Oasis hlb μelution plate 30 μm

To enrich acetylated peptides from the unbound IMAC peptides, the unbound peptides were lyophilized and dissolved in IAP buffer (50 mM MOPS, 50 mM NaCl, 10 mM Na2HPO4, pH ~7.2) according to the manufacturer’s instruction. The PTMScan Acetyl-Lysine Motif (Ac-K) Immunoaffinity Beads were purchased from Cell Signaling (Prod, 13416). The beads were washed three times with 1 ml IAP, ~5 μl 100% beads were used for each sample for the immunoaffinity enrichment^{7 (link)} under end-end mixing for 1 h at 4 °C. The beads were then washed with two times 1 ml IAP, 1× IAP plus 0.05% RapiGest SF (Waters) surfactant, followed by three times 1 ml PBS washes; the lysine-acetylated peptides were eluted with two times 40 μl 0.15% TFA. The eluted acetylated peptides were desalted via Oasis HLB μElution plate 30 μM (Waters). The enriched peptides from both IMAC and acetylated immunoaffinity were dried via speedvac for overnight, and they were dissolved in 0.1% TFA/water including iRT peptides. Both eluates were combined to a final volume of 8 μl for LC–MS analysis.