Abstract
Highly sensitive and quantitative analytical methods are essential for metabolomics. In this report, we introduce an analytical method focused on endogenous phosphorus metabolites, using nanoflow liquid chromatography-electrospray ionization tandem mass spectrometry (nanoLC-ESI-MS/MS) and culture-derived isotope-tagged metabolites as global internal standards for quantitative metabolomics. The nanoLC-ESI-MS/MS method employing a stone-arch microcolumn with amino propyl silica gel achieved good separation of phosphorus metabolites with forty- to hundred-fold increase of sensitivity compared with semimicro flow LC-ESI-MS. The quantitative reproducibility of the nanoLC-ESI-MS has been improved to the point where it is useful for studies of cellular metabolism. Focused metabolomics using culture-derived internal standards was employed to monitor 184 phosphorus-related metabolic changes in cancer cells treated with metabolic enzyme inhibitors, methotrexate, fluorouracil, and gemcitabine. We found marked perturbations of cellular metabolism, of which many, though not all, were in line with the known biological activities of these drugs.
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