Submitted to: Mass Spectrometry
Publication Type: Peer reviewed journal
Publication Acceptance Date: 5/20/2001
Publication Date: N/A
Citation: Interpretive Summary: Neospora caninum is a protozoan parasite which is a major abortifacaent in dairy and beef cattle worldwide, as well as a cause of neurological disorders in dogs and horses. Multiple geographic and host isolates have been identified, and dif- ferences between the isolates have been noted in infectivity and virulence to experimental rodent hosts (Dubey,1999). In vitro studies have suggested that infection of cells by tachyzoites may be mediated by host cell-parasite surface protein interactions, with active penetration of host cells by tachyzoites using conoid-associated proteins (Hemphill, 1996). Differences in infectivity between geographic isolates may be related to dif- ferences in protein expression in the tachyzoite stage. The total expressed protein component, or proteome, of the tachyzoite stage of N. caninum has not been described. Major efforts in the field of parasite proteomics (Banks et al.,2000; Chambers et al.,2000; Gavaghan,2000) have been undertaken in an attempt to relate genomic information to protein products which may have important roles in infection, development, transmission, and resistance to parasite pathogens of humans and livestock (Alaiya et al.,1999; Edgar et al.,2000; Barrett et al.,2000; Gutierrez,2000). In this study, we have used 2-dimensional gel electrophoresis and MALDI- TOF peptide mass fingerprinting to develop protein maps of 6 Neospora isolates. The data were used to compare protein profiles of the 6 Neospora tachyzoite isolates to determine differences in protein expression between the isolates.
Technical Abstract: Six isolates from the genus Neospora (N. caninum NC-1, NC-2, NC- 3, NC-Illinois, and NC-Liverpool, and N. hughesi) were collected from in vitro cell culture, and proteins were extracted using a chaotropic detergent solution. Two-dimensional electrophoresis was carried out on the solubilized proteins using immobilized pH gradient (3-10) strips in the first dimension and 4-12% gradient slab gels in the second dimension. After Sypro Ruby Red staining of focused gels, 2-D electrophoresis protein maps of the 6 Neospora isolates were produced, and 80 protein spots were landmarked and excised from the gels and subjected to in-gel digestion with lysyl endopeptidase. Resulting peptides were analyzed by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) peptide mass fingerprinting for protein identification. The mass spectra data generated were used for comparison of protein profiles to elucidate strain and species differences in protein expression from the 6 Neospora tachyzoite isolates. Three proteins were further analyzed by MS, and 2 were identified based on comparisons of generated fragment ion mass data in database searches.