Orbitrap elite instrument

Mouse intestinal epitelial cells were lysed with an AFA Focused-Ultrasonicator (Covaris). Histones were purified from the lysates (Active Motif, Cat. No. 40026) and desalted by off-line reversed phase chromatography on an Agilent 1200 tower using a Jupiter 5 μm C4 300 Å Column 150 × 2 mm (Phenomenex). The resulting peak area was used to estimate concentration against histone preparations of known concentration. Desalted histones were lyophilized and spiked 1:1 with histones isolated from 5×10⁶ HeLa cells grown in RPMI 1640 SILAC heavy arginine (¹³C6 ¹⁵N4) medium (Cambridge Isotope Labs), followed by treatment with NHS-propionyl synthesized at neutral pH and digestion with trypsin (Promega). Peptides were lyophilized and treated again to obtain a homogenous population of derivatized lysines and new N-termini, followed by high resolution – high mass accuracy LC-MS/MS in an Orbitrap Elite instrument (Thermo Scientific) equipped with a nanoACQUITY UPLC tower with a 1×100mm HSS T3 1.8 μm column (Waters). Mass spectrometry data were interpreted using Mascot Distiller (Matrix Science) for identification, followed by manual verification. The peak area was processed using Skyline Software v1.4.0.422 (University of Washington) to quantify histone peptides bearing specific post-translational modifications.