SILAC-Based Quantification of Lysine Acetylation

For SILAC experiments, THP-1 or U937 cells were cultured in RPMI 1640 deficient in L-arginine and L-lysine, and supplemented with 10% dialyzed FBS. Two linages of cells were cultured in light medium (L-arginine (Arg 0) and L-lysine (Lys 0)) and heavy medium (L-arginine 13C6-15N4-HCl (Arg 10) (#89990, Thermo) and L-lysine 13C6-15N2-HCl (Lys 8)) (#88209, Thermo). Prior to infection, cells were treated with PMA (Phorbol 12-myristate-13-acetate) (#P1585, Sigma-Aldrich) for 24 h, then heavy-labeled cells were infected at 90% confluency with S. Typhimurium 14028S at MOI 100, while light-labeled cells were mock infected. After incubation for 1 h at 37°C in a 5% CO₂ atmosphere, extracellular bacteria were killed at 100 mg/ml gentamicin for 2 h, and then switched to medium containing 25 mg/ml of gentamicin for the remainder of the experiment. Cells were lysed at indicated time points in lysis buffer containing 8M urea, 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM ethylene diamine tetra acetic acid (EDTA), 2 g/l Aprotinin, 10 g/l Leupeptin, 1×protease inhibitor cocktail (#CW2200, Cwbio), and 5 mM sodium butyrate. Protein concentration was determined using a BCA protein assay and 5 mg proteins in two states mixed at equal ratio. Samples were digested with trypsin, and peptides were enriched for lysine acetylation using the anti-KAc antibodies noncovalently coupled to protein A agarose beads (#13416, Cell Signaling Technology). Desalted peptides were analyzed by online nanoflow liquid chromatography tandem mass spectrometry (LC-MS/MS) using a Q Exactive mass spectrometer. Heavy arginine (Arg10) and lysine (Lys8) were selected for SILAC quantification. The eXtracted Ion Current (XIC) of all isotopic clusters associated with the identified amino acids sequence in the light label cluster was summed up, and calculated the ratio between two heavy and light label partners. Then the ratio was normalized, the median of the total ratio population was shifted to 1 and data was analyzed using the MaxQuant software version 1.3.0.5. We used Significance A to assess the significance of outlier ratios. Only peptides with average 1.5-fold change of acetylation in two states and significance A values less than 0.05 were considered up or down regulated. The score at lysine acetylation of proteins greater than 20 is considered to be reliable.