Peptides were injected onto a homemade 30 cm C18 column with 1.8 μm beads (Sepax), with an Easy nLC-1000 HPLC (Thermo Fisher), connected to a Q Exactive Plus mass spectrometer (Thermo Fisher). Solvent A: 0.1% formic acid in water, Solvent B: 0.1% formic acid in acetonitrile. The gradient began at 3% B and held for 2 min, increased to 30% B over 13 min, increased to 70% over 2 minutes and held for 3 mins, then returned to 3% B in 2 min and re-equilibrated for 8 min, for a total run time of 30 min. For peptides isolated from the single gel band corresponding to the majority of proteins, the gradient began at 3% B and held for 2 min, increased to 30% B over 41 min, increased to 70% over 3 min and held for 4 min, then returned to 3% B in 2 min and re-equilibrated for 8 minutes, for a total run time of 60 minutes. The Q Exactive Plus was operated in data-dependent mode, with a full MS1 scan followed by 8 data-dependent MS2 scans.
