The secondary metabolites of WT and ΔAfLaeA strains were analyzed by an LC-MS assay in this study. WT and ΔAfLaeA strains were cultured using TYGA liquid medium for 7 days, respectively. The fermentation broths were collected by filtration using a vacuum filter pump. Then, the fermentation broths were extracted three times by mixing them with the same volume of ethyl acetate. The extracts were evaporated under vacuum and dissolved in chromatography-grade methanol (SK, Korea). Finally, the samples were filtered through a 0.22-μm filter and subjected to LC-MS (Thermo Scientific Ultimate 3000; Thermo Fisher Scientific, USA). The metabolic profiles of the WT and ΔAfLaeA strains were compared using Thermo Xcalibur software (Thermo Fisher Scientific). Untargeted metabolomics was performed using Compounds Discoverer 3.0 software (Thermo Fisher Scientific).
Compounds discoverer 3
Manufactured by Thermo Fisher Scientific
Sourced in United States
Compounds Discoverer 3.0 is a software product developed by Thermo Fisher Scientific. The core function of this software is to analyze and interpret data from chemical compounds and molecular structures.
Automatically generated - may contain errors
Lab products found in correlation
Thermo xcalibur software, by Thermo Fisher Scientific (2 mentions)
Thermo scientific ultimate 3000, by Thermo Fisher Scientific (1 mentions)
Thermo high resolution q exactive focus mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Dionex ultimate 3000 uhplc system, by Thermo Fisher Scientific (1 mentions)
Quercetin, by Merck Group (1 mentions)
Kaempferol, by Merck Group (1 mentions)
Epicatechin, by Merck Group (1 mentions)
Isorhamnetin, by Merck Group (1 mentions)
4 protocols using compounds discoverer 3
1
Metabolic Profiling of WT and ΔAfLaeA Strains
2
Comparative Metabolomics of Fungal Strains
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Three hyphal mass discs (7 mm) of each strain were incubated in PD broth for 7 days at 28 °C and 180 rpm, followed by mass collection through vacuum filtration [44 (link)]. The biomass was dried, weighed (WT, 3.14 g; ΔAosec22, 2.89 g), and used to prepare extracts of the same concentration. Ethyl acetate (250 mL) was added to extract the fermentation broth and ultrasonic extraction was performed for 40 min. The fermentation broth was allowed to stand for 12 h and the crude extract was concentrated in ethyl acetate under vacuum [44 (link)]. The crude extract was dissolved in 500 µL of analytic-grade methanol and the solution was filtered through a 0.22 µm membrane filter for liquid chromatography–mass spectrometry (LC–MS) analysis. The metabolic profiles of the WT and mutant strains were compared using the Thermo Xcalibur software (Thermo Fisher Scientific). Untargeted metabolomics was performed using Compounds Discoverer 3.0 software (Thermo Fisher Scientific) [49 (link)].
3
Metabolomic Analysis of Fungal Strains
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The WT and mutant strains were inoculated into PD broth at 28°C and 180 rpm for 5 days, and then the fermentation broth was extracted using ethyl acetate and dried under a vacuum. The dried crude extract samples were dissolved into methanol, and the volume was fixed to 1 mL according to the dry weight of mycelia of each sample and the mass volume ratio. The experiment was repeated three times independently and analyzed by LC-MS using a Thermo Scientific Dionex Ultimate 3000 UHPLC system with a Thermo high-resolution Q Exactive focus mass spectrometer (Thermo, Bremen, Germany). Untargeted metabolomics analysis was performed using Compounds Discoverer 3.0 software (Thermo Fisher Scientific, Carlsbad, CA).
4
Profiling Flavonoid Metabolites in Brassica Seed Coats
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Seed coats were harvested at 20, 30, and 40 DAF from individual plants of ZY821, RNAi-BnA09myb47a, GH06, and BnA09MYB47a-overexpressing lines. The raw flavonoids were extracted from the flash-frozen fresh seed coats (100 mg fresh weight), and subjected to UPLC − HESI − MS/MS analysis25 (link),37 (link). Flavonoid metabolites were identified by retention times, mass spectrometry, and public information25 (link),35 (link)–37 (link). The contents of flavonoid metabolites were calculated using the standard curves of available standards: epicatechin, quercetin, kaempferol, and isorhamnetin (Sigma, Aldrich, Shanghai, China). Metabolomics analysis was also performed using Compounds Discoverer 3.0 software (Thermo Fisher Scientific, CA, USA). The Raw MS data have been deposited in MetaboLights under accession number MTBLS6703 (https://www.ebi.ac.uk/metabolights/search ). Chemical structure depiction was retrieved from the PubChem database (https://pubchem.ncbi.nlm.nih.gov ). All analyses were conducted in triplicate, and the values were represented as means ± SD of three replicates. Statistical significance was based on Tukey’s test, and P ≤ 0.05 was considered to be statistically significant.
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