The proposed studies are designed to evaluate new methods using a mass spectral readout to provide sensitive, detection of selected proteins, as well as methods to monitor post-translational modification events of targeted antigens. The proposed assay involves an antigen capture step mediated by immobilized antibody (immuno) and an analytical step involving mass spectral analysis of bound antigen (proteomics). The goal of the project is to develop rapid sensitive methods of antigen capture from complex mixtures of unrelated proteins in a maimer that permits the subsequent precise molecular weight determination of the bound antigen using time of flight mass spectrometry. The ability to distinguish subtle variation in the size of a targeted antigen will allow analysis of post-translational modification events for any targeted antigen to be achieved. In addition, the ability to obtain semi-quantitative data based on the area under a specific molecular weight peak on the mass spectral read-out will be critically evaluated. The proof of concept studies will focus on a number of properties of the secreted streptococcal cysteine protease SpeB which is known to post-translationally modify the surface anti-phagocytic M protein, and degrade the secreted bacterial plasminogen activator SK.
