TY - JOUR
T1 - A high-performance liquid chromatography-tandem mass spectrometry method for quantitation of nitrogen-containing intracellular metabolites
AU - Lu, Wenyun
AU - Kimball, Elizabeth
AU - Rabinowitz, Joshua D.
N1 - Funding Information:
The authors thank David Botstein for motivating this line of research and the Lewis-Sigler Institute and Department of Chemistry at Princeton University for funding these efforts. The also thank Jie Yuan, Robert Moder, Sunil Bajad, Melisa Gao, Sanford Silverman, Celeste Peterson, Thomas Silhavy, and John T. Groves for their valuable comments, suggestions, and technical assistance.
PY - 2006/1
Y1 - 2006/1
N2 - 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 13C-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 13C-labeled standard extract, and determining the 12C/13C-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.
AB - 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 13C-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 13C-labeled standard extract, and determining the 12C/13C-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.
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U2 - 10.1016/j.jasms.2005.09.001
DO - 10.1016/j.jasms.2005.09.001
M3 - Article
C2 - 16352439
AN - SCOPUS:29844441918
SN - 1044-0305
VL - 17
SP - 37
EP - 50
JO - Journal of the American Society for Mass Spectrometry
JF - Journal of the American Society for Mass Spectrometry
IS - 1
ER -