Computational protocol: Identification of proteins differentially expressed by Chlamydia trachomatis treated with chlamydiaphage capsid protein VP1 during intracellular growth

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Protocol publication

[…] Protein identification was performed by means of the MaxQuant software with integrated Andromeda search engine (v1.3.0.5). The tandem mass spectra were searched against Chlamydia trachomatis serovar E (strain E/11,023) UniProt reference proteome database (926 sequences) concatenated with a reverse decoy database and protein sequences of common contaminants. Trypsin/P was specified as the cleavage enzyme allowing up to two missing cleavages, three modifications per peptide, and five charges. Mass error was set to 6 ppm for precursor ions and 0.02 Da for fragment ions. Oxidation on Met and acetylation on protein N-terminal were specified as variable modifications. The FDR (False Discovery Rate) thresholds for protein, peptide, and modification site were specified as 0.01. The minimum peptide length was set as 6. [...] The label-free quantification for identified proteins was performed with the Max LFQ software integrated into the MaxQuant software. In addition to the parameter settings mentioned above, LFQ quantification was enabled by checking “Match between runs,” “LFQ”, and “iBAQ.” All the other parameters in MaxQuant were set to their default values. After MaxQuant processing, the proteinGroup.txt file was loaded and analyzed by the Perseus software (v1.5.0.31). Briefly, the LFQ intensity values of two samples were transformed logarithmically. The missing LFQ value was replaced by normal distribution to simulate the background LFQ intensity level for undetected protein abundance values. The relative protein ratio was calculated by exponentially transformation of difference value between logarithmically transformed LFQ values of two samples. […]

Pipeline specifications

Software tools MaxQuant, Andromeda, Perseus
Application MS-based untargeted proteomics
Organisms Cavia porcellus
Diseases Chlamydia Infections, Infection, Sexually Transmitted Diseases