Mass spectrometry analysis

Peptides were resuspended in sample loading buffer (2% acetonitrile and 0.05% TFA) and separated on an UltiMate 3000 RSLCnano HPLC system (ThermoFisher) hyphenated to a hybrid quadrupole-orbitrap mass spectrometer Q Exactive HF-X (ThermoFisher). First, the peptides were desalted on a reverse-phase C18 pre-column (Dionex, 5 mm × 0.3 mm ID) for 3 min. Subsequently the pre-column was switched in line with the analytical column (300 mm × 0.075 mm ID) packed in-house with ReproSil-Pur C18 AQ 1.9 μm reversed-phase resin (Dr. Maisch GmbH). Solvent A consisted of 0.1% formic acid in water, and solvent B consisted of 80% acetonitrile and 0.1% formic acid in water. Peptides were eluted using a linear gradient of 5–42% B over 166 min at a flow rate of 300 nL/min. The temperature of the pre-column and the column was maintained at 50 °C. The mass spectrometer was operated in Top30 data-dependent acquisition mode, where the most intense 30 precursors within the m/z range of 350–1500 were selected for MS2 fragmentation with a Normalized Collision Energy of 28%. MS2 spectra were acquired in the Orbitrap at a resolution setting of 15,000 FWHM (Full Width Half Maximum), a maximum ion Trap Fill Time of 60 ms and an AGC target of 1.0e⁵, respectively. Dynamic Exclusion was set to 45 s.

MS/MS spectra were searched against a UniProtKB/Swiss-Prot human database containing 134,921 protein entries supplemented with 245 frequently observed contaminants collated with the Andromeda search engine²⁴. Precursor and fragment-ion mass tolerances were set to 6 and 20 ppm, respectively, after initial recalibration. Protein N-terminal acetylation and methionine oxidation were allowed as variable modifications. Cysteine carbamidomethylation was defined as a fixed modification. Minimum peptide length was set to seven amino acids, with a maximum of two missed cleavages. The false discovery rate was set to 1% at both the peptide and the protein level by using a forward-and-reverse concatenated decoy database approach. For SILAC quantification, multiplicity was set to two-channel (Lys + 0/Arg + 0, Lys + 8/Arg + 10) labeling. At least two ratio counts were required for peptide quantification, and the “re-quantify” option was enabled. All raw files and MaxQuant search results have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD014297²⁵.