GNPS - Integrated Omics-Based Discovery of Novel Genes, Secondary Metabolite Clusters, and Small Molecules in Penicillium spp. with Disparate Fungal Lifestyles

Four fungal strains, P. expansum R19, P. expansum Pe21, P. chrysogenum 404, and P. chrysogenum 413, were evaluated. Flasks containing 50 mL of potato dextrose broth (PDB) were individually inoculated with 100 uL of conidial suspensions of the fungi. Cultures were grown for 7 days, shaking at 25C. Filter-sterilized (0.2 um) spent growth medium (50 mL from three independent cultures) was centrifuged for 30 minutes at 15,000 x g. Chloroform was added to 3 mL medium at a 1:1 ratio before centrifuging again at 10,000 x g for 10 minutes. The supernatant was transferred to a tube containing 3 mL 60% methanol and vortexed for 2 minutes, then mixed with 5 mL of ethyl acetate. The top organic layer was transferred to a 15 mL conical tube and evaporated to dryness with continual flow of nitrogen gas. Sample residuals were resuspended in 100 uL 50% methanol/0.1% formic acid. Twenty uL from each sample was pooled to make a quality control (QC) sample. Three samples of potato dextrose broth (PDB) without fungus were processed similarly but were not included in the QC. Five uL of the samples were separated on a 40 C heated, 150 x 2.1 mm Hypersil GOLD VANQUISH HPLC column with 1.9 um particles coupled to a Vanquish HPLC pump controlling a 10-minute linear gradient from 0% to 95% acetonitrile and 0.1% formic acid at a flow rate of 0.2 mL per minute. Eluent was electrosprayed at 3.5 kV positive polarity into an Exploris 240 mass spectrometer using an internal mass calibrant. Sheath gas was 35, auxiliary gas was 7, and sweep gas was 1. The ion transfer tube temperature was 325 C and the vaporizer temperature was 275 C. Advanced peak determination, mild trapping, and internal mass calibration was enabled. Default charge state was one and the expected peak width was 6 sec. AcquireX software was used to create a background ion exclusion list from a blank sample consisting of 50% methanol/0.1% formic acid and an inclusion ion list from the QC sample. Subsequently, five injections of QC were performed to generate MS2 spectra. After each of those injections, the resolved ions were appended to the exclusion list for the subsequent injection. Survey scans were recorded in the Orbitrap at 60,000 resolution over a mass range of m/z 70-800. The RF lens was 70%. Monoisotopic precursor selection was enabled, the minimum intensity was 5,000, charge states were filtered to one and automated dynamic exclusion was enabled. Twenty precursors selected within a 1.0 Da isolation window were fragmented by high energy collision-induced dissociation (30%, 50%, 70% normalized stepped collision energy), and fragment ions were resolved in the Orbitrap at 30,000 resolution. Next, all test samples were analyzed alongside four intermittent injections of QC and blank consisting of 50% methanol/0.1% formic acid. Survey scans were recorded in the Orbitrap at 120,000 resolution over a mass range of m/z 70-800. The RF lens was 70%. The samples also were analyzed in negative ion mode at -2.5 kV but with the same other settings.