Optimized sampling protocol for mass spectrometry-based metabolomics in Streptomyces

Abstract In quantitative metabolomics studies, the most crucial step was arresting snapshots of all interesting metabolites. However, the procedure customized for Streptomyces was so rare that most studies consulted the procedure from other bacteria even yeast, leading to inaccurate and unreliable metabolomics analysis. In this study, a base solution (acetone: ethanol = 1:1, mol/mol) was added to a quenching solution to keep the integrity of the cell membrane. Based on the molar transition energy (ET) of the organic solvents, five solutions were used to carry out the quenching procedures. These were acetone, isoamylol, propanol, methanol, and 60% (v/v) methanol. To the best of our knowledge, this is the first report which has utilized a quenching solution with ET values. Three procedures were also adopted for extraction. These were boiling, freezing–thawing, and grinding ethanol. Following the analysis of the mass balance, amino acids, organic acids, phosphate sugars, and sugar alcohols were measured using gas chromatography with an isotope dilution mass spectrometry. It was found that using isoamylol with a base solution (5:1, v/v) as a quenching solution and that freezing–thawing in liquid nitrogen within 50% (v/v) methanol as an extracting procedure were the best pairing for the quantitative metabolomics of Streptomyces ZYJ-6, and resulted in average recoveries of close to 100%. The concentration of intracellular metabolites obtained from this new quenching solution was between two and ten times higher than that from 60% (v/v) methanol, which until now has been the most commonly used solution. Our findings are the first systematic quantitative metabolomics tools for Streptomyces ZYJ-6 and, therefore, will be important references for research in fields such as 13C based metabolic flux analysis, multi-omic research and genome-scale metabolic model establishment, as well as for other Streptomyces.