A FDR < 3.0% to peptide matches above homology or identity threshold was considered significant. selleck products For Mascot searches, the parameters used were trypsin as the enzyme of choice and one missed cleavage, ± 1 Da for the precursor mass, ± 0.5 Da for the fragment ion mass. Oxidation of methionines along with N-terminal
acetylation of proteins, N-terminal formylation, deamidation and cyclization of glutamine (pyro-glutamate) were allowed as possible modifications whereas alkylation of cysteines (carbamidomethylcysteines) was set as constant modification. Identification was considered valid for Mascot protein scores greater than 30 and a significance threshold of p < 0.05. If a protein 'hit' was identified by only one peptide, the MS/MS data was to exhibit a clear spectrum with sequence tags that matched at least three consecutive y or b fragment ion series. Finally, a good correlation between the experimental and theoretical molecular mass and pI was also considered for positive identifications. Putative signals for protein export were predicted SRT2104 supplier using SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/), LipoP 1.0 (http://www.cbs.dtu.dk/services/ LipoP/), TatP 1.0 (http://www.cbs.dtu.dk/services/TatP/) and SecretomeP 2.0 (http://www.cbs.dtu.dk/services/SecretomeP/). Potential transmembrane domais
were predicted with TMHMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). Molecular weight (M r) and pI of secreted proteins was calculated with the Expasy compute pI/Mw tool (http://www.expasy.ch/tools/pi_tool.html).
Statistical analysis Spot intensity differences obtained from comparative 2DE gel images of M. bovis BCG strains Moreau and Pasteur were statistically analyzed by one-way ANOVA with Student’s t-test to determine significant differences among group means. Statistical analysis was carried out using the data obtained from 4 different sets of independent biological samples. A p-value ≤0.05 was considered as statistically significant. Acknowledgements We thank Rodrigo Mexas (Laboratório de Produção e Tratamento de Imagem, IOC/FIOCRUZ) for his precious contributions and the FIOCRUZ/PDTIS 2DE and Mass Spectrometry platform Methane monooxygenase facilities (Dr. J. Perales and André Ferreira). Carolina Zavareze (FAP) kindly provided the Sauton culture medium and the BCG Moreau vaccine strain. This work received financial support from the WHO/TDR Special Programme for Research Training in Tropical Diseases and the following Brazilian agencies: CNPq, FAPERJ and PDTIS/FIOCRUZ. Electronic supplementary material Additional file 1: Figure S1 – PCR confirmation of the genetic identity of the BCG strains used. (PDF 275 KB) Additional file 2: Table S1 – M. bovis BCG Moreau culture filtrate proteins identified by MS/MS (PDF 1 MB) Additional file 3: Table S2 – Predicted localization of identified proteins.