Anti acetyl lysine beads

DL-dithiothreitol (DTT), iodoacetamide (IAA), trifluoroacetic Acid (TFA), acetonitrile (ACN), and methanol were purchased from Sigma. Trypsin was purchased from Promega. C18 SPE Cartridge was purchased from Waters (Milford, MA, USA). Anti acetyl-Lysine beads were purchased from Cell Signaling Technology. Ultrapure water was prepared from a Millipore purification system. An Ultimate 3000 nano UHPLC system coupled with a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) with an ESI nanospray source was used.

Protein (20 mg) was reduced with 10 mM DTT (dithiothreitol) at 37°C for 2 h. After cooling, 50 mM IAA (iodoacetamide) was added for alkylation at room temperature in the dark for 30 min. Add five times the volume of ddH₂O, dilute the urea concentration to 1.5 M, add Trypsin Gold (Promega) at a ratio of 20:1, and digest at 37°C for 16–18 h. After digestion, SPE C18 column (Waters Inc., Milford, MA, United States) was used to peptide desalted and then lyophilized with centrivap vacuum concentrator (Labconco Inc., Kansas City, MO, United States). The lysine-acetylated peptides were enriched by immunoaffinity using anti-Ac-K antibody beads [PTMScan Acetyl-Lysine Motif (Ac-K) Kit, Cell Signal Technology], as previously described (Bouchut et al., 2015 (link)). Briefly, the peptides were mixed with anti-acetyl-lysine beads (Cell Signaling Technology) for 2.5 h at 4°C in MOPS IAP buffer (50 mM MOPS, 10 mM KH₂PO₄, and 50 mM NaCl in 1MTris, pH = 7.0) and then centrifuged for 30 s at 3,000 g at 4°C. Finally, the peptides were eluted with 0.15% TFA. The peptides were desalted using a peptide desalting rotating column (Thermo Fisher Scientific, Germany) before MS identification.