Protein Identification and Label Free Quantitation

DDA raw MS/MS spectra was processed with MaxQuant (version 1.6) for protein identification and quantitation (Cox and Mann, 2008). Peptide identification by the Andromeda search engine was based on the in-house POTbaseMS database (Moreno et al., 2018). The following parameters were applied to the analysis: 10 ppm peptide mass tolerance; 0.8 Da MS/MS tolerance; Trypsin was specified as enzyme and a maximum of two missed cleavages were allowed; a decoy database search with a 1% FDR cutoff on the peptide and protein level; carbamidomethylation of cysteine was set as a fixed modification, while the oxidation of methionine was set as variable modification. The “label-free quantification” and “match between runs” settings were also highlighted in the software. Valid peptides were expected to have a minimum length of six amino acids. Peptide quantitation was performed for proteins identified with at least two peptides (a minimum of one unique and one razor) unmodified peptide. Peptides intensity was taken and further normalized by LFQ algorithm. Known contaminants and reversed hits were removed from the analysis.