HIGH RESOLUTION MASS SPECTROMETRY STRATEGIES FOR THE IDENTIFICATION OF SMALL AND LARGE BIOACTIVE MOLECULES.

Abstract The aim of the present thesis was to set-up and to apply an analytical approach based on proteomics for the identification of bioactive macromolecules contained in two complex bioactive matrices, bovine colostrum and panax ginseng, that could be exploited for nutraceutical and pharmaceutical applications. The approach starts with proteome characterization, followed by bioinformatics analysis aimed not only at addressing the biological activity of the identified proteins and at establishing protein-protein interaction, but also at predicting the bioactivity of the digested peptides arising from gastro-intestinal digestion. The knowledge generated by such an approach permits not only the production of a safe and high quality/standardized bioactive matrix, but also the set-up of suitable analytical protocols aimed at analyzing qualitatively and quantitatively the bioactive proteins. The goal was reached through an integrated analytical strategy based on a proteomic and bioinformatics approach and by applying innovative techniques for sample preparation, including affinity chromatography and the combinatorial peptide library technology (CPLL or PM, ProteoMiner), aimed at reducing the dynamic range of protein concentrations. In the first part of the work, the proteomic approach was used to generate an exhaustive protein data bank of bovine colostrum, including the minor protein components. By using a bioinformatics approach, a cluster of bioactive proteins effective for treating wound healing was identified. An integrated analytical strategy was then set-up for the quality control of the bioactive protein components of bovine colostrum (part II). Asian ginseng (Panax ginseng C. A. Meyer), one of the most highly regarded of herbal medicines in the Orient, was selected as the second matrix to be studied; a comprehensive proteomic analysis was carried out together with a bioinformatics approach for protein/peptide bioactive constituents (part III). Overall, this work contributes not only to mapping the Bovine Colostrum and Panax Ginseng proteomes, but also to setting-up a suitable analytical platform aimed at achieving a high standardization and an in-depth quality profiling of bovine colostrum in the manufacture of food supplements. The identification of proteins characterized by innovative functions through proteomic and bioinformatic approaches also permits the exploration of novel biological functions of Bovine Colostrum and Panax Ginseng. This innovative approach can be applied to the further exploration of various natural sources of bioactive macromolecules, which are claimed to have health benefits.