Saliva is a protein rich fluid bathing the mouth that has multiple functions: digestion, lubrication, antimicrobial for example. As we age many people experience xerostomia or dry mouth either as a regular consequence of the ageing process or through intake of medication. Many studies of the composition of saliva have been made (e.g. from our group 1-4). Few have looked at changes in complexes in saliva however previous work by Moffa et al (5) has demonstrated that changes in the partners for anti-fungal salivary protein histatin-5 has consequences for the anti-fungal properties of histatin-5. There is very little information on how saliva changes over the life course, and particularly for protein complexes. Understanding of the function of complexes and how those may change could help with maintenance of healthy oral functions into older age.
The aim of this project is to develop methods to explore protein complexes in saliva and to discover how those change across the life course in healthy human saliva donors. The objectives will be to:
• Develop mass spectrometry-based analysis of protein complexes in saliva using offline size exclusion chromatography to separate endogenous complexes
• Develop quantitative methods to explore how the protein complexes in saliva change with age and or medication
• Verify protein complexes through complementary techniques, such as Western blotting and co-immunoprecipitation
• Develop methods to understand how changes in complexes contribute to changes in saliva function, such as rheology and antibacterial assays
Methodology:
Saliva will be sourced from the Dentistry Research Tissue Bank at the Birmingham Dental Hospital, under existing ethical approval.
Saliva complexes will be explored using a mass spectrometry-based approach after isolation using size exclusion chromatography. Preliminary analysis in our labs suggests complexes can be detected following methods developed by Kirkwood et al (6). Size exclusion columns, such as Superose 12 10/300 GL, are already available in the labs and access to mass spectrometers will be through the Advanced Mass Spectrometry Facility (AMSF) at the University of Birmingham. The AMSF manages use of 4 high resolution mass spectrometers, such as the QExactive and Eclipse insturments, which will be appropriate for this research.
Quantitative analysis using heavy labelled cross-linking chemistry (such as BS3 (bis(sulfosuccinimidyl)suberate)) will be investigated and developed. Cross linking mass spectrometry is a rapidly expanding field: previous work using BS3 isomers has shown conformational changes in proteins in biological samples (7). Novel search engines for processing data have been published (eg 8) and also established software such as Proteome Discoverer have built in functions. The utility of these with the data generated will be evaluated to find the optimum methods.
Further characterisation of proteins will be made through techniques such as Western blotting, co-immunoprecipitation, functional assays and assessment of post translational modifications (eg 9). Saliva proteins have a wide range of functions and therefore there is scope for many different assays in the later stages of the research.
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