Acclaim pepmap100 c18 3 μm

Each sample of digested peptides was desalted with a ZipTip^® with 0.6 μL resin bed volume (Millipore, Burlington, MA, USA) according to the manufacturer’s instructions. The eluent was dried and resuspended in 0.1% formic acid, and was analyzed using a Q Exactive HFX mass spectrometer connected to an Easy nLC 1200 ultra high-pressure chromatography system (Thermo Scientific, Waltham, MA, USA). The samples were loaded onto a reverse-phase nano-trap column (75 μm interior diameter × 2 cm, Acclaim PepMap100 C18 3 μm, 100 Å, Thermo Fisher Scientific, Waltham, MA, USA) with mobile phase A (0.1% formic acid and 2% acetonitrile), and separated over an EASY-Spray column, (ES803A, 75 μm i.d. × 50 cm C18 2 μm, 100 Å, Thermo Fisher Scientific, Waltham, MA, USA) using a gradient (2% to 32% over 60 min) of mobile phase B (0.1% formic acid, 80% acetonitrile) at a flow rate of 250 nL/min. The mass spectrometer was operated in positive ion mode with a capillary temperature of 275 °C and a potential of 2100 V applied to the emitter. All the data were acquired with the mass spectrometer operating in automatic data dependent switching mode. A high-resolution (60,000) MS precursor ion scan (350–1500 m/z range) was performed to select the 10 most intense ions for the subsequent fragmentation and MS/MS analysis using HCD (NCE 29 at 15,000 resolution) each duty cycle.