Fourier transform-ion cyclotron resonance-mass spectrometry (FTICR-MS) is capable of acquiring unmatched quality of isotopologue data for stable isotope resolved metabolomics (SIRM). This capability drives the need for a continuous ion introduction for obtaining optimal isotope ratios. Here we report the simultaneous analysis of mono and dinucleotides from crude polar extracts by FTICR-MS by adapting an ion-pairing sample preparation method for LC–MS analysis. This involves a rapid cleanup of extracted nucleotides on pipet tips containing a C18 stationary phase, which enabled global analysis of nucleotides and their 13C isotopologues at nanomolar concentrations by direct infusion nanoelectrospray FTICR-MS with 5 min of data acquisition. The resolution and mass accuracy enabled computer-assisted unambiguous assignment of most nucleotide species, including all phosphorylated forms of the adenine, guanine, uracil and cytosine nucleotides, NAD+, NADH, NADP+, NADPH, cyclic nucleotides, several UDP-hexoses, and all their 13C isotopologues. The method was applied to a SIRM study on human lung adenocarcinoma A549 cells grown in [U-13C] glucose with or without the anti-cancer agent methylseleninic acid. At m/z resolving power of 400,000, 13C-isotopologues of nucleotides were fully resolved from all other elemental isotopologues, thus allowing their 13C fractional enrichment to be accurately determined. The method achieves both high sample and high information throughput analysis of nucleotides for metabolic pathway reconstruction in SIRM investigations.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Fourier transform ion cyclotron-mass spectrometry
Heteronuclear single quantum coherence
Pentose phosphate pathway
Precalculated exact mass isotopologue search engine
Stable isotope resolved metabolomics
Annesley, T. M. (2003). Ion suppression in mass spectrometry. Clinical Chemistry, 49, 1041–1044.
Coulier, L., Bas, R., Jespersen, S., Verheij, E., van der Werf, M. J., & Hankemeier, T. (2006). Simultaneous quantitative analysis of metabolites using ion-pair liquid chromatography: Electrospray ionization mass spectrometry. Analytical Chemistry, 78, 6573–6582.
Desiderio, D. M., & Beranova-Giorgianni, S. (2000). Mass spectrometry of the human pituitary proteome: Identification of selected proteins. Rapid Communications in Mass Spectrometry, 14, 161–167.
Dodbiba, E., Breitbach, Z. S., Wanigasekara, E., Payagala, T., Zhang, X. T., & Armstrong, D. W. (2010). Detection of nucleotides in positive-mode electrospray ionization mass spectrometry using multiply-charged cationic ion-pairing reagents. Analytical and Bioanalytical Chemistry, 398, 367–376.
Edwards, J. L., & Kennedy, R. T. (2005). Metabolomic analysis of eukaryotic tissue and prokaryotes using negative mode MALDI time-of-flight mass spectrometry. Analytical Chemistry, 77, 2201–2209.
Fan, T. W.-M. (2010). Metabolomics-edited transcriptomics analysis (Meta). In C. A. McQueen (Ed.), Comprehensive toxicology (pp. 685–706). Oxford: Academic Press.
Fan, T. W.-M., & Lane, A. N. (2008). Structure-based profiling of Metabolites and isotopomers by NMR. Progress in NMR Spectroscopy, 52, 69–117.
Fan, T. W.-M., & Lane, A. N. (2011). NMR-based stable isotope resolved metabolomics in systems biochemistry. Journal of Biomolecular NMR, 49, 267–280.
Fan, T., Bandura, L., Higashi, R., & Lane, A. (2005). Metabolomics-edited transcriptomics analysis of Se anticancer action in human lung cancer cells. Metabolomics Journal, 1, 325–339.
Fan, T. W.-M., Kucia, M., Jankowski, K., Higashi, R. M., Rataczjak, M. Z., Rataczjak, J., et al. (2008). Proliferating Rhabdomyosarcoma cells shows an energy producing anabolic metabolic phenotype compared with primary myocytes. Molecular Cancer, 7, 79.
