Computational protocol: Profiling the Trypanosoma cruzi Phosphoproteome

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Protocol publication

[…] Proteins were identified by automated database searching (Mascot Daemon, Matrix Science) against a T. cruzi protein sequence database, containing 19,615 protein sequences downloaded from GeneDB, and a ‘decoy database’, prepared by sequence reversing each entry of the amino acid sequence from the genome annotation. This database was complemented with frequently observed contaminants (porcine trypsin, Achromobacter lyticus lysyl endopeptidase and human keratins) and their reversed sequences as well. Search parameters specified a MS tolerance of 5 ppm, a MS/MS tolerance at 0.5 Da and full trypsin specificity, allowing for up to three missed cleavages. Carbamidomethylation of cysteine was set as a fixed modification and oxidation of methionines, N-terminal protein acetylation and N-pyroglutamate were allowed as variable modifications. The peak list generation for Mascot searches, as well as protein validation, protein grouping and phosphosite localization, were done using MaxQuant . The FDRs were calculated based on the number of reverse hits from the searches against the decoy database. Peptides were required to have at least 6 amino acids in length, and a FDR of 0.01 was applied at the levels of peptides, proteins and phosphorylation sites. As described previously, a separate FDR calculation is necessary for substoichiometric modifications (e.g. phosphotyrosines), decreasing false positives and therefore avoiding overestimating the occurrence of such modifications in the sample . This calculation is incorporated at MaxQuant platform. Only the phosphorylation sites PTM with a score higher than 0.75 and a delta score higher than 5 were considered. The comparison against the data described here and the publication from Nakayasu was done using only the phosphorylation sites that have been localized by that paper for just one position.To make our data accessible to the scientific community, we uploaded our study to the data repositories PHOSIDA (, Proteome Commons ( and TriTrypDB (, enabling researchers to access information about the phosphorylation sites identified here. […]

Pipeline specifications

Software tools Mascot Server, MaxQuant
Databases PHOSIDA TriTrypDB GeneDB
Application MS-based untargeted proteomics
Organisms Trypanosoma cruzi
Chemicals Phosphopeptides