Analysis of carboxylic acid metabolites from the tricarboxylic acid cycle in Bacillus subtilis cell extract by capillary electrophoresis using an indirect photometric detection method.

ABSTRACT

With a growing interest in metabolome analysis, there is a need for developing robust methods for analysis of intracellular metabolites profiles in real samples like e.g., bacteria cell. Due to their weak absorbance properties, tri- and dicarboxylic acids from TCA cycle (citric, isocitric, 2-oxoglutaric, succinic, fumaric, malic) as well as carboxylic acid metabolites from glycolysis pathway, urea cycle and metabolism of amino compounds (formic, pyruvic, lactic, acetic, glutamic) were analyzed by capillary electrophoresis (CE) with indirect UV detection. Using 4 mM 2,6-pyridinedicarboxylic acid as a highly UV absorbing carrier electrolyte, 0.2 mM cetyltrimethylammonium bromide, 10% ethylene glycol and 10% acetonitrile, pH 3.5, carboxylic acids metabolites were analyzed in Bacillus subtilis cell extract from two different cultures glucose and malate. CE with an electrokinetic injection mode achieved limits of detection in the range of 13-54 ppb (1.12-10(-7) - 5.96-10(-7) M). The reproducibility and linearity of method was investigated with RSD for migration time less than 1.3% and acceptable correlation coefficients. The optimized CE method was used to compare metabolome content of cell extract derived from two different culture media containing either glucose or malate as a carbon source. The changes in carboxylic acid metabolites profile were observed depending from used culture medium. Carboxylic acid concentrations ranged in cell extract from malate culture from 59 to 0.5 microM for lactate and citrate, respectively, and in cell extract from glucose culture from 133 to 0.5 microM for glutamate and citrate, respectively. Appropriate concentrations of carboxylic acid in the single bacterium cell were estimated at mM and sub-mM levels.