Mass spectrometric analysis of DSB-containing chromatin
Chromatin samples were isolated as described above with the following modifications: 100μl of LSS extracts were supplemented with sperm nuclei (5,000 sperm/μl) and incubated for 10 min at 21ºC before addition of PflMI restriction endonuclease (0.05U/μl) and incubated for an additional 60 min. Chromatin was isolated through sucrose cushions as described above and extensively digested with 100U of micrococcal nuclease for 30 min at 37ºC (NEB). Chromatin proteins were reduced with DTT (5mM, 30 min 60ºC) and alkylated with iodoacetamide (15mM, 30 min RT) before fractionation on SDS-PAGE. “in-gel” digestion was performed with proteomic-grade trypsin (Promega) at 5 ng/μl for 16 hrs at 37ºC. The resulting digestion peptides were extracted from the gel pieces with 1:2 (vol/vol) 5% formic acid: acetonitrile solution. The resulting peptides were labeled with isobaric mass tags (ITRAQ 4-plex, Sciex), combined, purified with in-house made STAGE tips 35, resuspended in 0.5% acetic acid and loaded onto a home-packed reverse phase C18 column (75 μm I.D.). The peptides were separated using a linear gradient (0 % to 42 % acetonitrile, 0.5 % acetic acid, 120 min, 150 nL/min) and directly sprayed into an LTQ-Orbitrap-Velos mass spectrometer for analysis (Thermo). The repetitive analytical cycle incorporated a high-resolution mass scan in the Orbitrap (resolution = 30,000) followed by tandem MS scans of the five most intense peaks observed in each Orbitrap mass spectrum. Peptides were identified and quantified using Proteome Discoverer software (Thermo, Version 1.4).