Iap buffer

Adapted from a previously published protocol, protein (3.6 mg) was prepared from normal (n=5) and AH liver explants (n=4) and were ground by mortar and pestle under liquid nitrogen prior to homogenization ^{33 (link)}. Protein samples were spiked with 50 ng of acetylated bovine serum albumin (BSA) as an internal standard, trypsin-digested overnight, acidified using trifluoroacetic acid, and purified via Sep-Pak® C18 Classic Cartridges (Waters, #WAT051910). An aliquot of 54 μg was evaporated to dryness and stored at −80°C for general protein quantitation (see below). Each remaining sample was frozen at −80°C for 4 hours and lyophilized for 48 hours. Peptides were then incubated for 2 hours at 4°C with immunoaffinity beads conjugated to acetyl-Lys antibody (Cell Signaling, #13416). After incubation, supernatants were removed and the beads were washed 2 times with IAP buffer (Cell Signaling, #9993) and 3 times with LC-MS grade water (Honeywell). Peptides were eluted from the beads with 0.15% trifluoroacetic acid twice and purified on Pierce® C18 Spin tips (Thermo Scientific, #84850), evaporated to dryness, and stored at −80°C.