NUTRITIONAL PEPTIDOMICS: DISCOVERY, QUANTIFICATION, AND FUNCTIONAL ANALYSIS OF PLANT PROTEIN DERIVED PEPTIDES

NUTRITIONAL PEPTIDOMICS: DISCOVERY, QUANTIFICATION, AND FUNCTIONAL ANALYSIS OF PLANT PROTEIN DERIVED PEPTIDES Sector CHIM/10 - Food chemistry Introduction and aims of thesis The study of bioactive peptides is a central issue in the development of innovative therapies. The increased attention for fresher and ‘greener’ foods and nutraceuticals possessing health-preventing or health-promoting properties makes bioactive peptides suitable candidates for a new era of pharmaceutical products. Analysing and understanding nature and bioactivity of nutritional peptides, typically delivered from parent food proteins, means comprehending an important level of environmental regulation of the human genome: diet is the environmental factor having the most profound life-long influence on health. Although a remarkable progress has been done in protein analysis, as a consequence of proteomic research, and in small molecule analysis, as a consequence of drug discovery/development initiatives, the field of nutritional peptidomic is still quite unexplored and some drawbacks should be addressed. The pharmacological applications of bioactive peptides depend primarily on their ability to be absorbed in order to exert their bioactivity. In addition, it is very likely that peptide sequences are subjected to structural alterations before performing their final activity in vivo due to different events, such as the attack of gastrointestinal enzymes, brush border peptidases, absorption through the intestinal barrier, and attack of intracellular peptidases in the intracellular absorption. Therefore, all of these different aspects about the bioavailability have attracted a growing interest in the last years. In addition, the possibility of the peptides breakdown during the gastrointestinal digestion is one of the most important factor to be considered when evaluating food-derived peptides for the promotion of human health. Chemical stability is also crucial for proper assay development, since these peptides could lose stability when placed in solution or in biological fluids or even before absorption. Once they are delivered, the biodistribution of bioactive peptides may be sometimes hampered as a result of proteolytic attack, primarily due to the action of brush border peptidases overexpressed at the microvilli surface of intestinal cells. However, before evaluating aspects such as the bioavailability, the optimization of hydrolytic conditions and the chemical identification of protein hydrolysates are other important aspects to be highlighted. The broad variety of physiological activities attributed to protein hydrolysates are determined by the type, number, position, and properties of amino acids present in the sequence of bioactive peptides. The optimization of the hydrolytic conditions for the obtainment of bioactive hydrolysates was also addressed in this work. Since peptides derive from proteins, the integration of peptidomics and proteomics methodologies permitted the enlargement of proteomic databank, which may facilitate the improvement of peptidomics platform libraries. In this context, advanced analytical techniques such as those based on mass spectrometry (MS) have emerged as indispensable and irreplaceable tools in the discovery, identification, quantification and functional analysis of bioactive peptides arising from proteolysis. Among the toolkit of techniques developed to investigate proteins at the proteome-wide scale, MS has gained popularity especially because of its ability to handle the hierarchical complexity associated with the biological systems. In addition, MS-based approaches coupled to cell culturing and bioinformatics tools set a new standard in peptide research. Based on these premises, the aim of my PhD project was to set-up and apply MS strategies in order to evaluate: I. The absorption at intestinal level of peptic and tryptic hydrolysates from lupin protein using an in vitro model based on Caco-2 cells, providing for the first time this kind of data on peptides from this seed. II. The modulation of protein-protein interaction (PPI) of proprotein convertase subtilisin/kexin type 9 (PCSK9) with the low density lipoprotein receptor (LDL-R) in HepG2 cells induced by lupin peptides as well as the quantification by MS of the absorbed peptides that had been predicted to be the best inhibitors of the PCSK9-LDLR PPI by molecular modeling. III. The absorption and metabolism of authentic samples of peptides from soy proteins using Caco-2 cells. IV. The extensive investigation of the hempseed proteome including the identification of minor protein components by CPLL methodology. V. The production of some hydrolysates from hempseed protein endowed of hypocholesterolemic properties using different proteases. VI. The enlargement of apricot seed protein databank through an extensive proteome characterization followed by an in silico driven approach for the prediction of the peptides released by simulated gastrointestinal digestion. General conclusion. The results obtained in this work allowed the enlargement of the bioactive-peptide library platform adding a new dimension to the potential health benefits derived from protein from the seeds of different plants of agronomic importance. In details, a growing body of reports on novel peptide sequences and function-structure relationships contributed to the improvement of plant protein and peptide knowledge. Information obtained from characterizing structural components of plant hydrolysates offers useful technological and functional implications for food ingredient formulation or pharmacological use.