LC-MS/MS analysis

A nanoflow UHPLC instrument (Easy nLC, Thermo Fisher Scientific) was coupled on-line to a Q Exactive mass spectrometer (Thermo Fisher Scientific) with a nanoelectrospray ion source (Thermo Fisher Scientific). Peptides were loaded onto a C18 reversed-phase column (25 cm long, 75 μm inner diameter) and separated with a linear gradient of 5–35% buffer B (100% acetonitrile in 0.1% formic acid) at a flow rate of 300 nL/min over 240 min. MS data were acquired using a data-dependent Top15 method dynamically choosing the most abundant precursor ions from the survey scan (400–1400 m/z) using HCD fragmentation. Survey scans were acquired at a resolution of 70,000 at m/z 400. Unassigned precursor ion charge states as well as singly charged species were excluded from fragmentation. The isolation window was set to 3 Da and fragmented with a normalized collision energy of 27. The maximum ion injection times for the survey scan and the MS/MS scans were 20 ms and 60 ms, respectively, and the ion target values were set to 1e6 and 1e5, respectively. Selected sequenced ions were dynamically excluded for 30 seconds. Data were acquired using Xcalibur software (Thermo).