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Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis
Published on: November 17, 2011
Simultaneous isotope dilution quantification and metabolic tracing of deoxyribonucleotides by liquid chromatography
Rostislav Kuskovsky1, Raquel Buj2, Peining Xu1
1AJ Drexel Autism Institute, Drexel University, 3020 Market St Suite 560, Philadelphia, PA, 19104, USA.
Abstract:
Quantification of cellular deoxyribonucleoside mono- (dNMP), di- (dNDP), triphosphates (dNTPs) and related nucleoside metabolites are difficult due to their physiochemical properties and widely varying abundance. Involvement of dNTP metabolism in cellular processes including senescence and pathophysiological processes including cancer and viral infection make dNTP metabolism an important bioanalytical target. We modified a previously developed ion pairing reversed phase chromatography-mass spectrometry method for the simultaneous quantification and 13C isotope tracing of dNTP metabolites. dNMPs, dNDPs, and dNTPs were chromatographically resolved to avoid mis-annotation of in-source fragmentation. We used commercially available 13C15N-stable isotope labeled analogs as internal standards and show that this isotope dilution approach improves analytical figures of merit. At sufficiently high mass resolution achievable on an Orbitrap mass analyzer, stable isotope resolved metabolomics allows simultaneous isotope dilution quantification and 13C isotope tracing from major substrates including 13C-glucose. As a proof of principle, we quantified dNMP, dNDP and dNTP pools from multiple cell lines. We also identified isotopologue enrichment from glucose corresponding to ribose from the pentose-phosphate pathway in dNTP metabolites.
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