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Paleoproteomics for cultural heritage conservation: biomolecular analysis of ancient Egyptian paint binders

Periodic Reporting for period 1 - EGYPTOMICS (Paleoproteomics for cultural heritage conservation: biomolecular analysis of ancient Egyptian paint binders)

Reporting period: 2017-10-01 to 2019-09-30

A wide part of the artistic production of ancient Egypt civilization is surprisingly still available to us and it is carefully preserved in Egyptian collections spread worldwide. Protein-based materials such as animal glue, milk and egg have been mentioned in classical sources to be used as binding media and adhesives by ancient Egyptians, but a limited number of analytical studies have been conducted so far, leaving several questions open such as: were certain proteins preferred for specific applications? Are there differences/similarities among similar objects from different periods and geographical areas? Can we discriminate original from modern proteins? The correct identification of the proteinaceous paint binder, as well as the characterisation of its molecular damage, are fundamental for understanding the technology behind artistic painting in ancient cultures and for the development of proper conservation treatments compatible with the original materials. In addition, preserving and investigating the museum’s collections is fundamental for the benefit of present and future generations.

The project’s aim is to shed light on ancient Egyptian artists’ technique by identifying the biological species of origin, as well as molecular damage, of proteins used as binding media and adhesive in ancient Egypt painted artefacts dating 3000 BC - 600 AD by applying state-of-the-art high resolution mass spectrometry-based proteomic strategies (palaeoproteomics). The three main objectives were to (i) identify the animal species used to produce proteinaceous binding media and adhesive; (ii) investigate objects of different nature (material, purpose etc.), periods and geographical area to evaluate trends/differences in protein use; and (iii) study the ancient protein degradation profile to discover undocumented modern conservation treatments.
To achieve the above mentioned objectives, a total of 93 micro-samples from 34 different objects, including wooden coffins, cartonnages, and mural paintings, have been obtained from the Egyptian collections of six European and one American museum in order to increase the objects heterogeneity. Collaborations have been personally established with: the museum of mediterranean and near Eastern antiquities (Stockholm, Sweden), the Vatican Museums (Rome, Italy), the Ny Carlsberg Glyptotek (Copenhagen, Denmark), the British Museum (London, UK), the Petrie Museum (London, UK), the Manchester Museum (Manchester, UK) and The Metropolitan Museum of Art (New York, USA). All objects were carefully selected after discussion with curators and conservators and samples were collected by the conservator, or by myself, in correspondence of the areas of interest. This phase required multiple trips to each museum.

All 93 samples have been screened by Fourier transform infrared spectroscopy (FTIR) to investigate the general composition, and a total of 41 samples, the ones showing the possible presence of proteins, have been selected and analyzed by mass spectrometry-based proteomics. Sample preparation included: protein residues extraction, digestion and then analysis of the tryptic peptides by nanoflow liquid chromatography tandem mass spectrometry (nLC-MS/MS). Protein identification was performed with the MaxQuant software. The resulting proteins and their modifications were compared with data from control samples prepared using the same workflow.

Animals such as cattle, domestic pig, horse/donkey and sheep/goat were used to extract collagen and glues show a different nature according to the specific application (e.g. adhesive vs paint binder). In addition the glue was prepared from both bones or soft tissues, it seems again depending on the artistic application. Besides collagens and one case of egg proteins, specific plant seed proteins were unexpectedly identified in two completely unrelated objects. This is the first analytical evidence of the use of seed extracts from two specific plants that were known to be used for other purposes only according to ancient sources. The results demonstrate how palaeoproteomics provides new evidence to advance understanding of the use of specific protein sources which can not be identified with other analytical techniques.

All results have been discussed personally with curators and conservators in order to better correlate the analytical results with what is already known in the Egyptology literature, and a final report for each object have been sent to the museums. In addition the results of the research have been presented at several international conferences:

C. Granzotto et al., ‘Paleoproteomic analysis of binding media and adhesives in ancient Egypt’, oral presentation, Ancient Proteins @20, Copenhagen, August 20th-22nd, 2018.
C. Granzotto et al., ‘Which is the species? Paleoproteomics applied to painted artifacts from ancient Egypt’, oral presentation, EAA, Barcelona, September 5th-8th, 2018.
C. Granzotto et al., ‘Palaeoproteomic analysis of paint binders and adhesives in ancient Egypt’, poster, Gordon Research Conference: Scientific Methods in Cultural Heritage Research, Barcelona, July 22nd-27th, 2018.
C. Granzotto et al., Palaeoproteomic analysis of paint binders and adhesives in ancient Egypt’, poster, ISBA, Jena, September 18th-21st, 2018

I hereby confirm that I have always acknowledged EU funding of my work throughout the fellowship and will continue to do so when presenting or publishing work derived from results from this fellowship in the future.
The application of proteomics to cultural heritage materials has already proved to be extremely informative and it is under expansion. However only few museums have access to these state-of-the-art techniques and the most traditional analytical techniques used for protein analysis do not allow to retrieve the level of information that can be obtained by mass spectrometry based proteomics. The results of this research have therefore open a new insight into the artists’ materials used in ancient Egypt, as well as the animal species available at that time, thus complementing the archaeological and art historical knowledge. As an example, by applying palaeoprotoemics, it was possible to finally identify the nature and the species of origin of protein-based materials that were only observed 10 years ago on a wall painting from the collection of the British Museum.

All collaborators agreed to publish the project outcome in a single scientific paper, to be submitted to a high impact-factor journal, which will include all samples analyzed in order to present to the scientific and art community this comprehensive study of ancient Egyptian artists’ technique. The paper will be open access and all MS raw data will be publicly available and deposited to the ProteomeXchange Consortium via the PRIDE partner repository. The paper is still in process due to the early termination of the project.
Nebamun wall painting (The British Museum, EA37977)