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Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry for metabolic flux analyses using isotope‐labeled ethanol
Author(s) -
Hollemeyer Klaus,
Velagapudi Vidya R.,
Wittmann Christoph,
Heinzle Elmar
Publication year - 2007
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.2840
Subject(s) - chemistry , acetaldehyde , mass spectrometry , ethanol , chromatography , desorption , analytical chemistry (journal) , detection limit , matrix (chemical analysis) , matrix assisted laser desorption/ionization , flux (metallurgy) , adsorption , organic chemistry
We describe a novel method for the determination of the concentration and labeling degree of ethanol originating from 1‐ 13 C‐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 [ 13 C 2 ]‐ethanol as internal standard. The relative signal intensities of the unlabeled ethanol derivative as well as of [1‐ 13 C]‐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‐ 13 C]‐ethanol originating from 1‐ 13 C‐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.