Evosep one

LC-MS instrumentation consisted of a breadboard Evosep One coupled to an LTQ Orbitrap for the more than 2000 HeLa injection experiment, and the Evosep One production version coupled to an Q Exactive HF-X Orbitrap (Thermo Fisher Scientific) for all other experiments. Purified peptides were separated on the HPLC columns with 3 μm Reprosil-Pur C18 beads (Dr. Maisch, Ammerbuch, Germany) and dimensions indicated below in Fig. 6B. On the LTQ Orbitrap MS, data were acquired with a Top6 data dependent shotgun method and with a Top12 method for the Q Exactive HF-X instrument. On the Q Exactive HF-X Orbitrap, the target value for the full scan MS spectra was 3 × 10⁶ charges in the 300–1650 m/z range with a maximum injection time of 50 ms and a resolution of 60,000 at m/z 200. Fragmentation of precursor ions was performed by higher-energy C-trap dissociation (HCD) with a normalized collision energy of 27 eV (24 (link)). MS/MS scans were performed at a resolution of 15,000 at m/z 200 with an ion target value of 5 × 10⁴ and a maximum injection time of 25 ms. Dynamic exclusion was set to 15 s to avoid repeated sequencing of identical peptides.

Samples for label-free quantification were process as described above. Both phosphoproteome and proteome peptides were analyzed with a “20 samples per day” method on the EvoSep One instrument and analysed on Q-Exactive Exploris 480 instrument (Thermo Fisher Scientific) running a high-resolution MS1 data-independent acquisition method. Full MS spectra were collected at 120 000 with AGC target of 3x10ˆ6 or maximum injection time of 50 ms. Scan range from 400-1000 m/z was used. The MS2 spectra were obtained with 60,000 resolution, with a AGC target of 10e5. 75 windows windows of 8 m/z were used with a MS1 scan every 200 m/z. The raw files were the analysis with Spectronaut 16 software.

Digests were loaded onto Evotips (Evosep, Odense, Denmark) according to the manufacturer’s instructions (Bache et al., 2018 (link)). Evotips were soaked in isopropanol and sequentially washed with 20 μl acetonitrile with 0.1% formic acid, equilibrated with 20 μl water with 0.1% formic acid (solvent A), loaded with 20 μl sample, washed with 20 μl solvent A and loaded with 100 μl solvent A. Tips were centrifuged for 60 s at 700 × g between each loading step. The Evotips were submerged in 0.1% formic acid on a 96-wells plate during sample loading to prevent drying. After the last step, tips were centrifuged for 20 s at 700 × g. LC was performed by the Evosep One (Evosep, Odense, Denmark) using the manufactures separation method of 11.5 min (100 samples/day) (Bache et al., 2018 (link)). The Evosep One was coupled to an Orbitrap mass spectrometer (Q Exactive HF Hybrid Quadrupole-Orbitrap, Thermo Fisher Scientific, Bremen, Germany). The Q Exactive HF system was operated in PRM mode using the following parameters: 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 60,000 at 200 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.