RP-LC was performed using a Dionex RSLC nano HPLC (Thermo Scientific). Peptides were injected onto a 75 μm × 2 cm PepMap-C18 pre-column and resolved on a 75 μm × 50 cm RP- C18 EASY-Spray temperature controlled integrated column-emitter (Thermo) using a four-hour multistep gradient from 5% B to 35% B with a constant flow of 200 nL min-1 as described previously 59,60. The mobile phases were: 2% ACN incorporating 0.1% FA (Solvent A) and 80% ACN incorporating 0.1% FA (Solvent B). The spray was initiated by applying 2.5 kV to the EASY-Spray emitter and the data were acquired on a Q-Exactive Orbitrap (Thermo Scientific) under the control of Xcalibur software in a data dependent mode selecting the 15 most intense ions for HCD-MS/MS.
