Abstract
Metabolite extraction is considered as one of the most important for quality controlling steps in metabolomic research. In the present study, optimal extraction protocol for fungal intracellular metabolites was investigated. Two extraction protocols with different solvent systems, methanol extraction and biphasic extraction under different pH conditions were compared. Number of detected peaks, sample-to-sample variation, and throughput of whole process were taken into account as criteria. Extracted metabolites were analyzed by gas chromatography-mass spectrometry (GC-MS) and principal component analysis (PCA). The extraction protocol using methanol as extraction solvent provided the highest number of detected peaks (~ 300 peaks) with the ease and speed, suggesting that the methanol-extraction method was suitable for intracellular metabolites extraction from P. chrysosporium. However, depending on the nature of metabolites of interest, the biphasic extraction method under different pH conditions is suitable for a targeted analysis.
Highlights
Metabolome is complex because of the highly chemically and physically diverse nature of metabolites and of often occurring the multiplication of metabolic pathways for some particular metabolites
To obtain as much information about the metabolome as possible, using of productive sample preparation, merged with selective extractions associated with different analytical techniques and combined with the efficient mathematical interpretation of the data are crucial to examine [1,2]
P. chrysosporium was grown on PDA slant for 7 days at 37°C, 1% conidial suspension was inoculated to HCLN medium (20 mL in a 200-mL Erlenmeyer flask) and grown at 37°C under static conditions
Summary
Metabolome is complex because of the highly chemically and physically diverse nature of metabolites and of often occurring the multiplication of metabolic pathways for some particular metabolites. To obtain as much information about the metabolome as possible, using of productive sample preparation, merged with selective extractions associated with different analytical techniques and combined with the efficient mathematical interpretation of the data are crucial to examine [1,2]. Mass spectrometry coupled with chromatography has become the preferred technique available for the quantification of metabolome. These analytical techniques possess high sensitivity, selectivity and simultaneous quantification of many different metabolites and require only a small sample volume for analysis [3,4]. The number and amounts of metabolites extracted must be maximized, while the degradation of metabolites during sample preparation process and the analytical variation should be minimized [5]. Gas chromatography-mass spectrometry (GC-MS) is one of the most commonly used analytical techniques with a high liability and a capability of high-throughput and automated analysis
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