I d hilic column

Tissue extracts were analyzed on 6550 iFunnel QTOF mass spectrometer (Agilent Technologies) interfaced with 1290 UPLC system (Agilent Technologies). Samples were analyzed using a Luna Aminopropyl, 3 μm, 150 mm × 1.0 mm I.D. HILIC column (Phenomenex). The mobile phase was composed of A = 20 mM ammonium acetate and 40 mM ammonium hydroxide in 95% water and B = 95% acetonitrile. The remaining 5 % were acetonitrile or water, respectively. The linear gradient elution from 100% B (0–5 min) to 100% A (50–55 min) was applied (A = 95% H2O, B = 95% ACN, with appropriate additives). A 10 min re-equilibration time was applied for HILIC, to ensure the column re-equilibration and maintain the reproducibility. The flow rate was 50 μL/min, and the sample injection volume was 5 μL. ESI source conditions were set as follows: dry gas temperature 200 °C and flow 11 L/min, fragmentor 380 V, sheath gas temperature 300 °C and flow 9 L/min, nozzle voltage 500 V, and capillary voltage −2500 V in ESI negative mode. The instrument was set to acquire over the m/z range 50–1000, with the MS acquisition rate of 2 spectra/s.

Tissue extracts (8 brain regions × 5 biological replicates) were analyzed on 6550 iFunnel QTOF mass spectrometer (Agilent Technologies) interfaced with 1290 UPLC system (Agilent Technologies). Samples were analyzed using a Luna Aminopropyl, 3 µm, 150 mm × 1.0 mm I.D. HILIC column (Phenomenex). The mobile phase was composed of A = 20 mM ammonium acetate and 40 mM ammonium hydroxide in 95% water and B = 95% acetonitrile (Ivanisevic, et al., 2013 (link)). The linear gradient elution from 100% B (0–5 min) to 100% A (50–55 min) was applied (A = 95% H2O, B = 95% ACN, with appropriate additives). A 10 min postrun was applied for HILIC, to ensure the column re-equilibration and maintain the reproducibility. The flow rate was 50 µL/min, and the sample injection volume was 5 µL. ESI source conditions were set as follows: dry gas temperature 200 °C and flow 11 L/min, fragmentor 380 V, sheath gas temperature 300 °C and flow 9 L/min, nozzle voltage 500 V, and capillary voltage −2500 V in ESI negative mode. The instrument was set to acquire over the m/z range 50–1000, with the MS acquisition rate of 2 spectra/s. For the MS/MS of selected precursors the default isolation width was set as narrow (~ 1.3 m/z), with a MS acquisition rate at 2 spectra/s and MS/MS acquisition at 3 spectra/s. MS/MS data were acquired at the collision energy of 20 V and of 40 V.