Mass spectrometry

Online LC‐MS/MS was performed on a Dionex UltiMate 3000 RLSCnano (Thermo Fisher Scientific, Bremen, Germany) system coupled to an LTQ‐Orbitrap Velos ETD (Thermo Fisher Scientific). Peptides were loaded onto a 150 mm Acclaim PepMap100 C₁₈ column (LC Packings, Sunnyvale, CA, USA) in formic acid (0.1% (v/v)), and separated over a 90 min. linear gradient from 3.2% to 44% mobile phase B (acetonitrile with formic acid (0.1% (v/v)) with a flow rate of 350 nl/min. The column was then washed with 90% mobile phase B before re‐equilibrating at 3.2% mobile phase B. The column was maintained at 35°C. The LC system was coupled to an Advion Biosciences TriVersa NanoMate source (Ithaca, NY, USA) which infused the peptides with a spray voltage of 1.7 kV. Peptides were infused directly into the mass spectrometer. The mass spectrometer performed a full FT‐MS scan (m/z 380–1600) and subsequent collision‐induced dissociation (CID, 35% normalized collision energy NCE) MS/MS scans of the three most abundant ions followed by higher energy collisional dissociation (HCD 55 NCE) of the same three ions. Analysed ions were placed on an exclusion list for 60 s. The CID and HCD spectra were used for peptide identification and quantification respectively. Each SCX set (i.e. the four SCX fractions from each sample) was run in sequence followed by a blank and repeated in triplicate.