All peptide samples were measured in a single-shot manner in a Q-Exactive HF-X hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific) after peptide separation by high-performance liquid chromatography (nanoLC 1200, Thermo Fisher Scientific) using a 50 cm reversed-phase column (made in house, packed with 1.9 µm C18 ReproSil particles). Peptides were eluted over a 90-minute-gradient from 0% to 95% buffer B (0.1% formic acid and 80% ACN) with a flow rate of 300 nL/minute.
Full scans were obtained from 300 to 1650 m/z with a target value of 3 × 106 ions at a resolution of 60,000 at 200 m/z. The fifteen most intense ions (Top15) of each full scan were fragmented with higher-energy collisional dissociation (HCD) (target value 1 × 105 ions, maximum injection time 120 ms, isolation window 1.4 m/z, underfill ratio 1%), and fragments were detected in the Orbitrap mass analyzer at a resolution of 15,000 at 200 m/z.
Full scans were obtained from 300 to 1650 m/z with a target value of 3 × 106 ions at a resolution of 60,000 at 200 m/z. The fifteen most intense ions (Top15) of each full scan were fragmented with higher-energy collisional dissociation (HCD) (target value 1 × 105 ions, maximum injection time 120 ms, isolation window 1.4 m/z, underfill ratio 1%), and fragments were detected in the Orbitrap mass analyzer at a resolution of 15,000 at 200 m/z.
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