Ultimate 3000lc

Analyte separation was performed by liquid chromatography in an Ultimate 3000LC combined with Q Exactive MS (Thermo) and screened with a ESI–MS (targeted MS/MS mode). The LC system is composed of a Thermo Hyper gold C18 (100 × 2.1 mm 1.9 μm) with an Ultimate 3000LC. The mobile phase is composed of solvent A (0.1% formic acid–5% acetonitrile–water) and solvent B (0.1% formic acid–acetonitrile) with a gradient elution (0–1.5 min, 100–80% A; 1.5–9.5 min, 80–0% A; 9.5–14.5 min, 0% A; 14.5–14.6 min, 0–100% A; and 14.6–18 min, 100% A)^{27 (link)}. The flow rate of the mobile phase was 0.3 mL min⁻¹. The column temperature was 40 °C, and the sample manager temperature was set at 4 °C^{26 (link)}. The mass spectrometry parameters in ESI+ and ESI− mode are listed as follows: ESI+: Heater Temp 300 °C; Sheath Gas Flow rate, 45 arb; Aux Gas Flow Rate, 15 arb; Sweep Gas Flow Rate, 1 arb; spray voltage, 3.0 kV; Capillary Temp, 350 °C; S-Lens RF Level, 30%. ESI-: Heater Temp 300 °C, Sheath Gas Flow rate, 45 arb; Aux Gas Flow Rate, 15 arb; Sweep Gas Flow Rate, 1 arb; spray voltage, 3.2 kV; Capillary Temp, 350 °C; and S-Lens RF Level, 60%^{26 (link),27 (link)}.
At the beginning of the sequence, we run four QC samples to avoid small changes in both chromatographic retention time and signal intensity. The QC samples are also injected at regular intervals (every ten samples) throughout the analytical run.

For method development, a Thermo Scientific UltiMate 3000 LC-system coupled to a Thermo Scientific Q Exactive focus hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) was used. In order to detect the PlTXs, electrospray ionisation in positive was used. The spray voltage in positive ionisation mode was set at 3.5 kV. The capillary temperature was set at 260 °C. For infusion experiments, PlTX was infused directly into the Mass spectrometer together with a flow of 0.1 mL min⁻¹ mobile phase containing 50% acetonitrile and additives. A full MS scan event of 500 to 1400 m/z with a resolution of 70,000 full width at half maximum (FWHM) was acquired. In order to obtain fragmentation spectra of PlTX, fragmentation spectra were also acquired. The so called MS² scans were obtained by selecting mass 898 ([M + H + 2Li]³⁺) with an isolation window of 4 Da. As collision gas nitrogen was used. The normalized collision energy (NCE) was set at 20 during fragmentation. Then after fragmentation, the ions were scanned from 187 to 1850 m/z with a resolution set at 70,000 FWHM. The automatic gain control representing the maximum capacity of the C-trap was set at a maximum of 10⁶ ions or a maximum injection time of 200 ms for the full scan and a maximum of 2 × 10⁵ ions or a maximum injection time of 200 ms for the MS² scans were allowed.

Metabolomics profiling was performed by Ultimate 3000 LC combined with Q Exactive MS (Thermo Fisher Scientific, CA, United States) as reported in our recent publication (Aleidi et al., 2021 (link)). Briefly, extracted metabolites were first separated using an ACQUITY UPLC HSS T3 (100 × 2.1 mm 1.8 μm) and a mobile phase composed of solvent A (0.05% formic acid-water) and solvent B (ACN) with a gradient elution over 16 min applied at 300 μl/min flow rate. MS spectra were acquired in full MS scan in the range m/z 50–1,500, with 25,000 enhanced mass resolution and a frequency 15 spectra per second. The capillary voltage was 3000 and 3200 V for positive and negative ionization modes, respectively. The fragmentation was achieved for MSMS experiments at 175 V, with nebulizer gas at 35 bsi, and gas temperature 450°C. Chromatographic and MS parameters (under positive and negative ionization modes) were kept as reported previously (Aleidi et al., 2021 (link)). Pooled samples prepared the quality control (QC) sample. A QC injection was performed every 10 LC-MS sample runs. In total, there were 18 QC samples injected and analyzed.