2.12. LC‐MS/MS analysis

LC‐MS/MS analysis was performed on a nanoAcquity UPLC system (Waters Inc.) connected to a Q Exactive mass spectrometer (Thermo Scientific) equipped with a Digital PicoView source (New Objective Inc., Littleton, MA). Solvent composition at the two channels was 0.1% formic acid for channel A and 0.1% formic acid, 99.9% acetonitrile for channel B. For each sample, 1 µL was injected. Peptides were trapped on a Symmetry C18 trap column (5 µm, 180 µm×20 mm, Waters Inc.) and separated on a BEH300 C18 column (1.7 µm, 75 µm×150 m, Waters Inc.) at a flow rate of 300 nL/min using a gradient from 5% to 35% B in 90 min, 60% B in 5 min and 80% B in 1 min. The mass spectrometer was operated in data‐dependent mode (DDA), acquiring full‐scan MS spectra (350−1500 m/z) at a resolution of 70.000 at 200 m/z after accumulation to a target value of 3.000.000, followed by higher‐energy collision dissociation (HCD) fragmentation on the twelve most intense signals per cycle. HCD spectra were acquired at a resolution of 35.000 using a normalized collision energy of 25 units and a maximum injection time of 120 ms. The automatic gain control (AGC) was set to 50000 ions. Charge state screening was enabled, and singly and unassigned charge states were rejected. Only precursors with intensity above 25,000 were selected for MS/MS. Precursor masses previously selected for MS/MS measurement were excluded from further selection for 40 s, and the exclusion window was set at 10 ppm. The samples were acquired using internal lock mass calibration on m/z 371.1010 and 445.1200.