2.7. Protein Identification

Andromeda search engine integrated in MaxQuant version 1.3.05 programme was used to identify the proteins in the Uniprot database (www.uniprot.org/downloads, November 2015) and supplemented with sequences of frequently observed contaminants. A mass tolerance of 6 ppm for the parental peptide and 0.5 Da for fragmentation spectra and a trypsin specificity allowing up to 2 mis-cleaved sites were set as the Andromeda search parameters. Fixed modifications of carboxyamidomethylation of cysteines and variable modifications of oxidation of methionine, deamidation of glutamine and asparagine were set. A minimal peptide length of 7 amino acids was set. MaxQuant performed an internal mass calibration of measured ions and peptide validation by the target decoy approach as described. Proteins and peptides with a better than 1% false discovery rate (FDR) were accepted if they had been identified by at least 2 peptides in one of the samples. Shared peptide sequences (razor peptides) were mapped to proteins by the principle of maximum parsimony in MaxQuant. Proteins were quantified by normalized summed peptide intensities computed as label free quantification (LFQ) values in MaxQuant 1.3.05  (Cox et al., 2014) LFQ data was generated in triplicate for all samples. LFQ data was generated in triplicate for all samples.