Histones were extracted in acid and chemically derivatized twice, digested with trypsin, followed two more rounds of derivatization and the peptides were desalted by using C18 stage-tips, as described earlier [42 (link)]. Samples were analyzed using an EASY-nLC nanoHPLC (Thermo Fisher Scientific) in a gradient of 0-35% 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 minutes at a flow-rate of 250 nL/min. Nano-liquid chromatography was coupled with a Q-Exactive mass spectrometer (Thermo Fisher Scientific). Full scan MS spectrum (m/z 290−1650) was performed in the Orbitrap (Thermo Fisher Scientific) with a resolution of 30,000 (at 400 m/z) with an AGC target of 1×10e6. The MS/MS events included both data-dependent acquisition and target, the latter for isobaric peptides to enable MS/MS-based quantification. The relative abundance of histone H3 and H4 peptides were calculated by using EpiProfile [43 (link)].
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Quantitative Histone Proteomic Analysis
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Corresponding Organization : University of Ostrava
Other organizations : Cornell University, NYU Langone Health, University of Pennsylvania, GlaxoSmithKline (United Kingdom)
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Variable analysis
independent variables
- Acid extraction of histones
- Chemical derivatization of histones (twice)
- Trypsin digestion of histones
- Desalting of peptides using C18 stage-tips
- Relative abundance of histone H3 and H4 peptides
- Gradient of 0-35% solvent B over 30 min and 34-100% solvent B over 20 min at a flow-rate of 250 nL/min during nano-liquid chromatography
- Full scan MS spectrum (m/z 290−1650) performed in the Orbitrap with a resolution of 30,000 (at 400 m/z) and an AGC target of 1×10e6
- MS/MS events included both data-dependent acquisition and target for isobaric peptides
- No positive or negative controls were explicitly mentioned in the protocol.
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