ABSTRACT: All chemicals were from Fisher Scientific unless otherwise noted. All solvents for extraction were HPLC grade, all solvents for mass spectrometry analysis were LCMS (Thermo Optima) grade. Streptomyces ansochromogenes subspecies pallens NRRL B-12018 was obtained from the ARS Culture Collection of the United States Department of Agriculture. A 30 mL Streptomyces ansochromogenes subspecies pallens NRRL B-12018 was inoculated from an ISP2 agar culture and cultivated in liquid ISP2 medium (4 g yeast extract, 10 g malt extract, 4 g D-glucose in 1 L deionized water, pH 7.3) for 11 days at 30 celsius at 225 rpm. The medium was then centrifuged in 50 mL centrifuge tube at 3200 g for 30 min. The cell pellet and supernatant were separated subsequently, and the supernatant (30 mL) was extracted three times with 50 mL ethyl acetate to remove lipophilic components. Then the supernatant was dried by rotary evaporation at 40 celsius. Finally, the dried supernatant was resuspended in 3.5 mL deionized water, centrifuged for 5 min at 16000 g, filtered with a Cytiva syringeless filter (0.2 micron mesh size, Cytiva, US503NPEORG). The filtered supernatant was immediately analyzed by liquid-chromatography-tandem mass spectrometry on a Thermo QExactive orbitrap mass spectrometer with a H-ESI-II ion source coupled to a Vanquish HPLC system. The LC parameters were as follows: Phenomenex Kinetex 2.6 micrometer C18 reverse phase 100 Ang. 150 x 3 mm LC column, solvent A: 20mM ammonium formate (pH 3.2), solvent B: 90% acetonitrile and 10% 200 mM ammonium formate (pH 3.2), column compartment temperature 30 celsius, injection volume 5 microliter, flow rate 0.5 mL/min, 0 min: 0% B, 5 min: 40% B, 4.5 min: 95% B, 5.5 min: 95% B, 6 min: 0% B, 10 min: 0% B. Electrospray ion source (Thermo H-ESI-II source) parameters were as follows: negative ion mode, sheath gas flow 35 psi, aux gas flow 3 psi, electrospray capillary temperature 320 celsius, electrospray capillary voltage 3.6 kV. MS parameters were as follows: full MS, resolution 70000, mass range 400-1200 m/z, dd-MS2 (data-dependent MS/MS), resolution 17500, AGC target 1x10^5, loop count 5, isolation width 1.0 m/z, collision energy 25 eV and dynamic exclusion 0.5 s. LC-MS data were analyzed with QualBrowser in the Thermo Xcalibur software package (v.4.3.73.11, Thermo Scientific).
Roland Kersten 
