A rapid and reliable method for metabolite extraction in yeast using boiling buffered ethanol

Area of Science:

• Biochemistry
• Metabolomics
• Yeast Genetics

Background:

• Accurate metabolite quantification is crucial for understanding cellular metabolism.
• Existing methods for yeast metabolite extraction can be time-consuming or inefficient.
• The stability of various metabolites during extraction is a significant challenge.

Purpose of the Study:

• To develop a simple, reliable, and efficient method for yeast metabolite extraction.
• To ensure the stability of a wide range of metabolites during the inactivation and extraction process.
• To enable quantitative analysis of metabolites from small amounts of yeast biomass.

Main Methods:

• Utilizing a boiling solution of 75% ethanol buffered with Hepes (70 mM, pH 7.5) at 80°C.
• Employing a rapid 3-minute incubation for yeast cells, with options for direct spraying or prior quenching in cold methanol.
• Concentrating extracts via vacuum evaporation and resuspending in water for analysis.

Main Results:

• Efficient inactivation of yeast metabolism within 3 minutes.
• High recovery and stability of diverse metabolites, including glycolytic intermediates, nucleotides, and organic acids.
• Quantitative metabolite analysis possible from less than 10 mg dry weight of yeast cells.

Conclusions:

• The developed method offers a fast and robust approach for yeast metabolite extraction.
• This technique is suitable for analyzing metabolites in mutants and for metabolic flux analysis.
• The method's applicability extends to other fungi, broadening its utility in biological research.