Peptides were separated on a Ultimate 3000 nano system (Dionex) by reverse-phase high-performance liquid chromatography, using a trapping column (PepMap 100, C18, 300 μm by 5 mm) equilibrated in loading buffer [3% (v/v) acetonitrile and 0.1% (v/v) TFA] at a flow rate of 9 μl/min for 7 min. Chromatographic separation was performed using an EASY-Spray C18 column (75 μm by 500 mm, 2-μm bead diameter) at a flow rate of 0.3 μl/min over a 30-min gradient from 3% buffer A [0.1% (v/v) formic acid]:97% buffer B [80% (v/v) acetonitrile and 0.1% (v/v) formic acid] to 20% buffer A:80% buffer B. Data were acquired using a Thermo Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific). All spectra were acquired in the Orbitrap in a data-dependent analysis mode using a top speed approach (3-s cycle time), with ions being subjected to higher-energy collisional dissociation (HCD) (normalized collision energy of 32%). MS1 parameters: 60K resolution at 200 mass/charge ratio (m/z); AGC, 4 × 105; maximum injection time, 50 ms; mass range, 350 to 2000; charge stated, 2+ to 6+. MS2 parameters: 30K resolution at 200 m/z; AGC, 5 × 104; maximum injection time, 54 ms. A dynamic exclusion window was applied for 60 s at a tolerance of 10 parts per million (ppm).

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