Poroshell ec c18

For the TPP analysis, peptides were dissolved in 10% formic acid and a volume corresponding to 2 μg of peptides was injected on a UHPLC 1290 system (Agilent) coupled to a Q exactive HF-X mass spectrometer (Thermo Fisher scientific). Peptides were trapped (Dr Maisch Reprosil C18, 3 μm, 2 cm × 100 μm) before being separated using an analytical column (Agilent Poroshell EC-C18, 2.7 μm, 50 cm × 75 μm). Trapping was performed for 5 min in buffer A (0.1% formic acid) at a flow rate of 0.005 ml/min. The following gradient was used for separation: 12 to 42% buffer B (80% acetonitrile +0.1% formic acid) in 95 min, 100% buffer B for 2 min, followed by 100% buffer A for 11 min. The flow was split to generate a final flow of 300 nl/min. The Q Exactive HF-X was operated in a data-dependent acquisition mode with positive ionization. Full MS spectra were acquired from 375 to 1500 m/z at 60,000 resolution, using an automatic gain control target value of 3 × 10⁶ charges and a maximum injection time of 20 ms. A maximum of 12 precursors were allowed to be fragmented. A dynamic exclusion of 18 s was used. MS2 fragmentation spectra were obtained with a fixed first mass of 120 m/z at 45,000 resolution, using an automatic gain control target of 1 × 10⁵ and a maximum injection time of 85 ms. Fragmentation was performed using HCD at a NCE of 32.

Data were acquired using an Ultimate3000 system (Thermo Fisher Scientific) coupled to an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific). Peptides were first trapped (Acclaim PepMap100 C18, 5 μm, 100A) before being separated on an analytical column (Agilent Poroshell EC-C18, 2.7 μm, 50 cm × 75 μm). Trapping was performed for 2 min in solvent A (0.1 M FA in water), and the gradient was as follows: 9–13% solvent B (0.1 M FA in 80% ACN) in 3 min, 13-44% in 95 min, 44–95% in 3 min, and finally 100% for 4 min. The mass spectrometer was operated in data-dependent mode. Full-scan MS spectra from m/z 375–1,600 were acquired at a resolution of 60,000 at m/z 400 after accumulation to a target value of 3 × 106. Up to 15 most intense precursor ions were selected for fragmentation. HCD fragmentation was performed at a normalized collision energy of 27 after accumulation to a target value of 1 × 105. MS/MS was acquired at a resolution of 30,000.

An Agilent 1200 HPLC apparatus coupled with an Agilent 6460 Jet Stream triple quadrupole (QQQ) mass spectrometer was used in this study. Using a chromatographic Agilent Poroshell EC-C18 column with 2.7 μm particles and dimensions of 100 × 2.1 mm ID after an Agilent Poroshell EC-C18 precolumn with 4.6 μm particles, separation of cannabinoids was achieved with a mobile phase of water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). The initial conditions were 34% of B held for 8 min; then, B was increased to 95% over 4 min and maintained for 1 min; then, B was reduced to 34% over 1 min and maintained for 6 min with an additional 3 min post-run. The flow rate was 0.2 mL/min, and the column temperature was maintained at 35 °C. Detection was performed in negative ion mode, and analytes were ionised by electrospray and monitored in multiple reaction monitoring (MRM) mode. Optimised mass spectrometer parameters were: gas temperature 300 °C, gas flow 5 L/min, nebuliser voltage 35 V, sheath gas temperature 250 °C at flow 11 L/min, and capillary and nozzle voltage 4000 V and 500 V, respectively. The MRM transition ions are shown in Table 1.

1,000 ng of peptides from each biological replicate were first injected onto an Agilent 1,290 Infinity UHPLC system on a 50-cm analytical column packed with C18 beads (Agilent Poroshell EC-C18, 2.7 μm, 50 cm × 75 μm) coupled online to an Orbitrap HF (Thermo Fisher Scientific). The LC-MS settings were used as in the article of Hidalgo-Gutiérrez et al., 2021 (link) (Hidalgo-Gutiérrez et al., 2021 (link)) with minor modifications. After 5 min of loading with 100% buffer A (H₂O with 0.1% formic acid), peptides were eluted at 300 nl/min with a 95-min gradient from 10 to 40% of buffer B (80% acetonitrile and 20% H₂O with 0.1% formic acid). For MS acquisition, we used an MS1 Orbitrap scan at 120,000 resolution, automatic gain control (AGC) target of 3 × 10⁶ ions and maximum inject time of 120 ms from 310 to 1,600 m/z; the 15 most intense ions were submitted to MS2 Orbitrap scan at 30,000 resolution, AGC target of 1 × 10⁵ ions and maximum inject time of 54 ms (isolation window of 1.4 m/z, NCE at 28% and dynamic exclusion of 16 s). The proteomic approach undertaken in this manuscript is presented visually in Figure 1.

The data were acquired with an UHPLC 1290 system (Agilent) coupled to a Q-Exactive HFX mass spectrometer (Thermo Fischer Scientific). The peptides were trapped (Dr Maisch Reprosil C18, 3 μM, 2 cm × 100 μM) for 5 min in solvent A (0.1% formic acid in water) before being separated on an analytical column (Agilent Poroshell, EC-C18, 2.7 μM, 50 cm × 75 μM). Solvent B consisted of 80% acetonitrile in 0.1% formic acid. The gradient was as follows: 5 min trapping, followed by 155 min gradient from 10% to 36% solvent B. Subsequently, 10 min of washing with 100% solvent B and 10 min re-equilibration with 100% solvent A. The mass spectrometer operated in data-dependent mode. Full scan MS spectra from m/z 375 to 1600 were acquired at a resolution of 60,000 to a target value of 3 × 10⁶ or a maximum injection time of 20 ms. MS/MS spectra were acquired at a resolution of 15,000. The top 15 most intense precursors with a charge state of two to five were chosen for fragmentation. The HCD fragmentation was performed at 27% normalized collision energy on selected precursors with 16 s dynamic exclusion at a 1.4 m/z isolation window after accumulation to 1 × 10⁶ ions or a maximum injection time of 50 ms.