Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for metabolic flux analyses using isotope-labeled ethanol.
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%.
Hollemeyer
Klaus
K
Biochemical Engineering Institute, Im Stadtwald, Bldg. A 1.5, Saarland University, D-66123 Saarbrücken, Germany. k.hollemeyer@mx.uni-saarland.de
Velagapudi
Vidya R
VR
Wittmann
Christoph
C
Heinzle
Elmar
E
D002247:Carbon Isotopes / Q000032:analysis; D000431:Ethanol / Q000032:analysis* / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D006019:Glycolysis / Q000502:physiology; D007553:Isotope Labeling / Q000379:methods; D008832:Microchemistry / Q000379:methods; D010427:Pentose Phosphate Pathway / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D002247:Carbon Isotopes; D029701:Saccharomyces cerevisiae Proteins; D000431:Ethanol; D005947:Glucose