Computational protocol: Definition of novel cell envelope associated proteins in Triton X-114 extracts of Mycobacterium tuberculosis H37Rv

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Protocol publication

[…] Experiments were performed on a Dionex Ultimate 3000 nano-LC system (Sunnyvale CA, USA) connected to a linear quadrupole ion trap-Orbitrap (LTQ-Orbitrap) mass spectrometer (Thermo Electron, Bremen, Germany) equipped with a nanoelectrospray ion source. The mass spectrometer was operated in the data-dependent mode to automatically switch between Orbitrap-MS and LTQ-MS/MS acquisition. Survey full scan MS spectra (from m/z 400 to 2,000) were acquired in the Orbitrap with resolution R = 60,000 at m/z 400 (after accumulation to a target of 1,000,000 charges in the LTQ). The method allowed sequential isolation of up to five of the most intense ions for fragmentation on the linear ion trap using collision induced dissociation at a target value of 100,000 charges.For accurate mass measurements the lock mass option was enabled in MS mode and the polydimethylcyclosiloxane (PCM) ions generated in the electrospray process from ambient air (protonated (Si(CH3)2O)6; m/z 445.120025) were used for internal recalibration during the analysis []. Target ions already selected for MS/MS were dynamically excluded for 30 seconds. General mass spectrometry conditions were: electrospray voltage, 1.9 kV Ion selection threshold was 500 counts for MS/MS, an activation Q-value of 0.25 and activation time of 30 ms was also applied for MS/MS.The obtained data was searched against the publicly available Tuberculist database version R10 http://genolist.pasteur.fr/TubercuList/ using MASCOT software version 2.1 (Matrix Science, UK). The database was in-house modified to include reversed sequences of the original ORFs in order to determine false-positive thresholds of the Mascot identification engine []. Tuberculist was preferred over secondary annotations performed by independent institutes because previous data from our group demonstrated that the Tuberculist annotation appear to be more reliable []. The criteria for the Mascot search were as follows: Cysteine carbamidomethylation was set as fixed modification, methionine oxidation and N-acetylation (protein) as variable modifications. Up to 3 missed cleavages were allowed. Peptide (precursor) ion mass tolerance was 15 ppm, and the fragment ion tolerance was 0.5 Da. Mascot scoring showed that p > 0.01 was equivalent to a score of 24. The criterion for a positive identification of proteins identified with at least 2 peptides was a minimal score of 24 for each peptide which represents a 1:10,000 false positive rate at protein level. The maximal score for a peptide from a reversed entry of the annotated M. tuberculosis H37Rv database was found to be 31 (data not shown). This was considered as a threshold for false-positive identifications, and all proteins identified in this study with only one peptide were based on a score higher than 37 (25:10,000). No false positive identifications were observed from the reversed database using these criteria. For visualization and validation of spectra, MSQuant version +1.4.2 was used. MSQuant is an open source tool available at http://msquant.sourceforge.net and is widely used for LC-MS/MS data analysis []. [...] Gene and protein sequences were obtained from Tuberculist http://genolist.pasteur.fr/TubercuList/ and BoviList http://genolist.pasteur.fr/BoviList/. Sequences alignments were done using the Blast 2 algorithm http://blast.ncbi.nlm.nih.gov/Blast.cgi. For prediction of lipoproteins, the LipoP algorithm was used http://www.cbs.dtu.dk/services/LipoP/. For detection of potential secreted proteins SignalP version 3.0 was used http://www.cbs.dtu.dk/services/SignalP/. […]

Pipeline specifications

Software tools MSQuant, LipoP, SignalP
Databases TubercuList Genolist
Applications MS-based untargeted proteomics, Protein sequence analysis
Organisms Mycobacterium tuberculosis, Mycobacterium tuberculosis H37Rv, Mycoplasma bovis
Diseases Tuberculosis