Identification of Leukotoxin and other vaccine candidate proteins in a Mannheimia haemolytica commercial antigen

Tucci, Paula; Estevez, Verónica; Becco, Lorena; Cabrera-Cabrera, Florencia; Grotiuz, Germán; Reolon, Eduardo; Marín, Mónica

Inactivated culture supernatant obtained from the industrial process was concentrated 20X by ultrafiltration (Vivaspin 500, 5 kDa, GE Healthcare) and analyzed by SDS-PAGE and coomassie blue staining. The most defined and intense bands were identified by peptide mass fingerprinting at Institut Pasteur Montevideo (Uruguay) using a 4800 MALDI TOF/TOF Analyzer (Applied Biosystems). Peptides from each sample were obtained by trypsin treatment (Sequencing Grade, Promega) overnight at 37°C. Peptide mass spectra were acquired as previously described (Lima et al., 2011) and MS/MS collision induced dissociation experiments of selected peptides were performed. Proteins were identified by NCBInr database searching with peptide m/z values using MASCOT search engine (Matrix Science) (Perkins et al., 1999) with the following searching parameters: monoisotopic mass tolerance, 0.08 Da; fragment mass tolerance, 0.45 Da and one missing tryptic cleavage allowed. Significant scores (p < 0.05) were used as criteria for positive protein identification.