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Systems Metabolic Engineering

Volume 985 of the series Methods in Molecular Biology pp 335-351

Date:

Nuclear Magnetic Resonance Methods for Metabolic Fluxomics

  • Shilpa NargundAffiliated withDepartment of Chemical and Biomolecular Engineering, University of Maryland
  • , Max E. JoffeAffiliated withDepartment of Chemical and Biomolecular Engineering, University of MarylandInterdisciplinary Graduate Program in the Biomedical and Biological Sciences, Vanderbilt University
  • , Dennis TranAffiliated withDepartment of Chemical and Biomolecular Engineering, University of Maryland
  • , Vitali TugarinovAffiliated withDepartment of Chemistry and Biochemistry, University of Maryland
  • , Ganesh SriramAffiliated withDepartment of Chemical and Biomolecular Engineering, University of Maryland Email author 

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Abstract

Fluxomics, through its core methodology of metabolic flux analysis (MFA), enables quantification of carbon traffic through cellular biochemical pathways. Isotope labeling experiments aid MFA by providing information on intracellular fluxes, especially through parallel and cyclic pathways. Nuclear magnetic resonance (NMR) and mass spectrometry (MS) are two complementary methods to measure abundances of isotopomers generated in these experiments. 2-D [13C, 1H] heteronuclear correlation NMR spectra can detect 13C isotopes coupled to protons and thus noninvasively separate molecules and atoms with a specific isotopic content from a mixture of molecular species. Furthermore, the fine structures of the peaks in these spectra can reveal scalar couplings between chemically bonded carbon atoms in the sample, from which isotopomer abundances can be quantified. This chapter introduces methods for NMR sample preparation and spectral acquisition of 2-D [13C, 1H] correlation maps, followed by a detailed presentation of methods to process the spectra and quantify isotopomer abundances. We explain the use of the software NMRViewJ for spectral visualization and processing, as well as MATLAB scripts developed by us for peak extraction, deconvolution of overlapping peaklets, and isotopomer abundance quantification. Finally, we discuss the applications of NMR-derived isotopomer data toward quantitatively understanding metabolic pathways.

Key words

Systems metabolic engineering Flux analysis NMR Isotope labeling