Quantitative Proteomic Analysis by Orbitrap MS

All MS analyses were performed on an Oribtrap Fusion Lumos (Thermo Fisher 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 ∼35 cm of Accucore resin (2.6 μm, 150Å, Thermo Fisher Scientific), and a linear gradient of Buffer A (97.4% H₂O, 2.5% ACN, 0.1% FA) to Buffer B (97.4% ACN, 2.5% H₂O, 0.1% FA), consisting of 2%–23% of Buffer B over 120 min at ∼500 nl/min was used for separation. Each analysis used the MultiNotch MS3-based TMT method (McAlister et al., 2014 (link)). For analysis with the Orbitrap Fusion Lumos mass spectrometer, the scan sequence began with an MS1 spectrum collected at 50,000 orbitrap resolution with an automatic gain control (AGC) target of 4x10⁵, max injection time of 50 ms, and mass range of 400-1500m/z, with centroid data collection. The 10 most intense ions were selected for MS2/MS3 analysis. MS2 analysis consisted of collision-induced dissociation (CID) with an AGC of 2x10⁴, normalized collision energy (NCE) 35%, maximum injection time 120 ms, and isolation window of 0.7Da. For MS3 acquisition, the precursors were fragmented by high energy collision-induced dissociation (HCD) and analyzed via the orbitrap (AGC 1x10⁵; NCE 65%, maximum injection time 150 ms; resolution was 50,000).