Orbitrap Mass Spectrometry Proteomic Pipeline

Mass spectrometric data were collected on an Orbitrap Fusion Lumos mass spectrometer coupled to a Proxeon NanoLC-1200 UHPLC. The 100 μm capillary column was packed with 35 cm of Accucore 50 resin (2.6 μm, 150Å; ThermoFisher Scientific). Peptides were separated using a 2.5 h gradient of 9∼35% acetonitrile gradient in 0.125% formic acid with a flow rate of ∼400nl min−1. The scan sequence began with an MS1 spectrum (Orbitrap analysis, resolution 120,000, 350−1400 Th, automatic gain control (AGC) target 5E5, maximum injection time 50 ms). SPS-MS3 analysis was used to reduce ion interference.⁷¹ (link)^,72 (link) The top 10 precursors were then selected for MS2/MS3 analysis. MS2 analysis consisted of collision-induced dissociation (CID), quadrupole ion trap analysis, automatic gain control (AGC) 1E4, NCE (normalized collision energy) 35, q-value < 0.25, maximum injection time 60 ms), and isolation window at 0.5. Following acquisition of each MS2 spectrum, we collected an MS3 spectrum in which multiple MS2 fragment ions are captured in the MS3 precursor population using isolation waveforms with multiple frequency notches. MS3 precursors were fragmented by HCD and analyzed using the Orbitrap (NCE 65, AGC 3E5, maximum injection time 150 ms, resolution was 50,000 at 400 Th).