Computational protocol: Conservation of Transit Peptide-Independent Protein Import into the Mitochondrial and Hydrogenosomal Matrix

Similar protocols

Protocol publication

[…] Raw files were processed with MaxQuant (version 1.4.1.2, Max Planck Institute for Biochemistry, Munich, Germany) for protein and peptide identification and quantification with default parameters if not otherwise stated. Searches were carried out using T. vaginalis protein sequences from TrichDB (release 1.3 from 26.5.2011 including 59,672 protein entries; ) applying the following parameters: Mass tolerance precursor (Orbitrap): 20 ppm first search, 4.5 ppm second search; mass tolerance fragment spectra (linear ion trap): 0.5 Da (linear ion trap); fixed modification: Carbamidomethyl (C), nicotin (K); variable modifications: Mthionine oxidation. Searches with protease-specific cleavage (depending on the used enzyme GluC, ArgC, maximum of two missed cleavage sites) were used with specific cleavage and in an alternative setting with N-terminal semispecific cleavage.For peptide and protein acceptance, the false discovery rate (FDR) was set to 1%, and only proteins with at least two identified peptides were used for protein assembly. Quantification was carried out using the label-free quantification algorithm implemented in MaxQuant using a minimal ratio count of 2 and the “match between runs” option enabled. Alternatively, raw files were further processed for protein and peptide identification using Proteome Discoverer (version 1.4.1.14, Thermo Scientific) connected to a Mascot server (version 2.4.1, Matrix Sciences, London, UK) with default parameters for spectrum selection. Searches were carried out using 59,672 protein entries and protein sequences from TrichDB (release 1.3 from 26.5.2011) applying the following parameters: Mass tolerance precursor (analyzed in the Orbitrap part of the instrument) 10 ppm, mass tolerance fragment spectra (analyzed in the linear ion trap) 0.4 Da, enzyme-specific cleavage with a maximum of one missed cleavage site and N- and C-terminal semispecific cleavage specificity, carbamidomethyl at cysteines and nicotine at lysines as fixed modification and methionine oxidation. For peptide and protein acceptance, the “Percolator” function with a Target FDR set to 1% and validation based on q-value was used. Only peptides with high confidence (FDR < 1%) were used for protein assembly. Protein grouping was enabled. Net charge of peptides was analyzed using the EMBOSS package pepstats (). […]

Pipeline specifications

Software tools MaxQuant, Proteome Discoverer, Mascot Server, EMBOSS
Databases TrichDB
Application MS-based untargeted proteomics
Organisms Saccharomyces cerevisiae, Trichomonas vaginalis