Mass Spectrometry-based Proteomics Protocol

Data were collected using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, San Jose, CA) coupled with a Proxeon EASY-nLC 1200 LC pump (Thermo Fisher Scientific). Peptides were separated on a 100 μm inner diameter microcapillary column packed with 35 cm of Accucore C18 resin (2.6 μm, 100 Å, Thermo Fisher Scientific). Peptides were separated using a 3 hours gradient of 6–22% acetonitrile in 0.125% formic acid with a flow rate of ~400 nL/min. Each analysis used an MS3-based TMT method as described previously described ^{40 (link)}.The data were acquired using a mass range of m/z 400 – 1400, resolution at 120,000, AGC target of 1 × 10⁶, a maximum injection time 100 ms, dynamic exclusion of 180 seconds for the peptide measurements in the Orbitrap. Data dependent MS² spectra were acquired in the ion trap with a normalized collision energy (NCE) set at 35%, AGC target set to 2.0 × 10⁵ and a maximum injection time of 120 ms. MS³ scans were acquired in the Orbitrap with an HCD collision energy set to 45%, AGC target set to 1.5 × 10⁵, maximum injection time of 200 ms, resolution at 50,000 and with a maximum synchronous precursor selection (SPS) precursors set to 10.