Scientific). Peptides were trapped on a 5 mm Pepmap 100 C18 column (300 μm i.d., 5μm particle size, Dionex) and fractionated on a 200 mm Alltima C18 column (300 μm i.d., 3 μm). Acetonitrile concentration in the mobile phase in 0.1% formic acid was increased from 5 to 18% in 88 minute, to 25% at 98 minute, 40% at 108 minute and to 90% in 2 minutes, at a flow rate of 400nL/minute. Peptides were electrospray into the mass spectrometer using an ion spray voltage of 5.5 kV and an interface heater temperature of 50°C. The MS survey scan range was m/z 350–1,250 acquired for 200 ms. The top 20 precursor ions were selected for 85ms per MS/MS acquisition, with a threshold of 100 counts. Dynamic exclusion was 16 s. Rolling CID function was activated, with an energy spread of 15 eV. The Human database used was UniProt_2015–02. Label-free quantification was activated with LFQ min. ratio count of 1. Match between runs with match time window of 0.7 minute and alignment time window of 4 minute were used for all analyses. For other parameters the default settings were used. All raw MS data were analyzed by MaxQuant software (version 1.5.2.8)