Fan, T. W., Lane, A. N., Higashi, R. M., Farag, M. A., Gao, H., Bousamra, M., et al. (2009). Altered regulation of metabolic pathways in human lung cancer discerned by 13C stable isotope-resolved metabolomics (SIRM)). Molecular Cancer, 8, 41.
Fan, T. W.-M., Yuan, P., Lane, A. N., Higashi, R. M., Wang, Y., Hamidi, A., et al. (2010). Stable isotope-resolved metabolomic analysis of lithium effects on glial-neuronal metabolism and interactions. Metabolomics, 6, 165–179.
Fan, T. W.-M., Lane, A. N., Higashi, R. M., & Yan, J. (2011a). Stable isotope resolved metabolomics of lung cancer in a SCID mouse model metabolomics, 7, 257–269.
Fan, T. W.-M., Tan, J. L., McKinney, M. M., & Lane, A. N. (2011b) Stable isotope resolved metabolomics analysis of ribonucleotide and rna metabolism in human lung cancer cells. Metabolomics doi:10.1007/s11306-011-0337-9.
Feng, H. T., Wong, N., Wee, S., & Lee, M. M. (2008). Simultaneous determination of 19 intracellular nucleotides and nucleotide sugars in Chinese Hamster ovary cells by capillary electrophoresis. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 870, 131–134.
Gangl, E. T., Annan, M., Spooner, N., & Vouros, P. (2001). Reduction of signal suppression effects in ESI-MS using a nanosplitting device. Analytical Chemistry, 73, 5635–5644.
Hinterwirth, H., Lammerhofer, M., Preinerstorfer, B., Gargano, A., Reischl, R., Bicker, W., et al. (2010). Selectivity issues in targeted metabolomics: Separation of phosphorylated carbohydrate isomers by mixed-mode hydrophilic interaction/weak anion exchange chromatography. Journal of Separation Science, 33, 3273–3282.
Inoue, K., Obara, R., Hino, T., & Oka, H. (2010). Development and application of an HILIC-MS/MS method for the quantitation of nucleotides in infant formula. Journal of Agricultural and Food Chemistry, 58, 9918–9924.
Kammerer, B., Frickenschmidt, A., Gleiter, C. H., Laufer, S., & Liebich, H. (2005a). MALDI-TOF MS analysis of urinary nucleosides. Journal of the American Society for Mass Spectrometry, 16, 940–947.
Kammerer, B., Frickenschmidt, A., Muller, C. E., Laufer, S., Gleiter, C. H., & Liebich, H. (2005b). Mass spectrometric identification of modified urinary nucleosides used as potential biomedical markers by LC-ITMS coupling. Analytical and Bioanalytical Chemistry, 382, 1017–1026.
Lane, A. N., Fan, T. W., & Higashi, R. M. (2008). Isotopomer-based metabolomic analysis by NMR and mass spectrometry. Biophysical Tools for Biologists, 84, 541–588.
Lane, A. N., Fan, T. W.-M., Xie, X., Moseley, H. N., & Higashi, R. M. (2009). Stable isotope analysis of lipid biosynthesis by high resolution mass spectrometry and NMR. Analytica Chimica Acta, 651, 201–208.
Lane, A. N., Fan, T. W.-M., Bousamra, M., I. I., Higashi, R. M., Yan, J., & Miller, D. M. (2011). Clinical applications of stable isotope-resolved metabolomics (SIRM) in non-small cell lung cancer. Omics, 15, 173–182.
Lu, W. Y., Clasquin, M. F., Melamud, E., Amador-Noguez, D., Caudy, A. A., & Rabinowitz, J. D. (2010). Metabolomic analysis via reversed-phase ion-pairing liquid chromatography coupled to a stand alone orbitrap mass spectrometer. Analytical Chemistry, 82, 3212–3221.
