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Supervisor: Dr. Ilaria Bonaduce

Title: Protein identification and localization in samples from polychrome works of art

Abstract: The research activity of this thesis is focused at developing, on one side,  a reliable protocol for protein localization in paint cross section, and, on the other, at improving analytical procedures based on mass spectrometry, including proteomics, to identify the biological source of proteinaceous media in samples from polychrome works of art.
Proteins localization. The most readily available method that gives insight into the stratigraphic structure and composition of a polychrome sample is the study of its cross section by optical microscopy (OM). Dyes, fluorescent or lanthanide stains are commonly used to map the proteinaceous materials in each layer of the stratigraphic structure. The limitations of tradition colorimetric stains have led to the development of a number of fluorescent stains with a wide variety of reactive groups that are available for conjugation to protein with highly specificity. FITC (fluoresceine-isothiocyanate ) and AMCA (NHS-succinimydyl-7-amino-4-methylcoumarin-3-acetic ester)  are some examples of fluorescent stains that direct conjugate to primary amines (-NH2) of the proteins by covalent binder. Flamingo and Sypro Ruby are other fluorescent stains commonly used to conjugate to cationic residues of proteins by a non-covalent interaction. Although very useful, these  fluorescent stains are prone to give false positive or negative results. In this thesis fluorescent stains will be systematically investigated in order to individuate the most effective protein affinity tag to localize casein, egg and animal glue paint binders, alone, with pigments or mixed to other organic media. This will be done by using spectroscopic techniques, including fluorescence spectroscopy and laser induced fluorescence spectroscopy (LIF). Sample fixation aimed at ensuring that the protein is not washed away during the labelling protocol, will be investigated as well. Finally possible chemical modifications of the most efficient fluorescent labels will be explored aimed at expanding the possibility of protein localization with other technique, including scanning electron microscopy (SEM) and laser ablation inductively coupled plasma mass spectrometry imaging. This would lead to a more reliable protein localization having removed possible biases due, among the others, to autofluorescence of the organic binder and fluorescence quenching.
Protein identification. The application of bottom-up strategies for proteomic analysis of proteins in art painting samples has expanded greatly in recent years. Nevertheless the characterization of degradation pathways and identification of specific markers which describe the ageing processes are fundamental to ensure a successful identification of a proteins, the knowledge of the modifications on the peptide sequence is actually very little. This will be investigate in the course of this research, as it would led to the production of an implemented database with more possibility of unambiguously identifying the biological source. To this aim, a combination of bottom up and top down proteomic approaches with high- performance liquid chromatography (HPLC) equipped with Orbitrap Fusion and Q-TOF analyzers will be used. These two proteomic approaches are in fact complementary. While the bottom up approach analyzes the sequence modifications of some peptides, the top down allows a better sequence coverage, thus integrating the bottom-up results.
Despite this, one the most critical step remains the proteins extraction and purification. The problem is especially difficult with respect to proteins extracted from aged samples and in presence of some pigments. It is well known fact that the solubility of proteinaceous materials decreases with time and that some cations form strong complexes with proteins. In this context, the use of detergents, urea or chatropic agents will be investigated as the most suitable tools to improve  protein extraction yields in sample collected from polychrome works of art.