Quantitative Proteomics of Human Samples

Proteomics sample preparation and analysis were performed as previously described.^{148 (link)} Briefly, protein precipitate was sonicated and reduced using DTT before being digested by sequence-grade trypsin and desalted. Peptides were TMT labelled using the TMT 10-plex isobaric label reagents (Thermofisher) and fractionated on a Waters XBridge BEH C18 (3.5 μm, 4.6 × 250 mm) reverse phase column using an Agilent 1100 HPLC system. Phosphopeptides were enriched using titanium dioxide coated magnetic beads. Fractionated samples and bead-enriched samples were individually loaded onto a C18 trap column (3 μm, 75μm × 2 cm, Thermo Fisher Scientific) connected in-line to a C18 analytical column (2 μm, 75 μm × 50 cm, Thermo EasySpray) using the Thermo EasyLC 1200 system. Peptides were ionized using a nanospray ion source into a Q-Exactive HF mass spectrometer (Thermo Fisher Scientific). MS/MS spectra were searched against the Uniprot human complete proteome FASTA database downloaded on 2018_10_26, using the MaxQuant software (Version 1.6.7.0) that integrates the Andromeda search engine.