Quantitation of intracellular taurine and hypotaurine

Cells were cultured in 10cm plate with total medium volume of 10 ml. Nearly 1–5×10⁶ cells were seeded for each plate. Different concentrations of reagents were added to disturb the intracellular concentrations of hypotaurine and taurine as necessary (Supplementary Figure S1). The cells were washed with cold phosphate buffered saline (PBS) in triplicate and 1ml cold water and methanol (v/v 79:21) were evenly spread onto each plate. The adherent cells were collected using a cell scraper and each sample was transferred into a 15ml centrifugation tube containing 2ml ice-cold chloroform. The mixture was sonicated briefly and then centrifuged in 4°C for 15 min at 12000g. Every 700μl supernatant was pipetted out and lyophilyzed at −50°C in a Labconco freeze dry system (Kansas City, MO). The protein layer for each sample was collected to dry until constant weight for intracellular taurine and hypotaurine was achieved. The concentrations were then normalized. For hypotaurine and taurine quantitation, an AccQTag Ultra Derivatization Kit from Waters (Milford, MA) was used. Every lyophilized sample was dissolved in 20μl methanol and water (21:79). 10μl of the mixture was used for derivatization according to the instructions provided by the manufacturer. Liquid chromatography separation was performed on a Waters ACQUITY Ultra performance LC system using a BEH C18 column (2.1mm×100mm, 1.7μm). Mobile phase A was 20mM ammonium formate dissolved in 0.5% formic acid and 1% acetonitrile water solution. Phase B was 1.6% formic acid dissolved in pure acetonitrile. The elution gradient of phase B was: 0-1.08min, 0.1% B; 1.08-11.48 min,0.1% −9.1% B; 11.48-16.3, 9.1%-21.2%B; 16.9-18.1 min, 59.6% B; 18.28-20min, 0.1%B. the flow rate was 0.35mL/min. The injection volume was 1μL. The column temperature was 55°C with sampler temperature of 15°C. Mass spectrometry analysis was accomplished on a Waters ACQUITY SQ Detector system scanned in positive SIR mode. The capillary voltage was 3 kV. Cone voltage was 30eV. The desolvation and cone temperature was 300°C and 120°C separately. The desolvation gas flow was 650L/h and cone gas flow was 50L/h.