Nanoflow liquid chromatography–tandem mass spectrometry (nLC–MS/MS)

The peptide mixture was resuspended in buffer containing 0.1% formic acid and loaded onto an Easy‐nLC system (Thermo Fisher Scientific), coupled online with an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific). Peptides were loaded into a picofrit 18 cm long fused silica capillary column (75 μm i.d., 360 μm o.d.) packed in‐house with reversed‐phase ReproSil‐Pur C18‐AQ 3 μm resin. The gradient length was 75 min. The gradient was from 2–26% buffer containing 100% ACN/0.1% formic acid at a flow rate of 300 nl/min. The MS method was set up in a data‐dependent acquisition (DDA) mode. For full MS scan, the mass range of 350–1200 m/z was analyzed in the Orbitrap at 120,000 FWHM (200 m/z) resolution and 5 × 10e5 AGC target value. HCD collision energy was set to 27, AGC target to 10e4, and maximum injection time to 120 ms. Detection of MS/MS fragment ions was performed in the ion trap in the rapid mode using the TopSpeed mode (3 s).