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Journal article
Simultaneous isotope dilution quantification and metabolic tracing of deoxyribonucleotides by liquid chromatography high resolution mass spectrometry
Analytical biochemistry, Vol.568, pp.65-72
01 Mar 2019
PMID: 30605633
Featured in Collection : UN Sustainable Development Goals @ Drexel
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.
•Deoxyribonucleoside mono-, di-, and tri-phosphates (dNTPs) are important bioanalytical targets.•LC-HRMS provides sufficient sensitivity and specificity for dNTP analysis in cellular metabolism studies.•13C and 15N labeling can be used orthogonally in dNTPs.•Simultaneous isotope dilution based quantification and isotope tracing with 13C labeled substrates is feasible.
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Details
- Title
- Simultaneous isotope dilution quantification and metabolic tracing of deoxyribonucleotides by liquid chromatography high resolution mass spectrometry
- Creators
- Rostislav Kuskovsky - Drexel UniversityRaquel Buj - Pennsylvania State UniversityPeining Xu - Drexel UniversitySamuel Hofbauer - University of PennsylvaniaMary T. Doan - Drexel UniversityHelen Jiang - Drexel UniversityAnna Bostwick - Drexel UniversityClementina Mesaros - University of PennsylvaniaKatherine M. Aird - Pennsylvania State UniversityNathaniel W. Snyder - Drexel University
- Publication Details
- Analytical biochemistry, Vol.568, pp.65-72
- Publisher
- Elsevier
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- A.J. Drexel Autism Institute
- Identifiers
- 991019167757604721
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- Collaboration types
- Domestic collaboration
- Web of Science research areas
- Biochemical Research Methods
- Biochemistry & Molecular Biology
- Chemistry, Analytical