Appropriate sampling for intracellular amino acid analysis in five phylogenetically different yeasts

Area of Science:

• Microbiology
• Metabolomics
• Biochemistry

Background:

• Microbial metabolome analysis requires precise intracellular metabolite quantification.
• Common sampling methods like methanol quenching and fast filtration need validation across different microbial species.

Purpose of the Study:

• To validate methanol quenching and fast filtration for intracellular amino acid analysis in five distinct yeast strains.
• To assess metabolite leakage during sampling protocols.

Main Methods:

• Tested methanol quenching (varying methanol concentrations) and fast filtration.
• Analyzed intracellular amino acid levels in Saccharomyces cerevisiae, Kluyveromyces marxianus, Pichia pastoris, Schizosaccharomyces pombe, and Zygosaccharomyces bailii.

Main Results:

• Negligible metabolite leakage was observed with 60% cold buffered methanol quenching across most yeasts and amino acids.
• Slightly increased leakage occurred with higher methanol concentrations.
• Fast filtration yielded consistent intracellular amino acid levels for all tested strains.

Conclusions:

• Both methanol quenching (at 60%) and fast filtration are valid, leakage-free methods for yeast amino acid metabolomics.
• These validated protocols support accurate intracellular metabolite profiling in diverse yeast species.