LC-MS/MS measurements and data processing

Peptides and phosphopeptides were loaded onto a 40 cm column (75 µm inner diameter) fused silica packed with 1.9 µM C18 ReproSil particles (Dr. Maisch GmBH) and maintained at 60 °C. A Dionex U3000 RSLC Nano HPLC system (Thermo Fisher Scientific) was interfaced with a Q Exactive HF X benchtop Orbitrap mass spectrometer using a NanoSpray Flex ion source (Thermo Fisher Scientific). Peptides were separated with a binary buffer system of 0.1% (v/v) formic acid (buffer A) and 80% (v/v) acetonitrile / 0.1% (v/v) formic acid (buffer B). For phosphoproteome and in vitro kinase assay analysis, peptides were eluted at 350 nL/min and separated with a gradient of 3–19% buffer B over 40 min, followed by 19–41% buffer B over 20 min. Peptides were analysed with a full scan (350–1400 m/z; R = 60,000 at 200 m/z) at a target of 3e6 ions, followed by up to ten data-dependent MS2 scans using HCD (target 1e5 ions; max. IT 50 ms; isolation window 1.6 m/z; NCE 27%; min. AGC target 2e4), detected in the Orbitrap mass analyser (R = 15,000 at 200 m/z). Dynamic exclusion (30 s) and Apex trigger (2–4 s) were switched on. For single-run proteome analysis, peptides were eluted at 300 nL/min, separated with a gradient of 5–30% buffer B over 2 h and analysed with a full scan (350–1400 m/z; R = 60,000 at 200 m/z) at a target of 3e6 ions, followed by up to 20 data-dependent MS2 scans using HCD (target 1e5 ions; max. IT 28 ms; isolation window 1.4 m/z; NCE 27%; min. AGC target 1e4), detected in the Orbitrap mass analyser (R = 15,000 at 200 m/z). Dynamic exclusion (30 s) was switched on. Raw MS data were processed using MaxQuant⁴⁸ (v1.6.6.0 and v1.6.0.9 for the proteome/phosphoproteome and in vitro kinase assays respectively), searching against the Human UniProt database (January, 2019), using default settings with the addition of ‘Phospho(STY)’ as a variable modification. The ‘Match between runs’ option was turned on for all analyses.