6533b7d6fe1ef96bd1266324

RESEARCH PRODUCT

Approches protéomiques en archéologie et héritage culturel : caractérisation de protéines anciennes préservées dans des coquilles de mollusques

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Archaeological mollusc shell artifacts and ecofacts are valuable sources to study past cultures and provide insights on how people exploited their environments. Mollusc shells were often used as raw materials to make personal ornaments and are abundantly found in archaeological sites. However, minute, heavily worked and/or fragmented shell ornaments are rarely identified at different taxonomic levels, due to limited availability of analytical approaches to determine taxon-specific (diagnostic) features. In recent years, proteomics, which exploits the high sensitivity of modern mass spectrometry techniques, has been successfully applied to the study of a variety of bioarchaeological remains, opening a new research field referred to as ‘palaeoproteomics’. While modern mollusc shells represent key study models in biomineralization research to investigate the molecular mechanisms of biocalcification and mineral deposition, palaeoproteomics on archaeological shells has never been carried out before. The challenges are mainly due to the intrinsic physico-chemical features of their substrates (including low protein content), and the paucity of protein sequences for reference.Therefore, this work presents a comprehensive proteomic-based investigation of archaeological mollusc shells, grounded in biomineralization research, and the first application of palaeoproteomics to shells. The main objective of this PhD was to investigate three key concepts: whether shell proteins carry taxonomic information, could be preserved over archaeological times scales and could be used as molecular barcodes for the taxonomic determination of ancient shell artifacts.High performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) was used for an in-depth proteomics characterisation of a selected model, Spondylus gaederopus, demonstrating that its shell-associated proteins are very different from other models and likely represent lineage-specific sequences. Peptide mass fingerprinting (PMF) by MALDI-TOF mass spectrometry was employed to obtain sequence information from thirty-four different molluscan taxa. Intracrystalline shell proteins displayed clearly distinct PMFs indicating that they may encode taxonomic information and could be used as molecular barcodes to identify archaeological mollusc shells. Accelerated aging experiments were performed to mimic the diagenesis of Spondylus shell and the stability of intracrystalline shell proteins was investigated by a combination of immunochemistry and quantitative TMT proteomics. We concluded that Spondylus represents a favourable system for protein preservation, as evidenced by the thermal stability of shell peptide sequences. However, the observed degradation patterns pinpointed the complexity of the whole diagenesis process, which does not entirely follow expected trajectories from theoretical models.Finally, a set of archaeological shell ornaments, recovered from different Mesolithic and Neolithic European prehistoric sites, were studied by proteomics coupled with structural, biomolecular and geochemical analyses. The studies showed that PMF by MALDI-TOF approach was not able to characterise these archaeological samples, which are very degraded or and/or have low protein contents, and the analysis of ancient shell proteins requires higher sensitivity, as offered by HPLC-MS/MS mass spectrometry. Indeed, palaeoproteomic analysis by HPLC-MS/MS of small “double-button” ornaments identified that they were made of freshwater mother-of-pearl shells, resolving the long-standing debate over their biological origin and provenance. The research carried out in this thesis highlights the immense potential of proteomics-based approaches to study archaeological and palaeontological shell remains.