Proteomics analysis

For each sample, a single stacking gel band containing all proteins was reduced with DTT, alkylated with iodoacetic acid and digested with trypsin. Extracted peptides were re-solubilized in 0.1% aqueous formic acid and loaded onto a Thermo Acclaim PepMap™ precolumn (ThermoFisher, 75 µM ID X 2 cm C18 3uM beads) and then onto an Acclaim™ PepMap™ EASY-Spray (ThermoFisher, 75 µM X 15 cm with 2 µM C18 beads) analytical column separation, using a Dionex Ultimate 3000 uHPLC at 250 nl/min with a gradient of 2–35% organic (0.1% formic acid in acetonitrile) over 2 h. Peptides were analyzed using a Thermo Orbitrap Fusion mass spectrometer operating at 120,000 resolution for MS1 with HCD sequencing at top speed (15,000 resolution) for all peptides with a charge of 2+ or greater. The raw data were converted to mgf format (Mascot generic format) for searching using the Mascot 2.5.1 search engine (Matrix Science) against human protein sequences (Uniprot 2020) or a protein FASTA database containing the wildtype and mutated forms of human MGP. The database search results were loaded onto Scaffold Q+ Scaffold_4.4.8 (Proteome Sciences) for statistical treatment and data visualization. Pinnacle (Optys Tech) was used to quantify all detected peptides using a MS1 quantification workflow (Targeted Quantification: Label Free DDA) wherein the peptide specific XICs from the raw mass spec data (.raw) were used to directly compare all identified peptide (*.dat) amounts across all experiments using precursor ion integrals (in counts). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (dataset identifier PXD043374). URL:https://www.ebi.ac.uk/pride/.