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.

Bacterial Metabolomics: Sample Preparation Methods

“…An optimized method might not be suitable for a similar bacteria strain implying the different aims and setup of the experiment [ 65 ]. Liu et al [ 66 ] highlighted the importance of clear sample preparation method designated for bacterial metabolomics in obtaining high-accuracy and high-reliability data analysis. Most metabolomic studies reported adopting the available or existing procedures conducted for other bacteria or yeast, resulting in inaccurate analysis and conclusion [ 66 ].…”

“…Liu et al [ 66 ] highlighted the importance of clear sample preparation method designated for bacterial metabolomics in obtaining high-accuracy and high-reliability data analysis. Most metabolomic studies reported adopting the available or existing procedures conducted for other bacteria or yeast, resulting in inaccurate analysis and conclusion [ 66 ]. A number of optimization studies on metabolite sample preparation methods have been reported on various species of bacteria [ 30 , 53 , 59 ].…”

“…Bacteria are grown in an appropriate culture medium to achieve sufficient cell density [ 52 ]. Quenching at a specific time and condition is done to stop all the enzymatic and metabolic activities of bacteria cells [ 55 , 66 , 71 ]. Harvesting is to separate the bacterial cells from the culture medium to obtain the intracellular metabolites (from the cell pellet) and extracellular metabolites (from the supernatant) [ 32 ].…”

“…As quenching solvents are likely to affect the membrane structure of the cells, the process must therefore be rapid [ 67 , 68 , 85 ]. The contact time of bacteria cells with quenching solvent should be kept to a minimum as time-dependent leakage may occur [ 66 ]. In addition, significant cellular changes may occur due to adherence of bacteria cells to the bottom of a centrifuge tube or surface of filter paper during harvesting or the rapid shift of temperature during quenching [ 52 ].…”

“…Lei et al [ 82 ] demonstrated that using a filter-based system with ethanol (−20°C) prior to liquid nitrogen quenching reduced metabolite leakage and produced highest recovery of almost all amino acids in isolating MRSA cell. An optimized quenching for Streptomyces ZYJ-6 using molar transition energy (ET) showed that isoamylol with base solution of acetone : ethanol (1 : 1) (5 : 1, v/v) at −30°C resulted in the least intracellular metabolite leakage compared to other solvents [ 66 ]. The base solution was added to the quenching solution to maintain the membrane's integrity of the cells, yet time-dependent leakage might still occur, highlighting the need to minimize the contact time between the bacterial sample and quenching solution [ 66 ].…”