Peptide mixtures were analyzed by online nanoflow LC-MS/MS using an EASY-nLC 1000 (Thermo Fisher Scientific, Odense, Denmark) connected to a quadrupole/Orbitrap mass spectrometer (Q Exactive HF, Thermo Fisher Scientific) through a nanoelectrospray ion source. The nano-LC system was operated in one column setup with EASY-Spray LC Columns (length, 50 cm; inner diameter, 75 μm) packed with C18 reversed-phase resin (2 μm). Solvent A was 0.1% FA in ddH2O, and solvent B was 80% ACN with 0.1% FA. Peptides were injected at a flow rate of 500 nl/min and separated with a gradient of 5 to 30% solvent B over 80 min, followed by a gradient of 30 to 60% for 10 min and 60 to 95% over 3 min at a flow rate of 250 nl/min. The Q-Exactive was set in the data-dependent mode to automatically switch between full-scan MS and MS/MS acquisition. MS experiments consisted of an Orbitrap full scan, followed by the acquisition of the 15 most intense peptide ions (top 15) detected in the survey MS scan. Mass spectrometer conditions for all experiments were as follows: full MS [Automatic Gain Control (AGC), 3 × 106; resolution, 60,000; mass/charge ratio (m/z) range, 375 to 1650; maximum ion time, 20 ms] and MS/MS (AGC, 15,000; maximum ion time, 80 ms; isolation width, 2 m/z with a dynamic exclusion time of 20 s). Singly charged ions and ions for which no charge state could be determined were excluded from the selection. Normalized collision energy was set to 28%. Spray voltage was set to 1.9 kV. No sheath and auxiliary gas flow. Heated capillary temperature was 275°C; S-lens radio frequency level is 50%.

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