Metabolic Profiling of Cultured Cells

Cells were cultured in 6-well plates to ~85% confluence and washed with 2 mL ice cold 1X Phosphate-Buffered Saline (PBS). The cells were then harvested in 300 µL freezing 80% acetonitrile (v/v) into 1.5 mL tubes and lysed by Bullet Blender (Next Advance) at 4 °C followed by centrifugation at 21,000 × g for 5 min at 4 °C. The supernatant was dried by speedvac and reconstituted in 7.5 µL of 66% acetonitrile and 2 µL was separated by a ZIC‐HILIC column (150 × 2.1 mm, EMD Millipore) coupled with a Q Exactive HF Orbitrap MS (Thermo Fisher) in negative detection mode. Metabolites were eluted within a 45 min gradient (buffer A: 10 mM ammonium acetate in 90% acetonitrile, pH = 8; buffer B: 10 mM ammonium acetate in 100% H2O, pH = 8). The MS was operated by a full scan method followed by targeted selected ion monitoring and data-dependent MS/MS (tSIM/dd-MS2). MS settings included full scan (120,000 resolution, 350–550 m/z, 3 × 106 AGC and 50 ms maximal ion time), tSIM scan (120,000 resolution, 1 × 105 AGC, 4 m/z isolation window and 50 ms maximal ion time) and data-dependent MS2 scan (30,000 resolution, 2 × 105 AGC, ~50 ms maximal ion time, HCD, Stepped NCE (50, 100, 150), and 10 s dynamic exclusion). Data were quantified using Xcalibur software (Thermo Fisher Scientific) and normalized by cell numbers. Ribonucleotide and deoxyribonucleotides were validated by authentic standards.