Chromatography was performed on an Easy LC 1000 nanoLC system (Thermo Fisher Scientific). The analytical nanoLC column was a pulled fused silica capillary (75 μm, intradermally), in-house packed to a length of 10 cm with 3 μm of C18 silica particles from Dr. Maisch. The peptide mixtures were loaded at 500 nl/min directly onto the analytical column. A binary gradient was used for peptide elution. Mobile phase A was 0.1% formic acid and 2% acetonitrile, whereas mobile phase B was 0.1% formic acid and 80% acetonitrile. Peptides were separated by a gradient elution at a flow rate of 300 nl/min, ramping from 5% B to 35% B in 60 min (50 min for SNO peptides and 120 min for nuclear extracts) and from 35% B to 100% B in additional 15 min; after 5 min at 100% B, the column was re-equilibrated at 2% B for 10 min before the following injection. MS detection was performed on a quadrupole-orbitrap MS Q Exactive (Thermo Fisher Scientific) operating in positive ion mode, with nanoelectrospray (nESI) potential at 1800 V applied on the column front end via a tee piece. Data-dependent acquisition was performed by using a top-12 method (top 8 for SNO peptide), where the 12 most abundant ions were automatically selected for higher-energy collisional dissociation fragmentation at normalized collision energy of 25%. Resolution (full width at half maximum), automatic gain control (AGC) target, and maximum injection time for full MS and MS/MS were 70,000/17,500, 1 × 106/1 × 105, and 20 ms/60 ms, respectively. MS/MS parameters for detection of SNO-peptides were as follows: resolution, 35,000; AGC target, 2 × 105; maximum injection time, 120 ms. MS full scan range was 350 to 1800 mass/charge ratio (m/z). Mass window for precursor ion isolation was 1.6 m/z. Ion threshold for triggering MS/MS events was 5 × 104. Dynamic exclusion was 30 s.

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