Journal
ANALYTICAL BIOCHEMISTRY
Volume 295, Issue 2, Pages 129-137Publisher
ACADEMIC PRESS INC
DOI: 10.1006/abio.2001.5183
Keywords
Escherichia coli; intracellular metabolites; ESI; extraction; LC-MS; quantification
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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.
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