Proteomics and protein activity profiling: an investigation into the salivary proteome and kinase activities in various systems using mass spectrometry
McAllister, Fiona E.
Protein identification and quantitation using mass spectrometry has evolved as the dominant technique for studying the protein complement of a system: cell, tissue or organism. The proteomics of body fluids is a very active research area as there is great potential for protein biomarker discovery; application of such technologies would revolutionise medical practice and treatment. Saliva, through its non intrusive nature of sampling, is an ideal body fluid for disease diagnosis, screening and monitoring. Gingivitis is a gum disease with symptoms including bleeding, swollen, and receding gums. After dental decay, gingivitis is estimated to be the most common disease worldwide, and around 40% of the population in the US are reported to have gingivitis. The end point goal of this project was to identify salivary biomarkers for gingivitis. This dissertation presents an investigation of: 1) the salivary proteome; 2) developments and applications of a mass spectrometry kinase assay; and 3) salivary biomarkers for gingivitis using proteomics and kinase activities. The soluble portion of the human salivary proteome (saliva supernatant) has been studied by several research groups but very few proteomic studies have been performed on the insoluble, cellular and bacterial portion of saliva. Presented here, is the first global proteomics study performed on the saliva residue and supernatant from the same test subject. A total of 834 and 1426 proteins were identified in the saliva supernatant and residue, respectively. A global analysis of protein complexes in saliva was also performed and is the first study, to date, of such an analysis. KAYAK (‘Kinase ActivitY Assay for Kinome analysis’) was further developed for its application on a number of cell types, tissue types, and a variety of organisms. Proof of concept work for in-gel kinase activity/kinase abundance correlation profiling using blue native gels was performed, and experiments using anion exchange chromatographic kinase activity/kinase abundance correlation profiling were performed to identify kinase-substrate pairs. KAYAK applications included the analysis of kinase activities in Saccharomyces cervisiae, Drosophila, mouse, and human saliva in which significant kinase activity was detected in the saliva supernatant, a novel finding. Finally, gingivitis was induced in patients, and the saliva samples were analysed using proteomics and kinase activity profiling. Although this work is ongoing, preliminary data indicate that there are increases in various inflammatory proteins, certain bacteria and also in the activity of particular kinases as a result of the induction of gingivitis. The overall study provided insights into the salivary proteome for both the human and bacterial complement, as well as discovering the presence of significant kinase activity in saliva. In the induced gingivitis study, almost half of all the proteins identified in the residue were from bacteria (1274 bacterial proteins, 198 species identified) and there may be more potential for biomarker discovery for certain diseases in the saliva residue than in the supernatant. A very large overlap was observed between the human proteins in the saliva supernatant and residue, indicating that many of the salivary proteins originate from lysed cells. The origin of the kinase activity in the saliva supernatant is not known but is also proposed to originate predominantly from lysed cells. A range of novel KAYAK applications have been investigated, demonstrating that KAYAK has a wide variety of future uses ranging from target compound evaluation in Pharmaceutical companies to patient testing in the clinic.