Study on sample preparation for the metabolomics of Metarhizium based on GC-MS

Objective  The metabolite pretreatment technology was optimized based on GC-MS to establish a rapid, accurate sample preparation protocol for metabolomics analysis in Metarhizium.

Method  Several sample preparation steps, including cell quenching, metabolite extraction, derivatization and detection were optimized, and the stability of this method was also determined.

Result  The quenching effect of 40% cold ethanol was better than that of other quenching solutions, and the recoveries of nucleic acid and protein of Metarhizium were 9.63% and 11.61% respectively. Total 109 metabolites were obtained by cold methanol, more than those by other methods. The longer derivation time was, the more metabolites could be obtained, and 1.5 h was the best. Too high initial temperature of gas chromatography was not conducive to acquisition of metabolites, and 50 ℃ was the best. The optimal sample preparation conditions were as follows: After quenching with 40% cold ethanol, the supernatant was extracted with 2 mL cold methanol. After centrifugation, the supernatant was dried with N₂, and then added with 80 μL 20 mg/mL methoxylamine hydrochloride pyridine solution. After severe oscillation for 30 s, the supernatant was reacted at 37 ℃ for 90 min, and cooled to room temperature. Then 80 μL BSTFA derivatization agent with 1%(φ) TMCS was added, the derivatization reaction continued for 1.5 h at 70 ℃, and solution cooled to room temperature.

Conclusion  The method is simple, convenient and reproducible, it is conducive to carry out more in depth studies on metabolic mechanisms, and provides references for related studies on metabolic groups of pathogenic microorganisms in agriculture and forestry.