks is a prerequisite for an efficient rational strain improvement ("metabolic engineering"). It is therefore necessary to accurately determine the concentration of a large number of reactants (i.e., metabolites, nucleotides, cofactors) in order to understand "in vivo" reaction kinetics. Quantification of intracellular concentrations of glycolytic intermediates and nucleotides in Escherichia coli K12 using a perchloric acid extraction and an LC-ESI-MS method was achieved. Intracellular metabolites (e.g., glucose 6-phosphate, fructose 1,6-bisphosphate, 6-phospho gluconate, acetyl-CoA, adenine nucleotides) were quantified under defined (glucose-limited steady-state) growth conditions. The method was verified by comparing the intracellular metabolite concentrations measured via LC-ESI-MS with enzymatic determinations. It is thus possible to identify and quantify more than 15 intracellular metabolites in parallel with a minimal amount of sample volume. (C) 2001 Academic Press.