High performance liquid chromatography coupled to tandem mass spectrometry and data analysis.

Fungal biomass was separated from supernatant by miracloth before 20-ml portions of culture supernatants were freeze-dried. Secondary metabolites (SMs) were extracted from 100-mg freeze-dried samples by resuspending them in 1 ml high-performance liquid chromatography (HPLC)-grade methanol and sonicating them for 1 h in an ultrasonic bath. Samples were filtered and dried under a nitrogen stream before being resuspended in 1 ml of HPLC-grade methanol. Next, 100-μl samples were diluted in 900 μl of methanol and passed through 0.22-μm filters into vials.

HPLC coupled to tandem mass spectrometry (MS/MS) analysis was performed using a Thermo Scientific QExactive Hybrid Quadrupole-Orbitrap mass spectrometer. Parameters were as follows: positive mode, +3.5 kV capillary voltage; 250°C capillary temperature; 50 V S-lens and an m/z range of 133.40 to 2,000.00. MS/MS was performed using a normalized collision energy (NCE) of 30 eV, and five precursors per cycle were selected. For the stationary phase, the Thermo Scientific Accucore C₁₈ 2.6-μm column (2.1 mm × 100 mm) was used. The mobile phase was carried out using 0.1% formic acid (A) and acetonitrile (B), and the following gradient was applied: 0 to 10 min, 5% B up to 98% B; hold for 5 min; 15 to 16.2 min, 95% B up to 5% B; hold for 8.8 min. The total run time was 25 min, and the flow rate was 0.2 ml min⁻¹ with 3-μl injection volume. Data analysis was conducted using the Xcalibur software, version 3.0.63 (Thermo Fisher Scientific).

Molecular networks were made using the Global Natural Products Social Molecular Networking (GNPS) website (https://ccms-ucsd.github.io/GNPSDocumentation/ from http://gnps.ucsd.edu). First, all MS/MS fragment ions within 17 Da of the precursor m/z were removed, and spectra were filtered by choosing only the top six fragment ions in the ±50-Da window for the entire spectrum. The precursor ion mass tolerance and the MS/MS fragment ion tolerance were set at 0.02 Da. Subsequently, networks were created where edges were filtered to have a cosine score higher than 0.6 and more than five matched peaks. Edges between two nodes were kept in the network only if each of the nodes appeared in each other’s respective top 10 most similar nodes. Finally, the maximum size of a molecular family was set at 100, and the lowest scoring edges were removed. Network spectra were then searched against the GNPS spectral libraries, and library spectra were filtered in the same manner as the input data. Matches between network spectra and library spectra were filtered to have a score higher than 0.6 and at least five matching peaks (66). GNPS data used in this work are available at https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=f815e5618b05433fb768299a351fb793 (72-h data).