Proteomic investigation and characterization of cystatin B interactome in saliva of patients with Alzheimer’s disease

Alzheimer’s disease is the most prevalent neurodegenerative disease in the elderly, characterized by accumulation in the brain of misfolded proteins, inflammation, and oxidative damage leading to neuronal cell death. By considering its diagnosis is still heavily based on the analysis of cerebrospinal fluid, the need of finding new non-invasive biomarkers has been pointed. In the context of biomarker discovery proteomics-based, mass spectrometry techniques represent one of the most used and powerful applications to study complex protein mixtures and to define protein profiles in different tissues and body fluids. According to literature, the possible use of saliva as a diagnostic tool has been explored in several oral and systemic diseases. Indeed, saliva comes across as one of the less invasive collectable biofluids suitable for the research of diseases’ biomarkers, especially due to the possibility to detect and measure many proteins and peptides which are expressed in tissues, cell-types and biofluids different from the oral cavity, including the brain. Thus, the aim of the present thesis has been to investigate the salivary proteome profile of Alzheimer’s disease patients in comparison with healthy controls, to evidence possible qualitative/quantitative variations associated with the disease (Part I). Having a cohort of healthy subjects age and sex matched was extremely important because salivary proteome composition is influenced by the process of aging as it has been proven from childhood to adulthood. However, little is known about the changes in saliva protein composition in advanced age. For this reason, a statistical comparison between a cohort of young adult controls and a cohort of old adult controls in relation with Alzheimer’s disease patients has been performed (Part II). To reach these goals, a top-down proteomic approach through HPLC-ESI-IT-MS and HPLC-ESI-high-resolution-MS/MS on the acid-soluble fraction of saliva has been chosen. Among the results obtained, the potential role of cystatins in neurodegeneration diseases appeared to agree with previous findings and we also observed the evidence of aggregates of cystatin B in saliva. Thus, we investigated and characterized the interactome of cystatin B in whole saliva for the first time through a Co-Immunoprecipitation assay followed by bottom-up proteomic approach using a nanoHPLC-ESI-high-resolution-MS/MS system (Part III).