Amazon speed mass spectrometer

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 ).

To measure the yield of CHD and 2-carboxamido-2-deacetyl-chelocardin (CDCHD), S. albus culture broths were acidified to pH 1–2 with 50% TFA, followed by extraction with 2 V of MeOH. The extract was centrifuged and analyzed by LC–MS. All measurements were performed on a Dionex Ultimate 3000 LC system using a Luna C-18 [2 (link)] HST, 100 × 2.0 mm, 2.5 µm column (Phenomenex). Separation of 1 µl sample was achieved by a linear gradient from (A) H₂O + 0.1% FA to (B) ACN + 0.1% FA at a flow rate of 500 µl/min and 45 °C. The gradient was initiated by a 0.5 min isocratic step at 5% B, followed by an increase to 95% B in 9 min to end up with a 1.5 min step at 95% B before re-equilibration with initial conditions. To achieve better separation of peaks, gradient was extended to 18 min. UV spectra were recorded by a DAD in the range from 200 to 600 nm. The MS measurement was carried on an amaZon speed mass spectrometer (Bruker Daltonics, Bremen) using the standard ESI source. Mass spectra were acquired in centroid mode ranging from 200–2000 m/z in positive ionization mode.

S. coelicolor::sal gene cluster cells were cultivated in 300-mL flasks containing 30 mL M1 medium supplemented with apr (25 μg mL⁻¹). The culture was grown at 30 °C with constant agitation at 180 rpm. After 13 days, the biomass was harvested by centrifugation, and 2% resin Amberlite XAD-16 was added to the supernatant before the resin was extracted with methanol. The received extracts were evaporated and dissolved in methanol and used for HPLC-MS analysis. The HPLC-MS measurement was performed on a Dionex Ultimate 3000 LC system utilizing a Waters Acquity BEHC-18 column (50 × 2 mm, 1.7-μm particle size). Separation of 2 μL sample was obtained using a linear gradient of A (water and 0.1% formic acid) and B (acetonitrile and 0.1% formic acid) at a flow rate of 600 μL min⁻¹ at 45 °C. The gradient was initiated by a 0.5-min isocratic step at 5% B followed by an increase to 95% B over 9 min and a final 1.5-min step at 95% B before reequilibration with initial conditions. UV spectra were recorded by a DAD from 200–600 nm. MS measurement was carried on an amaZon speed mass spectrometer (Bruker Daltonics, Bremen, Germany) using the standard ESI source. Mass spectra were acquired in centroid mode ranging from 200–2000 m/z in positive ionization mode with auto MS² fragmentation.