Luo, B., Groenke, K., Takors, R., Wandrey, C., & Oldiges, M. (2007). Simultaneous determination of multiple intracellular metabolites in glycolysis, pentose phosphate pathway and tricarboxylic acid cycle by liquid chromatography-mass spectrometry. Journal of Chromatography. A, 1147, 153–164.
Matuszewski, B. K., Constanzer, M. L., & Chavez-Eng, C. M. (2003). Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Analytical Chemistry, 75, 3019–3030.
Mei, H., Hsieh, Y. S., Nardo, C., Xu, X. Y., Wang, S. Y., Ng, K., et al. (2003). Investigation of matrix effects in bioanalytical high-performance liquid chromatography/tandem mass spectrometric assays: application to drug discovery. Rapid Communications in Mass Spectrometry, 17, 97–103.
Miura, D., Fujimura, Y., Tachibana, H., & Wariishi, H. (2010). Highly sensitive matrix-assisted laser desorption ionization-mass spectrometry for high-throughput metabolic profiling. Analytical Chemistry, 82, 498–504.
Moseley, H. N. (2010). Correcting for the effects of natural abundance in stable isotope resolved metabolomics experiments involving ultra-high resolution mass spectrometry. BMC Bioinformatics, 11, 139.
Moseley, H. N. B., Lane, A. N., Belshoff, A. C., Higashi, R. M., & Fan, T. W.-M. (2011). Non-steady state modeling of UDP-GlcNAc biosynthesis is enabled by stable isotope resolved metabolomics (SIRM). BMC Biology, 9, 37.
Murray, K. M. (2009). Glycoproteins, Harper’s Illustrated Biochemistry. New York: McGraw-Hill.
Murray, R. K., Bender, D. A., Botham, K. M., Kennelly, P. J., Rodwell, V. W., & Weil, P. A. (2009). Harper’s Illustrated Biochemistry. New York: McGraw-Hill.
Nelson, D. L., & Cox, M. M. (2005). Lehninger principles of biochemistry (4th ed.). New York: W.H. Freeman and Company.
Pingitore, F., Tang, Y. J., Kruppa, G. H., & Keasling, J. D. (2007). Analysis of amino acid isotopomers using FT-ICR MS. Analytical Chemistry, 79, 2483–2490.
Pluskal, M. G. (2000). Microscale sample preparation. Nat Biotech, 18, 104–105.
Shukla, A. & Majors, R. E. (2005) Micropipette tip-based sample preparation for bioanalysis. Lc Gc North America 23, 646.
Soo, E. C., Aubry, A. J., Logan, S. M., Guerry, P., Kelly, J. F., Young, N. M., et al. (2004). Selective detection and identification of sugar nucleotides by CE-electrospray-MS and its application to bacterial metabolomics. Analytical Chemistry, 76, 619–626.
We thank R. Burra and T. Xu for technical assistance, H. Moseley for valuable discussions, and J. Winnike for critical comments on the manuscript. This study was supported in part by NIH Grant numbers 1R01CA118434-01A2, 3R01CA118434-02S1, Kentucky Lung Cancer Research Program (LCRP) OGMB101380 (to TWMF), 1R21CA133668-01A2 (to ANL), University of Louisville CTSPGP grant numbers 20044 (to TWMF) and 20061 (to ANL), and Kentucky Lung Cancer Research Program (postdoctoral fellowship to PL). The FTICR-MS instrumentation was supported by NSF/EPSCoR grant number EPS-0447479 (TWMF).
Electronic supplementary material
Below is the link to the electronic supplementary material.
About this article
Cite this article
Lorkiewicz, P., Higashi, R.M., Lane, A.N. et al. High information throughput analysis of nucleotides and their isotopically enriched isotopologues by direct-infusion FTICR-MS. Metabolomics 8, 930–939 (2012). https://doi.org/10.1007/s11306-011-0388-y
- Stable isotope-resolved metabolomics (SIRM)
- Simultaneous detection
- FTICR MS
- Methylseleninic acid