The HPLC system was coupled to a Q Exactive Orbitrap MS (Thermo Fisher Scientific) with a nano-ES ion source. The TMT labelled peptides were separated by a C18 reverse-phase capillary column. The column was eluted using linear gradients of 2–35% acetonitrile in 0.1% formic acid at a constant flow rate of 300 nL/min for 220 min. The instrument was operated in the positive-ion mode with the ESI spray voltage set at 1.8 kV. The data were acquired in a data-dependent manner using the top 20 most abundant ions for Higher-energy C-trap dissociation fragmentation. The spectral data acquisition was performed using Thermo Xcalibur 3.0.63.
