2.8. Identification of Peptides and Proteins

The Mass spectrometry data (.raw) were searched against the human RefSeq database (RefSeq94 containing 113,658) appended with 116 frequently observed contaminants using Mascot (v2.2) and SEQUEST HT search algorithms through the Proteome Discoverer platform (v2.2, Thermo Scientific, Bremen, Germany). The search parameters for both algorithms included a maximum of one missed cleavage, oxidation of methionine, phosphorylation at serine, threonine, tyrosine as dynamic modifications and carbamidomethylation at cysteine, TMT 6 plex Lysine and TMT 6 plex N-terminal as static modifications. A precursor mass error tolerance of 10 ppm and a fragment mass error tolerance of 0.05 Da were considered during the analysis. The data were searched against a decoy database and peptides identified at a <1% false discovery rate (FDR) were considered further for protein identification. The phosphorylation probability at each S/T/Y site was calculated using the PTM-RS node in the Proteome Discoverer; peptides with more than 75% site localization probability were considered for further analysis.