LC/MS analysis for organic acids was performed using an Agilent Technologies
6540 Q-TOF equipped with a Jet Spray ESI source operated in negative ionization mode at a 3 Hz scan rate in high resolution mode across mass range 50 to 1,700 m/z. The source parameters were 150°C drying 76 gas, 12 L/min drying gas, nebulizer pressure of 45 psi, sheath gas temperature 325°C, sheath gas flow 12 L/min, capillary at 2,000 V, nozzle at 0 V, fragmentor 90 V, skimmer 35 V, octopole RF 750 V. Five microliter of each sample was injected using an HTC autosampler into a 2 μL loop supplied by an Agilent
1260 binary pump. The column was a 2.1 mm × 100 mm Waters
HSS-T3 C18 column with 1.8 μm packing. Buffer A was 20 mmol/L ammonium formate pH 2.9; buffer B was acetonitrile. Gradient separation began with 0.5% buffer B at 0.4 mL/min, held until 2.5 minutes, then buffer B was increased to 99.5% at 4 minutes. The stream was switched from the LC/MS to waste, and for regeneration, flow was increased to 0.5 mL/min at 5 minutes and 1 mL/min at 5.5 minutes, held until 8.5 minutes, and then decreased to 0.5 mL/min at 9 minutes. The column was re-equilibrated by a shift to 0.5% buffer B at 10.5 minutes, flow as decreased to 0.4 mL/min at 13 minutes, and held until 14 minutes. Other compounds were analyzed by ion-paired reverse phase analysis in negative mode on a 6550 Q-TOF.
Mirhadi S., Zhang W., Pham N.A., Karimzadeh F., Pintilie M., Tong J., Taylor P., Krieger J., Pitcher B., Sykes J., Wybenga-Groot L., Fladd C., Xu J., Wang T., Cabanero M., Li M., Weiss J., Sakashita S., Zaslaver O., Yu M., Caudy A.A., St-Pierre J., Hawkins C., Kislinger T., Liu G., Shepherd F.A., Tsao M.S, & Moran M.F. (2022). Mitochondrial Aconitase ACO2 Links Iron Homeostasis with Tumorigenicity in Non–Small Cell Lung Cancer. Molecular Cancer Research, 21(1), 36-50.
