LC–MS/MS analysis of the proteome and phosphoproteome

The eight proteomic samples and the eight samples enriched for phosphopeptides were analysed by LC–MS/MS on an RSLCnano system (Thermo Fisher Scientific, USA) coupled to a Q-Exactive HF mass spectrometer (Thermo Fisher Scientific, USA). The samples were first injected onto a cartridge trap column (PepMap 100, C18, 0.3 × 5 mm, Thermo Fisher Scientific, USA) for 3.3 min at a flow rate of 5 µL/min, 2% acetonitrile, 0.1% formic acid before switching in line with the main column. Separation was performed on a C18 nano column (ACQUITY UPLC M-class, Peptide CSH 130A, 1.7 µm 75 µm × 250 mm, Waters Corp, Milford, MA) at 260 nL/min with a linear gradient from 5–35% over 96 min. The LC mobile phases were as follow: A contained 0.1% (v/v) formic acid in water and B contained 0.1% (v/v) formic acid in 80% (v/v) acetonitrile. Mass spectra for the eluted peptides were acquired on a Q Exactive HF mass spectrometer in data-dependent mode using a mass range of m/z 375–1,500, resolution 120,000, AGC target 3 × 10⁶, maximum injection time 60 ms for the MS1 peptide measurements. Data-dependent MS2 spectra were acquired by HCD as a Top20 experiment with a normalized collision energy (NCE) set at 28%, AGC target set to 1 × 10⁵, 15,000 resolution, intensity threshold 1 × 10⁴ and a maximum injection time of 250 ms. Dynamic exclusion was set at 20 s to help capture phospho isomers and the isolation window set to 1.6 m/z.