Cipolletta, Brunella (2025) Development and application of innovative chemical methodologies for diagnostics in cultural heritage. [Tesi di dottorato]

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Abstract

The chemical investigation of cultural heritage (CH) materials represents one of the most challenging and interdisciplinary areas within analytical sciences.1 Works of art and archaeological artefacts are complex, multicomponent systems composed of organic and inorganic substances that have interacted and transformed over centuries. Understanding their molecular composition and degradation pathways is fundamental to reconstruct manufacturing and artistic techniques and to develop scientifically informed conservation strategies.2,3 However, CH materials pose major analytical challenges due to their heterogeneity, the extremely low concentration of target analytes, pervasive contamination and degradation, and strict sampling limitations imposed by the uniqueness and fragility of the artefacts.2,4 The overarching aim of this doctoral research was the development and application of innovative mass spectrometry (MS)–based analytical strategies for CH diagnostics, with particular emphasis on organic materials. The project was structured around two complementary objectives: (1) the identification of ancient proteins and organic molecules in historical and archaeological samples through highly sensitive, selective, and minimally invasive workflows, and (2) the chemical characterization of proteinaceous materials to elucidate degradation mechanisms, pigment–binder interactions, and the effects of manufacturing and environmental factors on long-term material evolution. In the first research line, several MS-based workflows were developed and applied to diverse case studies, including archaeological pottery residues, historical iron-gall inks, restoration adhesives5, and ancient proteinaceous fibers. These studies combined gas chromatography–mass spectrometry (GC–MS), liquid chromatography–tandem mass spectrometry (LC–MS/MS) proteomics, and complementary spectroscopic and microscopic techniques, demonstrating that multi-analytical strategies enable comprehensive molecular fingerprinting of complex historical matrices while maintaining minimal invasiveness. Tailored extraction and solubilization protocols were established for highly degraded and carbonized materials, enabling, for example, the molecular identification of ancient silk proteins from Vesuvian archaeological textiles. The research further advanced minimally invasive proteomics through the development of a novel workflow integrating minimally-invasive sampling using a trypsin-functionalized cellulose acetate sheet6,7 with direct MALDI–mass spectrometry imaging (MALDI–MSI). This approach established a new paradigm for non-destructive, spatially resolved proteomic analysis of surfaces and cross-sections of works of art. The second research line focused on the chemical characterization of proteinaceous materials to elucidate their degradation and ageing behavior. An analytical and computational pipeline was implemented to semi-quantitatively assess post-translational and diagenetic modifications in proteins and to visualize the distribution of chemical changes along their sequences. This approach supported the first proteomic investigation of pigment–protein binder interactions in casein-based paint reconstructions, revealing pigment-specific effects on casein degradation pathways. A limited proteolysis mass spectrometry (LiP–MS) method was further proposed to probe pigment-induced structural alterations, extending proteomic analysis toward conformational and reactivity studies in complex heritage materials. Complementary studies on collagen-based materials—including historical glues and novel bioadhesives—demonstrated that chemical treatments leave diagnostic molecular fingerprints detectable through MS-based proteomics. Comparative analyses linked specific modification patterns (deamidation, oxidation, backbone cleavage) to manufacturing chemistry and degradation conditions, providing a molecular framework for evaluating collagen’s adhesive performance and stability.8 Overall, this doctoral research advanced the analytical and interpretative framework for the molecular investigation of organic CH materials. By integrating multi-analytical approaches, minimally invasive proteomics, and advanced computational pipelines, it achieved significant progress in adapting MS methodologies to the unique constraints of heritage science.

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