Journal: Analytical and Bioanalytical Chemistry
Article Title: High-throughput Saccharomyces cerevisiae cultivation method for credentialing-based untargeted metabolomics
doi: 10.1007/s00216-023-04724-5
Figure Lengend Snippet: High-throughput sample generation using a D48-well plate. (1) Cultivation: yeast strains are cultivated in a D48 well plate in the presence of unlabelled and/or stable isotope-labelled substrates. (2) Normalization: before pelleting the cells, 200 μL aliquots of the cell cultures are transferred into a new D48 plate containing ISOTONE II (Beckman Coulter) solution for biovolume (μL/mL) determination using the Multisizer Z3 (Beckman Coulter). The latter is used to calculate the resuspension volume for the dried metabolite extracts prior to LC-MS analysis. (3) Cell pelleting: fast centrifugation treatment. (4) Quenching: the bottom of the plate is immerged in liquid nitrogen. (5) Metabolite extraction: the cell pellets are resuspended in pre-cooled MeOH:H 2 O (− 20 °C). After resuspension, − 20 °C pre-cooled MTBE is added for metabolite extraction. After the incubation step, the cell extracts of each well are transferred into 2-mL Eppendorf tubes. (6) Phase separation: the cell extracts are centrifuged in order to achieve a phase separation between the upper organic phase (MTBE) and the lower aqueous phase (MeOH:H 2 O). (7) Sample evaporation: the collected phases are evaporated overnight using a SpeedVac vacuum device (Labconco). (8) Sample reconstitution: samples are reconstituted, adapting the resuspension volume to the biovolume (μL/mL) for the purpose of normalization. (9) Untargeted LC-MS analysis: the biovolume-normalized samples are analysed by HILIC-MS (metabolomics) or RP-LC-MS (lipidomics). Abbreviations are reported in the abbreviation list
Article Snippet: After the incubation step, the cell extracts of each well are transferred into 2-mL Eppendorf tubes. (6) Phase separation: the cell extracts are centrifuged in order to achieve a phase separation between the upper organic phase (MTBE) and the lower aqueous phase (MeOH:H 2 O). (7) Sample evaporation: the collected phases are evaporated overnight using a SpeedVac vacuum device (Labconco). (8) Sample reconstitution: samples are reconstituted, adapting the resuspension volume to the biovolume (μL/mL) for the purpose of normalization. (9) Untargeted LC-MS analysis: the biovolume-normalized samples are analysed by HILIC-MS (metabolomics) or RP-LC-MS (lipidomics).
Techniques: High Throughput Screening Assay, Liquid Chromatography with Mass Spectroscopy, Centrifugation, Extraction, Incubation, Evaporation, Hydrophilic Interaction Liquid Chromatography, Reversed-phase Chromatography
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