Easy 1200 nano lc system

LC–ESI–MS/MS was performed on a Q-Exactive Plus mass spectrometer coupled with an EASY 1200 nano-LC System (Thermo Fisher Scientific, Bremen, Germany). LC–ESI–MS/MS settings were the same as those stated in a previous study (22 (link)). Briefly, 3 μg of the peptide mixture was first loaded onto a trap column (100 μm inner diameter, 20 mm long, 5 μm resin, C18, Dr. Maisch GmbH, Ammerbuch, Germany) in buffer A (0.1% formic acid in water). Reversed-phase high-performance LC was then performed with the EASY 1200 nano LC System (Thermo Fisher Scientific, Bremen, Germany) using a self-packed column (75 μm inner diameter, 150 mm long, 3 μm ReproSil-Pur C18 beads, 120 Å, Dr. Maisch GmbH, Ammerbuch, Germany), and the peptide mixture was separated with a linear gradient of buffer B (0.1% formic acid in 85% acetonitrile) at a flow rate of 300 nL/min controlled by IntelliFlow for 120 min. MS/MS data were acquired using a data-dependent top 20 method by dynamically choosing the most abundant precursor ions. The survey scans were selected with an isolation window of 1.6 Thomson and fragmented by higher energy collisional dissociation with a normalized collision energy of 27 eV. The maximum ion injection times for the survey and MS/MS scans were 50 ms, and the ion target values were set to 1e6 and 1e5, respectively. Selected sequenced ions were dynamically excluded for 60 s.