Global metabolite analysis of yeast: evaluation of sample preparation methods

TLDR

Sample preparation is a limiting step in microbial metabolomics, and eukaryotic and prokaryotic cells differ markedly in their responses to classical protocols, making it risky to apply a single method across diverse eukaryotes. We reviewed and evaluated the entire sample preparation workflow for yeast metabolite analysis. The study focused on current metabolomics needs, assessing six extraction methods for diverse intracellular metabolites and evaluating quenching, extraction, and concentration steps. We found that quenching yeast with cold methanol causes metabolite leakage, identified six extraction methods with varying efficacy, and observed losses during lyophilization and solvent evaporation, leading us to recommend quenching with cold methanol followed by pure methanol extraction and reduced‑pressure evaporation for high‑throughput analysis. © 2005 John Wiley & Sons, Ltd.

Abstract

Abstract Sample preparation is considered one of the limiting steps in microbial metabolome analysis. Eukaryotes and prokaryotes behave very differently during the several steps of classical sample preparation methods for analysis of metabolites. Even within the eukaryote kingdom there is a vast diversity of cell structures that make it imprudent to blindly adopt protocols that were designed for a specific group of microorganisms. We have therefore reviewed and evaluated the whole sample preparation procedures for analysis of yeast metabolites. Our focus has been on the current needs in metabolome analysis, which is the analysis of a large number of metabolites with very diverse chemical and physical properties. This work reports the leakage of intracellular metabolites observed during quenching yeast cells with cold methanol solution, the efficacy of six different methods for the extraction of intracellular metabolites, and the losses noticed during sample concentration by lyophilization and solvent evaporation. A more reliable procedure is suggested for quenching yeast cells with cold methanol solution, followed by extraction of intracellular metabolites by pure methanol. The method can be combined with reduced pressure solvent evaporation and therefore represents an attractive sample preparation procedure for high‐throughput metabolome analysis of yeasts. Copyright © 2005 John Wiley & Sons, Ltd.