5×1000 Projects

Thanks to the 5×1000 destinations to our Foundation, it was possible to activate the following projects:

Development and application of N-tail terminomics workflows for the characterization of proteolytic events of pathological relevance in ocular diseases

Reference Year 5X1000: 2022

Project Start: 01/01/2024

Project End: 31/12/2025

Project Summary – Abstract: Diseases of ophthalmological relevance, such as glaucoma, diabetic retinopathy, age-related macular generation (AMD), retinal detachment, as well as corneal diseases (e.g. Fuchs syndrome), have a significant impact on the patient’s quality of life, as well as on the social and economic costs borne by national health systems. Although the pathogenesis follows different trajectories, as well as the affected ocular tissues, the onset, but also the progression, of these diseases is shared by alterations in the remodeling of ocular extracellular tissues. However, as with many human pathologies affecting other anatomical districts, the molecular mechanisms underlying these processes are poorly understood. By virtue of these considerations, a fine characterization of endogenous proteolytic fragments, i.e. peptides released by enzymatic digestion of proteins, within ocular fluids, such as aqueous and vitreous humor, represents a promising approach to identify enzymatic degradation phenomena specific to the pathological phenomenon in question. In fact, the occurrence of proteolytic events of pathological relevance to ocular tissues, such as trabeculate, retinal layers, corneal tissues, can be reconstructed, with a reasonable margin of reliability, by examining the peptide fragments of the two fluids, aqueous and vitreous humour in the course of comparative analyses between samples isolated from subjects suffering from a specific disease and control patients (defined on the basis of the pathology under examination). In addition, exploring the repertoire of protein fragments in samples of a cellular nature, such as those identifiable in corneal endothelial sampling, can provide important parameters for correlating the origin of the peptide fragment and the tissue to which it belongs. By virtue of the reduced concentration of proteins, and peptides, within the fluids in question, experimental approaches with high sensitivity represent the only technological solution to provide an answer to the scientific problem posed. In this sense, mass spectrometry is undoubtedly the most suitable approach to obtain the widest possible coverage of biological data. Classic strategies (“shot-gun”) of proteomics for the characterization of the proteome of a biological matrix involve the fragmentation of the proteins contained in it by digestion with trypsin before injecting the sample into the mass spectrometer. This instrument then performs the characterization of the mass (and charge) of the peptides present in solution. Identification is followed by bioinformatics approaches necessary to trace the identification of proteins present in solution. However, this approach does not optimally distinguish peptide fragments derived from endogenous proteolysis from artificial tryptic proteolysis. In order to characterize the endogenous proteolytic fragments, specific proteomics workflows were then elaborated, such as the N-tail terminomics to which this project refers. Conceptually, the approach is based on the chemical labelling, in vitro, of all N-termini of proteins and peptides present in solution prior to trypsin digestion. In this way, endogenous peptide fragments can be chemically protected from further trypsin digestion events in vitro and characterized by conventional mass spectrometer approaches.

Date of receipt of 5×1000 funds: 28/09/2023

Total project cost: 29.703,73

Total 5×1000 shares: 29.703,73

Statement of allocation of resources 5×1000 2022: