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 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.
Buchholz
A
A
Institute of Biotechnology 2, Research Centre Juelich, 52425 Juelich, Germany.
Takors
R
R
Wandrey
C
C
D000105:Acetyl Coenzyme A / Q000032:analysis; D000227:Adenine Nucleotides / Q000032:analysis; D002853:Chromatography, Liquid / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry / Q000254:growth & development / Q000378:metabolism*; D005635:Fructosediphosphates / Q000032:analysis; D005942:Gluconates / Q000032:analysis; D019298:Glucose-6-Phosphate / Q000032:analysis; D006019:Glycolysis; D013058:Mass Spectrometry / Q000379:methods
D000227:Adenine Nucleotides; D005635:Fructosediphosphates; D005942:Gluconates; D019298:Glucose-6-Phosphate; D000105:Acetyl Coenzyme A; C029063:fructose-1,6-diphosphate; C008884:6-phosphogluconic acid