Orbitrap fusion tribrid ms system

Peptides were resuspended in 0.2% formic acid in water. Peptides were separated over a 25-cm EasySpray reversed phase LC column (75 μm inner diameter packed with 2 μm, 100 Å, PepMap C18 particles, Thermo Fisher Scientific). The mobile phases (A: water with 0.2% formic acid and B: acetonitrile with 0.2% formic acid) were driven and controlled by a Dionex Ultimate 3000 RPLC nano system (Thermo Fisher Scientific). Gradient elution was performed at 300 nl min⁻¹. For the FP ABPP experiment, the mobile phase was ramped to 3% B over 3 min, followed by a ramp to 35% B at 93 min, a ramp to 42% B at 103 min, and a wash at 95% B for 10 min. For the BMB034 ABPP experiment, the mobile phase was ramped to 5% B over 4 min, followed by a ramp to 25% B at 72 min, and a wash at 9% B for 6 min. Eluted peptides were analyzed on an Orbitrap Fusion Tribrid MS system (Thermo Fisher Scientific). Survey scans of peptide precursors were collected in the Orbitrap from 300–1500 m/z for the FP ABPP experiment or 350–1350 m/z for the BMB034 ABPP experiment. Monoisotopic precursor selection was enabled for peptide isotopic distributions, precursors of z = 2–5 were selected for data-dependent MS/MS scans for 2 s of cycle time.