Metabolite extraction from adherently growing mammalian cells for metabolomics studies: optimization of harvesting and extraction protocols

摘要: Trypsin/ethylenediaminetetraacetic acid (EDTA) treatment and cell scraping in a buffer solution were compared for harvesting adherently growing mammalian SW480 cells metabolomics studies. In addition, direct with solvent was tested. Trypsinated scraped pellets extracted using seven different extraction protocols including pure methanol, methanol/water, acetone, acetone/water, methanol/chloroform/water, methanol/isopropanol/water, acid–base methanol. The extracts analyzed by GC-MS after methoximation/silylation derivatization propyl chloroformate, respectively. metabolic fingerprints 25 selected metabolites amino acids intermediates of energy metabolism quantitatively determined. Moreover, the influence freeze/thaw cycles, ultrasonication homogenization ceramic beads on yield Pure acetone yielded lowest efficiency while methanol recovered similar metabolite amounts good reproducibility. Based overall performance, methanol/water chosen as suitable solvent. Repeated did not improve extraction. Trypsin/EDTA caused substantial leakage proving it inadequate Gentle subsequent resulted average sevenfold lower recovery quantified making latter one method choice to harvest extract from cells.