Enrichment of Lys-acetylated peptides

To enrich acetylated peptides, the tryptic peptides were dissolved in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, pH 8.0) followed by incubation with pre-washed agarose-conjugated anti-acetyllysine antibody beads (PTM Biolabs) at 4 °C for 12 h with gentle shaking¹⁴. The beads were washed four times with NETN buffer and twice with ddH₂O and the bound peptides were eluted with 0.1% trifluoroacetic acid¹⁴. The eluted fractions were combined and vacuum-dried. The resulting peptides were cleaned with C18 ZipTips (Millipore) according to the manufacturer’s instructions.