Skip to main content Skip to navigation

Protein complexes in saliva and how they change over the life course: implications of ageing on saliva function

Primary Supervisor: Dr Melissa Grant, School of Dentistry

Secondary supervisor: Dr Aneika Leney

PhD project title: Protein complexes in saliva and how they change over the life course: implications of ageing on saliva function

University of Registration: University of Birmingham

Project outline:

Background: 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.

References:

  1. Pasha S. et al. The Saliva Proteome of Dogs: Variations Within and Between Breeds and Between Species. Proteomics. 2018 Feb;18(3-4):1700293
  2. Davis I. J. et al. Longitudinal quantification of the gingival crevicular fluid proteome during progression from gingivitis to periodontitis in a canine model. J. Clin Periodontol. 2016 Jul;43(7):584-94
  3. Grant M. M. et al. Proteomic analysis of a noninvasive human model of acute inflammation and its resolution: the twenty-one day gingivitis model. J Proteome Res. 2010 Sep 3;9(9):4732-44.
  4. Leney A.c. & Heck A. J. R. Native Mass Spectrometry: What is in the Name? J Am Soc Mass Spectrom. 2017 Jan;28(1):5-13.
  5. Moffa E. et al. In Vitro Identification of Histatin 5 Salivary Complexes. PLoS One. 2015 Nov 6;10(11):e0142517
  6. Kirkwood K. J. et al. Characterization of Native Protein Complexes and Protein Isoform Variation Using Size-fractionation-based Quantitative Proteomics Mol Cell Proteomics. 2013 Dec; 12(12): 3851–3873.
  7. Belsom A. et al. Serum Albumin Domain Structures in Human Blood Serum by Mass Spectrometry and Computational Biology. Molecular & Cellular Proteomics 2016, 15: 1105–1116.
  8. Pirklbauer G. et al. MS Annika: A New Cross-Linking Search Engine. J. Proteome Res. 2021, 20, 5, 2560–2569
  9. Koro C. et al. Carbamylated LL-37 as a modulator of the immune response. Innate Immun. 2016 Apr;22(3):218-29

BBSRC Strategic Research Priority: Integrated Understanding of Health: Ageing

    Techniques that will be undertaken during the project:

    The student will work both in Drs Grant’s and Leney’s labs.

    The student will gain experience in saliva preparation and storage, protein complex isolation via size exclusion chromatography and protein identification via mass spectrometry, exploring both bottom up and top down techniques. The student will build skills in data analysis, particularly in mass spectrometry, quantitative mass spectrometry and cross-linking mass spectrometry. Further analysis of protein characterisation, including Western blotting may be developed.

    Contact: Dr Melissa Grant, University of Birmingham