Liquid chromatography coupled with multi-stage mass spectrometry (LC-MS3)
An Ultimate 3000 RSLC nano UHPLC was used for online fractionation, equipped with a 300 μm ID x 5 mm Acclaim PepMap μ-Precolumn (Thermo) and a 75 μm ID x 50 cm 2.1 μm particle Acclaim PepMap RSLC analytical column (Thermo). Loading solvent was 0.1% formic acid. Analytical solvent contained HPLC grade H2O, acetonitrile, and formic acid. Samples were loaded at 5 ml/min for 5 min in loading solvent before beginning the analytical gradient. Formic acid concentration was maintained at 0.1% during the analytical gradient while concentration of acetonitrile gradually increased. All separations were carried out at 55 °C. Mass spectrometry data were acquired using Orbitrap Lumos mass spectrometer (Thermo). TMT-based analysis used a MultiNotch MS3-based method (McAlister et al., 2014). MS1 scans surveyed 380-1500 Th, with resolution of 120,000, automatic gain control (AGC) target of 2 x 105, and maximum injection time of 50 ms. Ions that had the counts of 5 x 103 counts and above triggered MS2 analysis, with Quadrupole isolation at an isolation width of 0.7 Th, normalised collision energy (NCE) set to 35% for CID fragmentation, 1.5x104 AGC target, and 120 ms maximum injection time. Top 6 MS2 ions were selected for HCD fragmentation (NCE 65%) in MS3. MS3 resolution was 60,000, with an AGC target of 1 x 105 and a maximum accumulation time of 150 ms. The entire MS/MS/MS cycle had a target time of 3 sec. Dynamic exclusion was set to +/- 10 ppm for 70 sec.