LC-MS/MS analysis

Peptides were analyzed by LC-MS/MS using a Dionex UltiMate 3000 Rapid Separation LC (RSLC) systems and a linear ion trap—Orbitrap hybrid Elite mass spectrometer (Thermo Fisher Scientific Inc., San Jose, CA). Six-microliter peptide samples were loaded onto the trap column, which was 150 μm × 3 cm in-house packed with 3 μm ReproSil-Pur® beads (New Objective, Inc. Woburn, MA). The analytical column was a 75 μm × 10.5 cm PicoChip column packed with 3-μm ReproSil-Pur® beads. The flow rate was kept at 300 nL/min. Solvent A was 0.1% FA in water, and Solvent B was 0.1% FA in ACN. The peptide was separated on a 120-min analytical gradient from 5% ACN/0.1% FA to 40% ACN/0.1% FA. The mass spectrometer was operated in data-dependent mode. The source voltage was 2.40 kV, and the capillary temperature was 275 °C. MS1 scans were acquired from 400 to 2000 m/z at 60,000 resolving power and automatic gain control (AGC) set to 1 × 106. The top fifteen most abundant precursor ions in each MS1 scan were selected for fragmentation. Precursors were selected with an isolation width of 1 Da and fragmented by collision-induced dissociation (CID) at 35% normalized collision energy in the ion trap; previously selected ions were dynamically excluded from re-selection for 60 s. The MS2 AGC was set to 3 × 105.