MASS SPECTROMETRY BASED PROFILING OF TWO DIFFERENT BIOLOGICAL SYSTEMS FOR POSSIBLE CLINICAL APPLICATION.

Several techniques including two-dimensional electrophoresis, imaging, mass spectrometry, and bioinformatics are used in proteomics to identify, quantify, and characterize proteins for clinical applications. The main aim is to identify proteins involved in pathological processes and to understand how illness can lead to altered protein expression in order to develop new diagnostic and prognostic tests, to identify new therapeutic targets, and eventually to allow the design of individualized patient treatment. For this purpose this dissertation is divided into two main chapters. Chapter one concerns the development of a novel method to isolate exosomes. Normal human urine contains large numbers of exosomes, which are 40 to 100 nm vesicles that originate in multivesicular bodies from every renal epithelial cell type facing the urinary space. Exosomes are rich in potential biomarkers, especially membrane proteins such as transporters and receptors that may be up- or downregulated during disease states. Differential centrifugation methods are commonly used to purify exosomes from urine. Here, we developed a new method to isolate exosomes by solubilizing exosomes in 1 % Sarkosyl, an anphyphilic detergent, followed by a carbon fractionation. We used LC-MS/MS to profile the proteome of human urinary exosomes. Overall, the analysis unambiguously identified 1618 proteins, showing an enrichment of 61.3% in exosomal proteins, after performing a Gene Ontology analysis. Thus, our modified exosome precipitation method is a simple, fast, highly scalable, and effective alternative for the isolation of exosomes. It may facilitate either the identification of exosomal biomarkers from urine or the production of drug delivery devices. Chapter two concerns the identification of proteins in adhesive material of the capsalid Neobenedenia girellae by a proteomic approach based on de novo sequencing and database search to overcome the lack of information concerning the genome of these parasites. Glandular secretions were obtained by a new method, set up in our laboratory, which allowed collecting a small amount of secretion without any contamination from other tissues either from the parasites as well as from the skin of the host. The proteomic analysis reveals that the adhesive is mainly composed of cytoskeletal proteins (actin, keratin and tubulin) but contains also ATP-synthase, 78 kDa glucose regulated protein and albumin. This work reports for the first time the characterization of a novel bioadhesive material used by capsalid parasites to adhere to fish. Such information broadens our knowledge of the molecular mechanisms involved in adhesiveness of parasites to hosts. Moreover, it offers new clues in understanding the mechanism of stickiness and adhesion of cytoskeleton components, often involved in both physiological and pathological processes, including neurodegenerative diseases.