Quantitative Analysis of Histone PTMs

Approximately 20 μg of extracted histones was resuspended in 30 μl of 100 mM ammonium bicarbonate, at pH 8.0. Chemical propionylation derivatization, digestion and desalting of histones followed by analysis by LC–MS and MS/MS were performed as described previously [32 (link), 33 (link)]. In brief, purified peptides were loaded onto 75-μm-ID fused-silica capillary columns packed with 12 cm of C18 reversed-phase resin (Reprosil-pur 120 C18, aq-3 μm particles, Fisher Scientific). Peptides were separated using EASY-nLC nano-HPLC (Thermo Scientific, Odense, Denmark) and introduced into a hybrid linear quadrupole ion trap–Orbitrap mass spectrometer (ThermoElectron) and resolved with a gradient from 0 to 35% solvent B (A = 0.1% formic acid; B = 95% MeCN, 0.1% formic acid) over 30 min and from 34 to 100% solvent B in 20 min at a flow-rate of 250 nL/min. The Orbitrap was operated in data-dependent mode essentially as previously described [33 (link)]. Relative abundances of peptide species were calculated by chromatographic peak integration of full MS scans using EpiProfile [34 (link)]. Where necessary, peptide and PTM identity were verified by manual inspection of MS/MS spectra.