Histone extraction and analysis by LC-MS/MS

Histones were isolated from the nuclei of frozen cell pellets by acid extraction and derivatized with propionic anhydride similarly as described previously.⁹³ Briefly, cell pellets were resuspended in nuclear isolation buffer (NIB) containing 15 mM Tris pH 7.5, 15 mM NaCl, 60 mM KCl, 5 mM MgCl₂, 1 mM CaCl₂, and 250 mM sucrose supplemented with 1 mM DTT, 10 mM sodium butyrate, 500 μM AEBSF, 5 nM microcystin, and 0.2% NP-40 Alternative. After allowing lysis to proceed on ice for 10 mins, nuclei were pelleted at 500 × g for 5 mins at 4°C, washed twice in NIB without detergent, and then extracted in 0.4 N H₂SO₄ for 2–4 hrs at 4°C with rotation. Insoluble debris was pelleted by centrifugation at 3400 × g for 5 mins at 4°C and proteins were precipitated overnight on ice by adding 1 volume of trichloroacetic acid to 3 volumes of supernatant. Precipitated proteins were pelleted as above, washed in 0.1% HCl in acetone and then acetone, and allowed to air dry before resuspending in 100 mM NH₄HCO₃. For derivatization, 1 volume of 25% propionic anhydride in 2-propanol was added to 2 volumes of sample containing 5–20 μg of protein. Ammonium bicarbonate salt was also added for buffering purposes. After incubation for 15 mins at 37°C, samples were dried in a speed vac and derivatized a second time as above. Derivatized samples were then resuspended in 100 mM NH₄HCO₃ and digested overnight at room temperature with 1 μg trypsin per 20 μg of protein. After two additional rounds of derivatization, digested peptides were desalted with C18 stage tips for analysis by mass spectrometry.

Histone peptides were resolved with EasyLC 1000 or Dionex UltiMate 3000 LC systems fitted with 75 μm i.d. x 15–20 cm fused silica columns (Polymicro Tech) packed with ReproSil-Pur 120 C18-AQ (3 μm, Dr. Maisch GmbH) and connected in line with a mass spectrometer (Thermo Elite, Velos, Fusion, QE, or QE-HF). The chromatography conditions generally consisted of a linear gradient from 5 to 33% solvent B (0.1% formic acid in 80% acetonitrile) in solvent A (0.1% formic acid in water) over 45 mins and then 33 to 98% solvent B over 5 mins at a flow rate of 300 nL/min. The mass spectrometer was programmed for data-independent acquisition (DIA). One acquisition cycle consisted of a full MS scan, 8 DIA MS/MS scans of 50 m/z isolation width starting from 325 m/z, a second full MS scan, and 8 more DIA MS/MS scans to reach 1125 m/z. Typically, full MS scans were acquired in the Orbitrap mass analyzer across 300–1100 m/z at a resolution of 60,000 in positive profile mode with a maximum injection time of 100 ms and an AGC target of 2e5. MS/MS data from CID or HCD fragmentation was collected in the ion trap (when available) or the Orbitrap. These scans typically used an NCE of 30, an AGC target of 1e4, and a maximum injection time of 50 ms. Histone MS data, including isotope incorporation, were analyzed with EpiProfile⁹⁴ and further processed in R to filter for peaks with consistent retention times.