Metabolomics of Pichia pastoris: impact of buffering conditions on the kinetics and nature of metabolite loss during quenching.
Mass spectrometry-based metabolomic profiling is a powerful strategy to quantify the concentrations of numerous primary metabolites in parallel. To avoid distortion of metabolite concentrations, quenching is applied to stop the cellular metabolism instantly. For yeasts, cold methanol quenching is accepted to be the most suitable method to stop metabolism, while keeping the cells intact for separation from the supernatant. During this treatment, metabolite loss may occur while the cells are suspended in the quenching solution. An experiment for measuring the time-dependent loss of selected primary metabolites in differently buffered quenching solutions was conducted to study pH and salt concentration-dependent effects. Molecular properties of the observed metabolites were correlated with the kinetics of loss to gain insight into the mechanisms of metabolite leakage. Size and charge-related properties play a major role in controlling metabolite loss. We found evidence that interaction with the cell wall is the main determinant to retain a molecule inside the cell. Besides suggesting an improved quenching protocol to keep loss at a minimum, we could establish a more general understanding of the process of metabolite loss during quenching, which will allow to predict optimal conditions for hitherto not analysed metabolites.
Department of Chemistry, BOKU - University of Natural Resources and Life Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria.|Department of Biotechnology, BOKU - University of Natural Resources and Life Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria.
Matthias
M
Mattanovich
Department of Biotechnology, BOKU - University of Natural Resources and Life Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria.
Hannes
H
Russmayer
Austrian Centre of Industrial Biotechnology, Muthgasse 11, 1190 Vienna, Austria.|Institute of Applied Statistics and Computing, BOKU - University of Natural Resources and Life Sciences Vienna, Peter Jordan-Strasse 82, 1190 Vienna, Austria.
Theresa
T
Scharl-Hirsch
Department of Biotechnology, BOKU - University of Natural Resources and Life Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria.|Austrian Centre of Industrial Biotechnology, Muthgasse 11, 1190 Vienna, Austria.
Verena
V
Puxbaum
Department of Biotechnology, BOKU - University of Natural Resources and Life Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria.|Austrian Centre of Industrial Biotechnology, Muthgasse 11, 1190 Vienna, Austria.
Jonas
J
Burgard
Department of Biotechnology, BOKU - University of Natural Resources and Life Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria.|Austrian Centre of Industrial Biotechnology, Muthgasse 11, 1190 Vienna, Austria.
Diethard
D
Mattanovich
Department of Chemistry, BOKU - University of Natural Resources and Life Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria.|Austrian Centre of Industrial Biotechnology, Muthgasse 11, 1190 Vienna, Austria.
Stephan
S
Hann
D019149:Bioreactors; D002021:Buffers; D002473:Cell Wall / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D002853:Chromatography, Liquid; D003470:Culture Media / Q000737:chemistry; D005285:Fermentation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D055442:Metabolome; D055432:Metabolomics / Q000379:methods*; D000432:Methanol / Q000494:pharmacology*; D010843:Pichia / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D053719:Tandem Mass Spectrometry
D002021:Buffers; D003470:Culture Media; D012965:Sodium Chloride; D000432:Methanol
Pichia pastoris; metabolite loss; metabolomics; quenching; yeast