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

Anal Biochem. 2001 Aug 15;295(2):129-37. doi: 10.1006/abio.2001.5183.


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.

MeSH terms

  • Acetyl Coenzyme A / analysis
  • Adenine Nucleotides / analysis
  • Chromatography, Liquid / methods
  • Escherichia coli / chemistry
  • Escherichia coli / growth & development
  • Escherichia coli / metabolism*
  • Fructosediphosphates / analysis
  • Gluconates / analysis
  • Glucose-6-Phosphate / analysis
  • Glycolysis
  • Mass Spectrometry / methods


  • Adenine Nucleotides
  • Fructosediphosphates
  • Gluconates
  • Glucose-6-Phosphate
  • Acetyl Coenzyme A
  • fructose-1,6-diphosphate
  • 6-phosphogluconic acid