Orbitrap fusion tribid

Sixty million cells were lysed in 9 M urea/20 mM HEPES pH8.0/0.1% SDS and a cocktail of phosphatase inhibitors. Six milligrams of protein were reduced with 4.5 mM DTT, alkylated with 10 mM iodoacetamide and then digested with trypsin (1/50, w/w) overnight at 37°C. Metal oxide affinity chromatography (MOAC) was performed to enrich phosphorylated peptides and reduce the sample complexity prior to tyrosine-phosphorylated peptide immunoprecipitation as previously described (46 (link)). Peptides were analyzed in an Orbitrap FusionTM TribidTM mass spectrometer (Thermo Fisher). RAW files were processed in MaxQuant version 1.6.0.1 using default setting if not stated otherwise (47 (link)). Peptides and proteins were identified with a target-decoy approach in revert mode using Andromeda search engine integrated into the MaxQuant environment. The search was performed against the human UniProt FASTA databased (April 2016). Oxidized methionine, protein N-acetylation and serine/threonine/tyrosine phosphorylation were selected as variable modifications and carbamidomethyl cysteine was selected as fixed modification. Enzyme specificity was set to trypsin and up to two missed cleavages were allowed for protease digestion. The false-discovery rate (FDR) at the protein, peptide and modification levels was set to 1%. The “match between runs” option was enabled.