An Acclaim PepMap RSLC C18 column (75 µm i.d. × 25 cm; 2 µm particles, 100 Å, nanoViper) was used for LC separation, and gradient elution was performed using an EASY-nLC 1000 liquid chromatograph system (Thermo Fisher Scientific, San Jose, CA) with a flow rate at 300 nL/min. Mobile phase A was 2% acetonitrile with 0.1% formic acid in water, and mobile phase B was 2% water with 0.1% formic acid in acetonitrile. The analytical gradient lasted for 80 min, where the composition of solvent B changed from 5 to 35%, followed by a washing and equilibration step where solvent B increased to 95% in 5 min, was held for 2 min, and then returned to 2% B in 2 min and was held for 8 min.
An ESI-Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA) operated in positive ion mode was used for analysis. The ESI spray voltage was set at 2.5 kV. High-energy collision dissociation (HCD) fragmentation was performed at 32% of the normalized collision energy. The mass spectra were acquired in a data-dependent manner. Following a full scan in the mass range of m/z 350 to 1600, HCD MS/MS was performed on the first to the fourth most intense ions from the survey MS full scan.
An ESI-Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA) operated in positive ion mode was used for analysis. The ESI spray voltage was set at 2.5 kV. High-energy collision dissociation (HCD) fragmentation was performed at 32% of the normalized collision energy. The mass spectra were acquired in a data-dependent manner. Following a full scan in the mass range of m/z 350 to 1600, HCD MS/MS was performed on the first to the fourth most intense ions from the survey MS full scan.