Metabolites were extracted from randomly selected tissue samples by adding 1 mL of 80:20 methanol:water solution on dry ice. Samples were incubated at −80 °C for 4 h and centrifuged at 4 °C for 5 min at 21,000 × g. Supernatants were transferred into LoBind Eppendorf microcentrifuge tubes and the cell pellets were re-extracted with 200 μL ice-cold 80% MeOH, spun down and the supernatants were combined. Metabolites were dried at room temperature under vacuum and resuspended in water for injection.
Samples were randomized and analyzed on a Q-Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer coupled to an UltiMate 3000 UHPLC system (Thermo Scientific). The mass spectrometer was run in polarity switching mode (+3.00 kV/-2.25 kV) with an m/z window ranging from 65 to 975. Mobile phase A was 5 mM NH4AcO, pH 9.9, and mobile phase B was acetonitrile. Metabolites were separated on a Luna 3 μm NH2 100 Å (150 × 2.0 mm) column (Phenomenex). The flowrate was 300 μl/min, and the gradient was from 15% A to 95% A in 18 min, followed by an isocratic step for 9 min and re-equilibration for 7 min. All samples were injected twice for technical duplicates. Metabolites were detected and quantified as area under the curve based on retention time and accurate mass (≤5 ppm) using the TraceFinder 3.3 (Thermo Scientific) software.
Samples were randomized and analyzed on a Q-Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer coupled to an UltiMate 3000 UHPLC system (Thermo Scientific). The mass spectrometer was run in polarity switching mode (+3.00 kV/-2.25 kV) with an m/z window ranging from 65 to 975. Mobile phase A was 5 mM NH4AcO, pH 9.9, and mobile phase B was acetonitrile. Metabolites were separated on a Luna 3 μm NH2 100 Å (150 × 2.0 mm) column (Phenomenex). The flowrate was 300 μl/min, and the gradient was from 15% A to 95% A in 18 min, followed by an isocratic step for 9 min and re-equilibration for 7 min. All samples were injected twice for technical duplicates. Metabolites were detected and quantified as area under the curve based on retention time and accurate mass (≤5 ppm) using the TraceFinder 3.3 (Thermo Scientific) software.