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Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for metabolic flux analyses using isotope-labeled ethanol

Authors

  • Klaus Hollemeyer,

    Corresponding author
    1. Biochemical Engineering Institute, Im Stadtwald, Bldg. A 1.5, Saarland University, D-66123 Saarbrücken, Germany
    • Biochemical Engineering Institute, Im Stadtwald, Bldg. A 1.5, Saarland University, D-66123 Saarbrücken, Germany.
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  • Vidya R. Velagapudi,

    1. Biochemical Engineering Institute, Im Stadtwald, Bldg. A 1.5, Saarland University, D-66123 Saarbrücken, Germany
    Current affiliation:
    1. VTT Technical Research Centre of Finland, Espoo, Finland.
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  • Christoph Wittmann,

    1. Biochemical Engineering Institute, Im Stadtwald, Bldg. A 1.5, Saarland University, D-66123 Saarbrücken, Germany
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  • Elmar Heinzle

    1. Biochemical Engineering Institute, Im Stadtwald, Bldg. A 1.5, Saarland University, D-66123 Saarbrücken, Germany
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Abstract

We describe a novel method for the determination of the concentration and labeling degree of ethanol originating from 1-13C-labeling experiments. This method is suitable for high-throughput metabolic flux analysis because of the possible parallel sample preparation and fast final analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). In a closed vial containing culture supernatant, ethanol is enzymatically oxidized to acetaldehyde. The acetaldehyde formed evaporates and is readily trapped in a second enclosed but open vial containing acidified 2,4-dinitrophenylhydrazine (DNPH). The 2,4-acetaldehyde dinitrophenylhydrazone (Ac-DNPH) that is formed is insoluble under these conditions. This leads to a constant conversion rate of the acetaldehyde produced from ethanol after 14 h minimum incubation time. MALDI-TOFMS was used to quantify the formed Ac-DNPH with [13C2]-ethanol as internal standard. The relative signal intensities of the unlabeled ethanol derivative as well as of [1-13C]-ethanol were linearly related to the ethanol concentration within a range of 1 to 50 mM with a limit of detection of 0.6 mM, a range which is sufficient for flux analysis in microtiter plate fermentation experiments. The method allows the estimation of the [1-13C]-ethanol originating from 1-13C-labeling experiments of Saccharomyces cerevisiae strains. In experiments where the expected flux range was exceeded, unlabeled ethanol was determined with a linear range from 30 to 500 mM. Ethanol quantification using this method was compared with enzymatic analysis and exhibited differences of less than 3.3% on average. Comparison of flux partitioning ratios between glycolysis and the pentose-phosphate pathway (PPP) based on MALDI-TOFMS and gas chromatography (GC)/MS methods showed good agreement, with differences for ethanol and alanine labeling of only 4.3%. Copyright © 2007 John Wiley & Sons, Ltd.

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