Evaluation and Optimization of Metabolome Sample Preparation Methods for Saccharomyces cerevisiae

Abstract

Metabolome sampling is one of the most important factors that determine the quality of metabolomics data. The main steps in metabolite sample preparation include quenching and metabolite extraction. Quenching with 60% (v/v) cold methanol at -40 °C has been most commonly used for Saccharomyces cerevisiae, and this method was recently modified as "leakage-free cold methanol quenching" using pure methanol at -40 °C. Boiling ethanol (75%, v/v) and cold pure methanol are the most widely used extraction solvents for S. cerevisiae. In the present study, metabolome sampling protocols, including the above methods, were evaluated by analyzing 110 identified intracellular metabolites of S. cerevisiae using gas chromatography/time-of-flight mass spectrometry. According to our results, fast filtration followed by washing with an appropriate volume of water can minimize the metabolite loss due to cell leakage as well as the contamination by extracellular metabolites. For metabolite extraction, acetonitrile/water mixture (1:1, v/v) at -20 °C was the most effective. These results imply that the systematic evaluation of existing methods and the development of customized methods for each microorganism are critical for metabolome sample preparation to facilitate the reliable and accurate analysis of metabolome.

GC/TOF-MS-based metabolomic profiling in cultured fibroblast-like synoviocytes from rheumatoid arthritis

“…For the metabolic profiling of FLS, the sample preparation was performed with a modification to the previous studies [18,19]. After 5 mL of phosphate-buffered saline (PBS) buffer added to the cell culture medium, adherent FLS were gently detached using a rubber tripped cell scraper.…”

PHO13 deletion-induced transcriptional activation prevents sedoheptulose accumulation during xylose metabolism in engineered Saccharomyces cerevisiae

“…Metabolite extraction was conducted with slight modifications to previously described (Kim et al, 2013a). Briefly, cells were collected by fast vacuum-filtering 1 mL of yeast strains at mid-exponential growth phase (approximately at OD = 1) through a 30 mm diameter nylon membrane filter (0.45 μm pore size; Whatman, Piscataway, NJ) and then washed with 5 mL of water.…”

Influence of washing and quenching in profiling the metabolome of adherent mammalian cells: a case study with the metastatic breast cancer cell line MDA-MB-231

“…11 The commonly employed buffer additives are HEPES, 10,12 AMBIC, 13,14 tricine, 10,12,15 or NaCl. 16 Although the influence of these buffer additives in preserving the membrane integrity and therefore in minimising metabolite leakage is well studied for bacteria, 11,12,[17][18][19] fungi 20 and yeast, 10,15,21,22 they have not been as rigorously studied in mammalian cells. Due to basic differences in the cell envelope structure, the sampling methods cannot simply be adapted for mammalian cells.…”