Easy spray pepmap c18 analytical column

The peptide samples were analyzed by LC/MS/MS using a Waters nanoAcquity coupled to a Thermo Fusion Lumos mass spectrometer. Samples were injected onto a Thermo PepMap C18 trap column, washed, and then loaded onto an Easy Spray PepMap C18 analytical column (75 μM id × 25 cm, 2 μM particle size) (Thermo Scientific). The samples were separated over a 120 min method, where the gradient for separation consisted of 2%–25% mobile phase B at a 300 nl/min flow rate; mobile phase A was 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in 100% acetonitrile. MS1 orbitrap scans were collected at a resolution of 120,000 and 1e6 AGC target. The MS2 spectra were acquired either in the orbitrap or the linear ion trap depending on peak charge and intensity using a 3 s TopSpeed CHOPIN method (Davis et al., 2017 (link)). Orbitrap MS2 scans were acquired at 7500 resolution, with a 5e4 AGC, and 22ms maximum injection using HCD fragmentation with a normalized energy of 30%. Rapid linear ion trap MS2 scans were acquired using a 4e3 AGC, 250 ms maximum injection time, CID fragmentation set at 30%. Dynamic exclusion was set to 30 s and precursors with unknown charge or a charge state of 1 and ≥ 8 were excluded.