Tryptic Digestion of Histone Proteins

In-solution tryptic digestion of synthetic peptides or core histones was performed as previously described38 (link). Briefly, a total of 2 μg of HPLC purified histones H3 and H4 were subjected to propionylation by adding 200 μl of freshly prepared 2:1 (v/v) water: propionic anhydride (Sigma) mixture and vortexing the mixture for 1 h at room temperature. The samples were then dried in a Speed-vac at 4 °C. The dried samples were resuspended in 50 mM ammonium bicarbonate, vortexed for 2 min and subjected to a second round of evaporation at 4 °C. The samples were collected and resuspended in 100 μl of 50 mM ammonium bicarbonate and vortexed for 5 min to re-dissolve the proteins. Our digestion solution was prepared by adding 200 μl of 50 mM ammonium bicarbonate in a vial containing 20 μg of lyophilized trypsin (Promega). Roughly 0.5 μl of this solution was added to each histone sample and digested overnight at 37 °C. After digestion, samples were dried completely in a Speed-vac and then resuspended in 0.2% formic acid before LC–MS/MS analyses.