APAP-CYS Quantification in Liver

The APAP-CYS content in liver was measured by LC-MS/MS following the previously described method^{71 (link)}. Liver protein extract was filtered through a desalting column (Zeba spin column, Thermo Fisher) pre-equilibrated with 50 mM (NH₄)₂HCO₃ following the manufacturer’s instructions. An aliquot (45 µl) of the filtrate was mixed with 5 µl of protease type XIV (80 U/ml) (Sigma) and incubated for 24 hours at 37 °C to liberate APAP-CYS. After digestion, 5 µL of norbuprenorphine-d3 (100 µg/ml, Cerilliant Corp.) was added as an internal standard, followed by acetonitrile (300 µL) for precipitation. Dry pellet was obtained by evaporation at 13 psi, and was reconstituted in 100 µL of 2% acetonitrile buffer with 0.1% formic acid. The reconstituted solution was cleared by centrifugation and the supernatant were transferred to a clean vial for LC-MS/MS (Thermo TSQ Quantiva triple-quadrupole mass spectrometer coupled with a Shimadzu Nexera UHPLC). Free APAP-CYS (m.w. 270, Cayman #26388) was used for the quantification standard.