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Determination of Isotopologue and Tandem Mass Isotopologue Ratios Using Gas Chromatography Chemical Ionization Time of Flight Mass Spectrometry - Methodology and Uncertainty of Measurement

  • Teresa MairingerEmail author
  • Stephen Hann
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2088)

Abstract

The accurate and precise analysis of isotopologue and tandem mass isotopologue ratios in heavy stable isotope labeling experiments is a critical part of assessing absolute intracellular metabolic fluxes. Resulting from feeding the organism of interest with a specifically isotope-labeled substrate, the principal characteristics of these labeling experiments are the metabolites’ non-naturally distributed isotopologue patterns. For the purpose of inferring metabolic rates by maximizing the fit between a priori simulated and experimentally obtained labeling patterns, 13C is the preferred stable isotope of use.

The analysis of the obtained labeling patterns can be approached by different mass spectrometric approaches. Gas chromatography (GC) features broad metabolite coverage and excellent separation efficiency of biologically relevant isomers. These advantages compensate for laborious derivatization steps and the resulting need for interference correction for natural abundant isotopes.

Here, we describe a workflow based on GC-high resolution mass spectrometry with chemical ionization for the analysis of carbon-isotopologue distributions and some positional labeling information of primary metabolites. To study the associated measurement uncertainty of the resulting 13C labeling patterns, guidance to uncertainty estimation according to the EURACHEM guidelines with Monte-Carlo simulation is provided.

Key words

Gas chromatography Chemical ionization Isotopologue distribution Tandem mass isotopologue distribution Primary carbon metabolism 13C based metabolic flux analysis Measurement uncertainty 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of Natural Resources and Life Sciences—BOKU ViennaViennaAustria
  2. 2.Department of Environmental ChemistryEawag: Swiss Federal Institute of Aquatic Science and TechnologyDuebendorfSwitzerland
  3. 3.Austrian Center for Industrial BiotechnologyViennaAustria

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