Orbitrap Fusion Lumos MS Workflow

All MS analysis was performed on an Oribtrap Fusion Lumos (Thermo Fischer Scientific) coupled to a Proxeon nLC-1200 ultra-high pressure liquid chromatography (UPLC) pump (Thermo Fisher Scientific). Peptides were separated onto a packed 100 µM inner diameter column containing 0.5 cm of Magic C4 resin (5 μm, 100 Å, Michrom Bioresources) followed by 40 cm of Sepax Technologies GP-C₁₈ resin (1.8 μm, 120 Å) and a gradient consisting of 6–30% (ACN, 0.125% FA) over 125 min at ~450 nL/min. The instrument was operated in data-dependent mode with a 60 s (±7 ppm window) expiration time, with FTMS^{1 (link)} spectra collected at 120,000 resolution with an AGC target of 500,000 and a max injection time of 100 ms. The ten most intense ions were selected for MS/MS and precursors were filtered according to charge state (required > 1 z). Monoisotopic precursor selection was enabled. Isolation width was set at 0.7 m/z. ITMS^{2 (link)} spectra were collected at an AGC of 18,000, max injection time of 120 ms and CID collision energy of 35%. For the FTMS^{3 (link)} acquisition, the Orbitrap was operated at 30,000 resolution with an AGC target of 50,000 and a max injection time of 250 ms and an HCD collision energy of 55%. Synchronous-precursor-selection (SPS) was enabled to include 10 MS^{2 (link)} fragment ions in the FTMS^{3 (link)} spectrum.