A high-performance liquid chromatography-tandem mass spectrometry method for quantitation of nitrogen-containing intracellular metabolites

Abstract

A comprehensive method of quantifying intracellular metabolite concentrations would be a valuable addition to the arsenal of tools for holistic biochemical studies. Here, we describe a step toward the development of such method: a quantitative assay for 90 nitrogen-containing cellular metabolites. The assay involves reverse-phase high-performance liquid chromatography separation followed by electrospray ionization and detection of the resulting ions using triple-quadrupole mass spectrometry in selected reaction monitoring mode. For 79 of the 90 metabolites, the assay is linear with a limit of detection of 10 ng/mL or less. Using this method, 36 metabolites can be reliably detected in extracts of the bacterium Salmonella enterica, with the identity of each metabolite confirmed by the presence, on growing of the bacteria in (13)C-glucose, of a peak corresponding to the isotope-labeled form of the compound. Quantitation in biological samples is performed by mixing unlabeled test cell extract with (13)C-labeled standard extract, and determining the (12)C/(13)C-ratio for each metabolite. Using this approach, the metabolomes of growing (exponential phase) and carbon-starved (stationary phase) bacteria were compared, revealing 16 metabolites that are significantly down-regulated and five metabolites that are significantly up-regulated, in stationary phase.