Histone preparation and mass spectrometry analysis.

Extracted histones were chemically derivatized and digested to tryptic peptides to make them amenable for bottom-up mass spectrometry as described earlier (35). The derivatized samples were desalted prior to liquid chromatography/mass spectrometry (LC-MS) analysis using C₁₈ Stage-tips. For mass spectrometry, the peptides were separated using a 75-μm (inner diameter [ID]) by 17-cm Reprosil-Pur C18-AQ (3 μm; Maisch GmbH, Germany) nanocolumn fitted on an EASY-nLC nanoHPLC instrument (Thermo Scientific, San Jose, CA). The high-performance liquid chromatography (HPLC) gradient comprising 2% to 28% solvent B (A, 0.1% formic acid; B, 95% MeCN, 0.1% formic acid) over 45 min, from 28% to 80% solvent B in 5 min, 80% B for 10 min at a flow rate of 300 nl/min was used. This nLC was coupled online to an LTQ-Orbitrap Elite mass spectrometer (Thermo Scientific), and data-independent acquisition (DIA) was used to acquire data (36). Briefly, full-scan MS (m/z 300 to 1,100) was acquired in the Orbitrap with a resolution of 120,000 (at 200 m/z) and an AGC target of 5 × 10e5. Tandem mass spectrometry (MS/MS) was done in in centroid mode in the ion trap with sequential isolation windows of 50 m/z with an automatic gain control (AGC) target of 3 × 10⁴, a collision-induced dissociation (CID) collision energy of 35 and a maximum injection time of 50 ms. Data were analyzed using the in-house software, EpiProfile (37), wherein the peptide relative ratio was calculated using the total area under the extracted ion chromatograms of all peptides with the same amino acid sequence (including all of its modified forms) as 100%.