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).
Thermo scientific ultimate 3000
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Thermo Scientific Ultimate 3000 is a high-performance liquid chromatography (HPLC) system designed for analytical and preparative applications. It features a modular architecture that allows for customization to meet specific laboratory needs. The system is capable of delivering precise and reliable solvent delivery, sample handling, and detection capabilities.
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Lab products found in correlation
6 protocols using thermo scientific ultimate 3000
1
Metabolic Profiling of WT and ΔAfLaeA Strains
2
HPLC Analysis of Methylxanthines and Polyphenols in Cocoa
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Among the analytical techniques employed to separate and quantify the methylxanthines and the polyphenols in cocoa beans and cocoa by products, high-performance liquid chromatography (HPLC) is the most common technique, either in normal or in reverse phase (Ramli et al., 2001 ). The detection system most frequently employed are ultraviolet absorbance (Belšč ak et al., 2009 ; Ortega et al., 2010 ; Todorovic et al., 2015 ; Hernandez-Hernandez et al., 2018 (link)).
The HPLC analysis was carried out in a Thermo scientific ultimate 3000 equipped with a diode array detector, auto sampler and quaternary pump (Thermo Fisher Scientific Inc., Waltham, MA, USA). The separation of theobromine and caffeine were carried out using a Dionex (3 μm, 300 Å, 2.1 × 50 mm) at flow rate of 0.3 mL/min and a temperature of 25 °C. The mobile phase used was an aqueous solution of tetrahydrofuran (0.1% v/v) as eluent A and acetonitrile as solvent B in an isocratic run for 10 min. The separated analytes were monitored at 280 nm and were identified by comparing the retention times and spectral data to those of standards.
The HPLC analysis was carried out in a Thermo scientific ultimate 3000 equipped with a diode array detector, auto sampler and quaternary pump (Thermo Fisher Scientific Inc., Waltham, MA, USA). The separation of theobromine and caffeine were carried out using a Dionex (3 μm, 300 Å, 2.1 × 50 mm) at flow rate of 0.3 mL/min and a temperature of 25 °C. The mobile phase used was an aqueous solution of tetrahydrofuran (0.1% v/v) as eluent A and acetonitrile as solvent B in an isocratic run for 10 min. The separated analytes were monitored at 280 nm and were identified by comparing the retention times and spectral data to those of standards.
3
Metabolic Profiling of Fungal Strains
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WT and Δfus3 strains were inoculated into PD broth for 7 days at 28°C; then, mycelium and fermentation broth were separated by filtration using a vacuum filter pump. The isolated mycelium was further dried and weighed, and corresponding fermentation broth was prepared according to the same weight of the mycelium. The fermentation broth was added to an equal volume of ethyl acetate and left to stand for extraction for 12 h, and then the crude extract obtained using a rotary evaporator was dissolved in 1 mL of methanol.74 Extracts were used for subsequent LC-MS (Thermo Scientific Ultimate 3000, Thermo Fisher Scientific, USA) analysis. Metabolic profiles and untargeted metabolomics of WT and Δfus3 mutant strains were analyzed using Thermo Xcalibur software and Compounds Discoverer 3.0 software (Thermo Fisher Scientific), respectively.
4
Molecular Weight Analysis of Acetylated Lignin
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SEC of the acetylated lignin samples was performed on a size-exclusion chromatographic system (Thermo Scientific Ultimate 3000, ThermoFisher, Waltham, MA, USA) equipped with a UV detector set at 280 nm. The analyses were carried out at ambient temperature using THF as eluent at a flow rate of 1 cm3 min−1. The aliquots (100 µL) of each sample dissolved in THF (1.5 mg cm−3) were injected into Plgel 3 µm MIXED E 7.5 × 300 mm. The column specifications allow the separation by molecular weight up to 30 kDa. The SEC system was calibrated with polystyrene standards with the molecular weight in the range from 500 Da to 30 kDa. The chromatographic data were processed with the PSS (Polymer Standards Service) WinGPC Unity software. The acetylation of lignin was performed using acetic anhydride according to the procedure reported elsewhere30 .
5
HPLC-UV Analysis of Serum Metabolites
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Serum extract samples (20 μL) were also analyzed using an HPLC-UV system (Thermo Scientific UltiMate 3000, Thermo Fisher Scientific Inc., Waltham, MA, USA) with a Raptor AR C18 column (150 × 4.6 mm, 2.7 μm particle size) purchased from Restek Co., (Bellefonte, PA, USA). The isocratic mobile phase was composed of 0.05 mM KH2PO4 and 10% acetonitrile (v/v) at a flow rate of 1 mL/min and UV detection at 220, 240, 260, and 280 nm. Peak identification and plasma concentration of metabolites, calculated from peak areas and standard calibration curves, were determined using HPLC software (Dionex Corp., Sunnyvale, CA, USA, 7.3).
6
Formylglycine-Generating Enzyme Inhibitor Assay
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All peptides used for this study were obtained from Peptron Inc. (Daejeon, South Korea) with 99% purity (based on 220 nm LC chromatograms). Native substrate (100 μM) and inhibitor (at concentrations ranging from 50–250 μM) were added to room-temperature-equilibrated reaction buffer containing 20 μM formylglycine-generating enzyme (FGE), equimolar CuSO4, 2 mM dithreitol (DTT), 50 mM sodium chloride, and 2× protease inhibitors. The reaction was conducted at RT for 1 h, followed by the addition of 100 mM HCl to terminate the reaction. The reaction products were desalted and then analysed using a binary-pump Thermoscientific Ultimate 3000 HPLC system (Thermo Scientific, Waltham, Massachusetts, United States) fitted with a reverse phase SCINChrom C18G 3 μm column (150 × 4.6 mm; 3.6 μm particle size) (Seoul, USA) and a dual wavelength absorbance detector (220 nm and 280 nm).
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