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Abstract
BackgroundStable isotope tracing with ultra-high resolution Fourier transform-ion cyclotron resonance-mass spectrometry (FT-ICR-MS) can provide simultaneous determination of hundreds to thousands of metabolite isotopologue species without the need for chromatographic separation. Therefore, this experimental metabolomics methodology may allow the tracing of metabolic pathways starting from stable-isotope-enriched precursors, which can improve our mechanistic understanding of cellular metabolism. However, contributions to the observed intensities arising from the stable isotope's natural abundance must be subtracted (deisotoped) from the raw isotopologue peaks before interpretation. Previously posed deisotoping problems are sidestepped due to the isotopic resolution and identification of individual isotopologue peaks. This peak resolution and identification come from the very high mass resolution and accuracy of FT-ICR-MS and present an analytically solvable deisotoping problem, even in the context of stable-isotope enrichment.ResultsWe present both a computationally feasible analytical solution and an algorithm to this newly posed deisotoping problem, which both work with any amount of 13C or 15N stable-isotope enrichment. We demonstrate this algorithm and correct for the effects of 13C natural abundance on a set of raw isotopologue intensities for a specific phosphatidylcholine lipid metabolite derived from a 13C-tracing experiment.ConclusionsCorrection for the effects of 13C natural abundance on a set of raw isotopologue intensities is computationally feasible when the raw isotopologues are isotopically resolved and identified. Such correction makes qualitative interpretation of stable isotope tracing easier and is required before attempting a more rigorous quantitative interpretation of the isotopologue data. The presented implementation is very robust with increasing metabolite size. Error analysis of the algorithm will be straightforward due to low relative error from the implementation itself. Furthermore, the algorithm may serve as an independent quality control measure for a set of observed isotopologue intensities.
Highlights
Stable isotope tracing with ultra-high resolution Fourier transform-ion cyclotron resonance-mass spectrometry (FT-ICR-MS) can provide simultaneous determination of hundreds to thousands of metabolite isotopologue species without the need for chromatographic separation
Specific patterns of isotopologues are usually observed, which are interpreted within the context of known cellular metabolic pathways [3,4,5]
Equation 1 represents the relative distribution of carbon isotopologues from natural abundance only, as a sum of multinomial coefficients multiplied by the intensity of IM+0, the theoretically untainted 12C monoisotopic peak
Summary
Stable isotope tracing with ultra-high resolution Fourier transform-ion cyclotron resonance-mass spectrometry (FT-ICR-MS) can provide simultaneous determination of hundreds to thousands of metabolite isotopologue species without the need for chromatographic separation. This experimental metabolomics methodology may allow the tracing of metabolic pathways starting from stable-isotope-enriched precursors, which can improve our mechanistic understanding of cellular metabolism. The better than 1 ppm mass accuracy of state-of-the-art FT-ICR-MS is often high enough to provide mass-to-charge ratios (m/z) down to the 3rd and 4th decimal place for metabolites less than a few thousand Daltons This is accurate enough to distinguish relativistic mass differences between expected isotopes of CHONPS elements and unambiguously determine the isotope-specific molecular formula of an individual peak. The FT-ICR-MS’s high mass resolution allows for the direct detection or deconvolution of individual isotopologues or mass-equivalent sets of isotopomers for a given metabolite
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