Quantification of intracellular metabolites in Escherichia coli K12 using liquid chromatographic-electrospray ionization tandem mass spectrometric techniques

The quantitative comprehension of microbial metabolic networks is a prerequ isite 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 o rder to understand "in vivo" reaction kinetics. Quantification of intracell ular concentrations of glycolytic intermediates and nucleotides in Escheric hia 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 concentr ations measured via LC-ESI-MS with enzymatic determinations. It is thus pos sible to identify and quantify more than 15 intracellular metabolites in pa rallel with a minimal amount of sample volume. (C) 2001 Academic Press.