Peptide separation was performed with a Thermo RSLC Ultimate 3000 ultra-high pressure liquid chromatography system (Thermo Scientific) at 36°C. Solvent A was 0.1% formic acid in water, and solvent B was 0.1% formic acid in 80% acetonitrile. Peptides were loaded onto an Acclaim PepMap 100 C18 trap column (75 μm x 2 cm; Thermo Scientific cat# 165535) at a flow rate of 4 μL/min and washed with 100% solvent A for 10 minutes. Then, they were transferred to a Thermo Easy-Spray PepMap RSLC C18 column (75 μm x 50 cm with 2 μm particles, Thermo Scientific cat# ES803) and separated at a flow rate of 300 nL/min using a linear gradient from 5 to 50% solvent B in 20 min, followed by another linear gradient from 50 to 100% solvent B in 40 minutes. The column was washed with 100% solvent B for 30 minutes before being re-equilibrated with 5% solvent B for 25 minutes.

Eluted peptides were sprayed directly into a Thermo Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Scientific). Data were collected using data dependent acquisition. A survey full scan MS (from 350–1800 m/z) was acquired in the Orbitrap with a resolution of 120,000. The AGC target (Automatic Gain Control for setting the ion population in the Orbitrap before collecting the MS) was set at 4 x 105 and the ion filling time was set at 100 msec. The 25 most intense ions with charge state of 2–6 were isolated in a 3 sec cycle and fragmented using HCD (high-energy collision induced dissociation) with 35% normalized collision energy. Fragment ions were detected in the Orbitrap with a mass resolution of 30,000 at 200 m/z. The AGC target for MS/MS was set at 5 x 104, ion filling time at 60 ms, and dynamic exclusion at 30 sec with a 10 ppm mass window. Data were reported in *.raw format.

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