Streptomyces strains were grown in 10 mL of TSB for 1 day, and 1 mL of each culture was used to inoculate 50 mL of production medium. Cultures were grown for 7 days at 28 °C. Metabolites were extracted with ethyl acetate from the supernatant, evaporated, and dissolved in methanol. One μL of each sample was separated using a Dionex Ultimate 3000 UPLC (Thermo Fisher Scientific, Waltham, MA, USA), a 10-cm ACQUITY UPLC® BEH C18 column, 1.7 μm (Waters, Milford, MA, USA) and a linear gradient of 0.1% formic acid solution in acetonitrile against 0.1% formic acid solution in water from 5% to 95% in 18 min at a flow rate of 0.6 mL/min. Samples were analyzed using an amaZon speed mass spectrometer or maXis high-resolution LC-QTOF system (Bruker, USA). Data were collected and analyzed with the Bruker Compass Data Analysis software, version 4.1 (Bruker, Billerica, MA, USA). Monoisotopic mass was searched in the natural product database DNP (Dictionary of Natural Products [27 ]).
Maxis high resolution lc qtof system
Manufactured by Bruker
Sourced in United States
The MaXis high-resolution LC-QTOF system is a laboratory instrument designed for high-performance liquid chromatography (LC) and quadrupole time-of-flight (QTOF) mass spectrometry analysis. The system provides high-resolution, accurate-mass measurements for the identification and characterization of a wide range of compounds.
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Lab products found in correlation
Dionex ultimate 3000 uplc, by Thermo Fisher Scientific (3 mentions)
Amazon speed mass spectrometer, by Bruker (1 mentions)
Compass data analysis version 4, by Bruker (1 mentions)
Dionex ultimate 3000 uplc system, by Thermo Fisher Scientific (1 mentions)
Xcalibur v 3, by Thermo Fisher Scientific (1 mentions)
Compass data analysis v 4, by Bruker (1 mentions)
Acquity uplc beh c18 1.7 m column, by Waters Corporation (1 mentions)
Amazon speed, by Bruker (1 mentions)
Ltq orbitrap xl mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Multitron shaker, by Infors (1 mentions)
4 protocols using maxis high resolution lc qtof system
1
Metabolite Profiling of Streptomyces Strains
2
Ikarugamycin Production and Extraction from Streptomyces albus
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S. albus strains were grown in 10 mL of TSB for 1 day, and 1 mL of each culture was used to inoculate the 50 mL of production media. Cultures were grown for 5 days at 30 °C. Metabolites were extracted with ethyl acetate from the supernatant and the acetone:methanol (1:1) mixture from biomass. Extracts were evaporated and dissolved in methanol. 1 µL of each sample was separated using a Dionex Ultimate 3000 UPLC (Thermo Fisher Scientific, USA) and a 10-cm ACQUITY UPLC® BEH C18 column, 1.7 μm (Waters, USA) and a linear gradient of acetonitrile against 0.1% formic acid solution in water from 5% to 95% in 10 or 18 minutes at a flow rate of 0.6 mL/min. Samples were analyzed using an amaZon speed mass spectrometer or, when needed, maXis high-resolution LC-QTOF system (Bruker, USA). Pure ikarugamycin was used as standard in MS/MS experiments (Sigma-Aldrich, USA).
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S. albus strains were grown in 10 mL of TSB for 1 day, and 1 mL of each culture was used to inoculate the 50 mL of production media. Cultures were grown for 5 days at 30 °C. Metabolites were extracted with ethyl acetate from the supernatant and the acetone:methanol (1:1) mixture from biomass. Extracts were evaporated and dissolved in methanol. 1 µL of each sample was separated using a Dionex Ultimate 3000 UPLC (Thermo Fisher Scientific, USA) and a 10-cm ACQUITY UPLC® BEH C18 column, 1.7 μm (Waters, USA) and a linear gradient of acetonitrile against 0.1% formic acid solution in water from 5% to 95% in 10 or 18 minutes at a flow rate of 0.6 mL/min. Samples were analyzed using an amaZon speed mass spectrometer or, when needed, maXis high-resolution LC-QTOF system (Bruker, USA). Pure ikarugamycin was used as standard in MS/MS experiments (Sigma-Aldrich, USA).
3
Metabolomic Analysis of Streptomyces albus Strains
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Streptomyces albus 4N24 as well as the control strains Streptomyces albus De14 and Streptomyces albus subsp. chlorinus were cultivated in 15 mL TSB medium for 24 h at 28°C. Main cultures containing 50 mL of DNPM were inoculated with 1 mL of pre-culture. After 7 days of cultivation at 28°C, the secreted metabolites were extracted with ethyl acetate and butanol, followed by solvent evaporation. The dry extracts were solved in 1 mL methanol and 1 μL of the solved sample was separated using a Dionex Ultimate 3000 UPLC (Thermo Fisher Scientific, Waltham, MA, USA), and a 10-cm ACQUITY UPLC® BEH C18 column, 1.7 μm (Waters, Milford, MA, USA). The mobile phase was comprised of two solvents: formic acid solved in acetonitrile (0.1%) and formic acid solved in water (0.1%). Solvent concentrations varied in a linear gradient from 5 to 95% in 18 min at a flow rate of 0.6 mL/min. The UPLC system was coupled either to amaZon speed mass spectrometer or maXis high-resolution LC-QTOF system (Bruker, USA), allowing the mass spectrometry analysis of the extracts. The software Bruker Compass Data Analysis version 4.1 (Bruker, Billerica, MA, USA) was used for data analysis. Monoisotopic mass was searched in the natural product database DNP (Dictionary of Natural Products; Buckingham, 1993 ).
4
DNPM Production Optimization in S. aurantiacus
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S. aurantiacus LU19075 was grown in 20 mL of TSB in a 100 mL baffled flask for one day, and 1 mL of the culture was inoculated into 100 mL of DNPM production medium in a 500 mL baffled flask. Cultures were grown for 6 days at 28 °C and 180 rpm in an Infors multitron shaker (Infors AG, Basel, Switzerland). Metabolites were extracted from the supernatant with butanol, concentrated to dryness, and dissolved in 1 mL methanol (MeOH). One microliter was separated on a Dionex Ultimate 3000 UPLC system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an ACQUITY UPLC BEH C18 1.7 µm column (30, 50, or 100 mm, Waters Corporation, Milford, MA, USA) using a linear gradient of 5–95 vol% aqueous acetonitrile (ACN) with 0.1% formic acid (FA) at a flow rate of 0.6 mL/min and a column oven temperature of 45 °C. Sample analysis was carried out on a coupled PDA detector followed by an amaZon speed (Bruker, Billerica, MA, USA) for production control. High-resolution masses were obtained from either an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) or a MaXis high-resolution LC-QTOF system (Bruker, Billerica, MA, USA) using positive ionization mode and mass range detection of m/z 200 to 2000. Data analysis was performed using Compass Data Analysis v. 4.1 (Bruker) and Xcalibur v. 3.0 (Thermo Fisher Scientific).
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