Peptide Quantification via PRM LC-MS/MS

For each sample, 20 μl of digest was loaded onto Evotips (Evosep, Odense, Denmark) according to the instructions of the manufacturer 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 separation method of the manufacturer of 11.5 min (100 samples/day) (Bache et al., 2018 (link)). The Q Exactive HF system was operated in PRM mode. Samples were sequentially measured and the column was not rinsed with a blank in between measurements. The following settings were used during method validation, namely, 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 15,000 at 400 m/z. A normalized collision energy of 27% was used for all peptides. A retention time window of 2 min was used for each peptide.