Saliva is a complex mixture of proteins, lipids and carbohydrates in a physiological buffer containing millimolar concentrations of inorganic phosphate, divalent cations (calcium) and bicarbonate, which supports intermolecular complex formation, tooth mineralization, dental plaque formation, and passive immunity to pathogens of the oral cavity. We propose a four-year proteomics-based research program with the goal to identify and catalogue the components of salivary secretions and to annotate the catalogue with respect to the biochemical properties of the catalogue's inventory. To accomplish high coverage of the proteome, we will implement two parallel approaches to purify and identify individual salivary proteins. First, conventional protein fractionation will be coupled with either multi-dimensional liquid chromatography-tandem electrospray mass spectrometry (LC-MS/MS) or with 2-dimensional geI-MALDI-TOF mass spectrometry. Because the abundant salivary proteins will obscure the purification and identification of medium and low copy salivary proteins, a second purification technology, based on high-throughput immuno-affinity chromatography, will be implemented to complement and support the conventional mass spectrometry-driven proteomics approach. Specifically, we propose to create a comprehensive library of anti-saliva antibodies, using bacteriophage display, to build multiple probes for each salivary component. These antibody or immune affinity reagents will permit detection, as well as, purification of specific salivary proteins, which will, in turn, permit a high-throughput approach to the characterization of each protein's post-translational modification state, degree of interaction with other salivary components, and affinity for an in vivo tooth model (hydroxyapatite).
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