Proxeon nlc 1200

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.

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).