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Quantification of Intracellular Metabolites in Escherichia coli K12 Using Liquid Chromatographic-Electrospray Ionization Tandem Mass Spectrometric Techniques

✍ Scribed by Arne Buchholz; Ralf Takors; Christian Wandrey

Publisher
Elsevier Science
Year
2001
Tongue
English
Weight
142 KB
Volume
295
Category
Article
ISSN
0003-2697

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✦ Synopsis

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 steadystate) 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.

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