Bottom-up proteomics: investigating the potential of the salivary proteome as a source of Schizophrenia biomarkers, and exploration of the protein composition of the oral mucosal pellicle

The work presented in this thesis describes the bottom-up mass spectrometry-based techniques which have been applied to saliva samples in order to identify potential biomarkers of Schizophrenia and to characterize the protein composition of salivary film adhering to oral surface. Bidimensional electrophoretic analysis of 17 saliva samples from adult schizophrenic subjects and 17 healthy subjects, combined whit mass spectrometry allowed the identification of different glycolytic enzymes, such as phosphoglycerate kinase 1, glyceraldehyde-3-phosphate dehydrogenase and alpha enolase, with a reduced concentration levels in schizophrenic patients. The altered levels of the glycolytic enzymes may lead to an imbalance in the pathways involved in energy production and in a massive oxidative stress that appears to be a central feature in schizophrenia. In addition, also annexin A2 showed a reduced level in schizophrenic patients and its possible role as a ROS scavenging molecule, due to the presence of a -Cys reactive residue in its structure, may be relevant in schizophrenia since this disease is often associated with a high oxidative stress. Protein S100 A12, often found increased in the serum of patients suffering from neurodegenerative or inflammatory diseases, displayed an increased level in our analysis of schizophrenic saliva samples, in accordance with the data previously obtained by the top-down proteomic analysis on the acid-soluble fraction of saliva. Interleukin-36-α, a potent activator of various chemokines and cytokines such as IL-6, often present at higher concentration in the serum of schizophrenic patients, showed increased levels in our analysis of schizophrenic saliva samples. Oral mucosal pellicle is a thin layer of salivary proteins lining epithelial oral cells. This layer is involved in oral health by protection from bacterial colonization and maintaining lubrication and it also participates to taste perception. In terms of protein composition, current data based on experiments in vivo highlight the presence of specific salivary components such as S-type Cystatins, Mucins (5B and 7), CAVI and secretory IgA. However, to our knowledge, the mucosal pellicle composition has not been thoroughly described using a proteomic approach. To perform such a characterization, we used a cell model based on TR146 cells that are suitable as a model of oral epithelium. This cell line was stably transfected in order to express MUC1, which can improve MUC5B adhesion on buccal cells. This model presents the advantage of forming the mucosal pellicle if exposed to human saliva. The aim of our work was therefore to isolate and characterize the protein composition of the mucosal pellicle formed in vitro on both TR146 and TR146/MUC1 cells, and to compare by nano-LC MS/MS the mucosal pellicle proteome with the salivary proteome. In this work we present a suitable method for the in vitro isolation of the mucosal pellicle by “shaving” it from the cells using trypsin, which also detaches the cells from the culture plate but does not induce cellular lysis, allowing the subsequent separation of cells and the pellicle. Our analyses on pellicles proteome confirmed the presence of specific salivary proteins in the pellicle like MUC7, but also revealed the adsorption of other proteins onto cells such as BPI fold-containing family B member 1, which was not previously reported in the pellicle composition. To conclude this thesis presents the proteomic mass spectrometry analysis applied to the study of human saliva which demonstrates the versatility of mass spectrometry and has highlighted areas of clinical medicine and oral health where proteomics and a personalized biomedical approach could be further investigated.