LC-MS/MS (protein-level TurboID and peptide-level PerTurboID)

Samples were loaded onto Evotips (according to the manufacturer’s instructions). Following a wash with aqueous acidic buffer (0.1% formic acid in water), samples were loaded onto an Evosep One system coupled to an Orbitrap Fusion Lumos (Thermo Fisher Scientific). The Evosep One was fitted with a 15 cm column (PepSep) and a predefined gradient for a 44 min method was employed. The Orbitrap Lumos was operated in data-dependent mode (1 s cycle time), acquiring IT HCD MS/MS scans in rapid mode after an OT MS1 survey scan (R = 60,000). The MS1 target was 4E5 ions, whereas the MS2 target was 1E4 ions. The maximum ion injection time utilized for MS2 scans was 300 ms, the HCD normalized collision energy was set at 32, and the dynamic exclusion was set at 15 s.