Shotgun Proteomics Analysis by LC-MS/MS

Samples were analyzed by one-dimensional Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS) on a Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific). The LC-MS/MS analysis was done as previously described (Kleiner et al., 2017 (link)). For each run, ∼1200 ng of peptide was loaded onto a 5 mm, 300 μm ID C18 Acclaim PepMap100 pre-column (Thermo Fisher Scientific) using an UltiMate^TM 3000 RSLCnano Liquid Chromatograph (Thermo Fisher Scientific). Peptides were then separated on a 50 cm × 75 μm analytical EASY-Spray column packed with PepMap RSLC C18, 2-μm material (Thermo Fisher Scientific) using a 260-min gradient as described in Kleiner et al. (2017) (link). The column was heated to 45°C via an integrated heating module. The analytical column was connected via an EASY-Spray source to the Orbitrap Mass Spectrometer. In between each sample, two washes with acetonitrile and one blank were run to reduce and assess carry over. Eluting peptides were analyzed in the Orbitrap Mass Spectrometer as described by Petersen et al. (2016) (link). Roughly 140,000 MS/MS spectra were acquired per sample run (Supplementary Table 1).