Our goal is to work cooperatively with the U-01 consortium to make the first draft of the human salivary proteome. Our group brings a great deal of relevant expertise to this project. For example, we have been using mass spectrometry (MS) approaches to carry out a detailed structure-function analysis of the human salivary glycome. Additionally, we have already completed an MS-based proteome cataloguing project in which we studied the effects of hypoxia on placental cells. Due to our longstanding interest in salivary biology, we began to compile the human salivary proteome. Initial data suggest that the results will be very interesting as we identified novel components including an amazing array of peptides. Accordingly, we now propose the following work plan. Our overall strategy is to perform a detailed analysis of submandibular/sublingual and parotid saliva samples collected as the ductal secretions from a single (blood) type O, Le (a-b-) donor. Our previous work showed that these individuals have the fewest salivary glycoforms, greatly reducing the complexity of the analyses. The sample preparation methods we propose include complementary approaches for subfractionating proteins by molecular weight and charge. Strategies for depleting major salivary components and selectively purifying minor species are also an important part of our plan. Sample separation methods will include 2D SDS PAGE and high performance liquid chromatography (HPLC). For protein identification, we will employ MS-based approaches (e.g., MALDI TOF MS, LC MS/MS). We also propose strategies for cataloguing the phosphorylated and glycosylated species, as well as for identifying protein-protein interactions. As a first step toward investigating the biological activities of salivary proteins, we will use recently developed software programs to make predictions about the structure of the molecules in our catalogue, both individually and in complexes. Finally, we will begin to address the issue of individual variation; saliva samples collected from additional (blood) type O, Le (a-b-) donors will be analyzed by a subset of the aforementioned approaches together with differential labeling techniques. As data emerge from these studies we will collaborate with the UCSF Library/Center for Knowledge Management (CKM) to design a Web-based system for sharing this information with the research community. Experimental procedures and the data they produce (e.g., gel spots, chromatographic profiles) will be linked to mass spectra and other pertinent information. Thus, at the conclusion of the proposed experiments we will have made the first compilation of the human salivary proteome. We envision that the data from this project will have important relevance to on-going efforts to improve oral health.
