Parallel Reaction Monitoring Proteomics

Stored digests were thawed and loaded onto Evotips (Evosep, Odense, Denmark) according to the manufacturer’s instructions and as previously described (Bache et al., 2018 (link); Foudraine et al., 2019 (link)). LC-MS/MS was performed using the Evosep One (Evosep, Odense, Denmark) coupled to an Orbitrap mass spectrometer (Q Exactive HF Hybrid Quadrupole-Orbitrap, Thermo Fisher Scientific, Bremen, Germany). LC was performed using the manufacturer’s separation method of 11.5 min (100 samples/day) (Bache et al., 2018 (link)). The Q Exactive HF system was operated in parallel reaction monitoring (PRM) mode. The following settings were used: a quadrupole isolation window of 0.6 m/z units, an automatic gain control target value of 1 × 10⁶ ions, a maximum fill time of 150 ms and a resolving power of 30,000 at 400 m/z. A normalized collision energy of 27% was used for all peptides. Measurements were unscheduled in the peptide selection experiments. Measurements were scheduled with retention windows of 2 min for each peptide in the subsequent experiments. LC-MS/MS data was analyzed in Skyline daily 19.1 or later (MacCoss Lab Software, University of Washington, United States). The mass spectrometry proteomics data have been deposited to the n ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2019 (link)) partner repository with the dataset identifier PXD025363.