Simultaneous isotope dilution quantification and metabolic tracing of deoxyribonucleotides by liquid chromatography high resolution mass spectrometry

Rostislav Kuskovsky, Raquel Buj, Peining Xu, Samuel Hofbauer, Mary T. Doan, Helen Jiang, Anna Bostwick, Clementina Mesaros, Katherine Aird, Nathaniel W. Snyder

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)65-72
Number of pages8
JournalAnalytical Biochemistry
Volume568
DOIs
StatePublished - Mar 1 2019

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Deoxyribonucleotides
Liquid chromatography
Liquid Chromatography
Isotopes
Dilution
Mass spectrometry
Mass Spectrometry
Metabolites
Metabolism
Deoxyribonucleosides
Pentoses
Glucose
Pentose Phosphate Pathway
Metabolomics
Ribose
Virus Diseases
Reverse-Phase Chromatography
Chromatography
triphosphoric acid
Nucleosides

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Kuskovsky, Rostislav ; Buj, Raquel ; Xu, Peining ; Hofbauer, Samuel ; Doan, Mary T. ; Jiang, Helen ; Bostwick, Anna ; Mesaros, Clementina ; Aird, Katherine ; Snyder, Nathaniel W. / Simultaneous isotope dilution quantification and metabolic tracing of deoxyribonucleotides by liquid chromatography high resolution mass spectrometry. In: Analytical Biochemistry. 2019 ; Vol. 568. pp. 65-72.
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Simultaneous isotope dilution quantification and metabolic tracing of deoxyribonucleotides by liquid chromatography high resolution mass spectrometry. / Kuskovsky, Rostislav; Buj, Raquel; Xu, Peining; Hofbauer, Samuel; Doan, Mary T.; Jiang, Helen; Bostwick, Anna; Mesaros, Clementina; Aird, Katherine; Snyder, Nathaniel W.

In: Analytical Biochemistry, Vol. 568, 01.03.2019, p. 65-72.

Research output: Contribution to journalArticle

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AU - Kuskovsky, Rostislav

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AU - Xu, Peining

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AU - Doan, Mary T.

AU - Jiang, Helen

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AU - Snyder, Nathaniel W.

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