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High information throughput analysis of nucleotides and their isotopically enriched isotopologues by direct-infusion FTICR-MS

Abstract

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.

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Abbreviations

AXP:

Adenine nucleotides

FTICR-MS:

Fourier transform ion cyclotron-mass spectrometry

HSQC:

Heteronuclear single quantum coherence

MSA:

Methylseleninic acid

PPP:

Pentose phosphate pathway

PREMISE:

Precalculated exact mass isotopologue search engine

SIRM:

Stable isotope resolved metabolomics

THF:

Tetrahydrofolate

UDP-GlcNAc:

UDP-N-acetylglucosamine

UXP:

Uracil nucleotides

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Acknowledgments

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).

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Correspondence to Teresa W.-M. Fan.

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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

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Keywords

  • Nucleotides
  • Stable isotope-resolved metabolomics (SIRM)
  • Direct-infusion
  • Simultaneous detection
  • FTICR MS
  • Ion-pair
  • 13C-glucose
  • A549
  • Methylseleninic acid