Orbitrap Fusion Lumos NanoLC-MS/MS analysis

NanoLC/MS/MS analyses were performed on an Orbitrap Fusion Lumos (Thermo Fisher Scientific), connected to an Ultimate 3000 pump (Thermo Fisher Scientific) and an HTC-PAL autosampler (CTC Analytics). Peptides were separated on a self-pulled needle column (150 mm length × 100 μm ID, 6 μm opening) packed with Reprosil-C18 AQ 3 μm reversed-phase material (Dr Maisch). The flow rate was set to 500 nl/min. The mobile phase consisted of (A) 0.5% acetic acid and (B) 0.5% acetic acid in 80% acetonitrile. Three-step linear gradients of 5 to 10% B in 5 min, 10 to 40% B in 60 min, and 40 to 100% B in 5 min were employed. For TMT-labeled samples, synchronous precursor selection-MS3 (SPS-MS3) (86 (link)) was performed. The MS scan range was m/z 375 to 1500. MS scans were performed by the Orbitrap with r = 120,000, MS/MS scans were performed by the Ion Trap in Turbo mode, and MS3 scans were performed by the Orbitrap with r = 15,000. Auto gain control was set to 4.00 × 10⁵, 1.00 × 10⁴, 5.00 × 10⁴ for MS, MS/MS, and MS3, respectively. The normalized CID collision energy was set to 35.