State of the art and challenges

Arguably, most of the cultural heritage objects produced using organic materials have high content of protein residues. The application of proteomics analysis based on high-resolution mass spectrometry (MS) allows accurate and relatively affordable sequencing of any protein residue present in quantities above femtomoles, with no need to pre-define a target. As a consequence it is not only possible to confidently identify the protein complex present in a sample, but also the biological species, and in some cases even the tissue, originally used as the source of the material for the production of ancient objects. Proteomics technology also allows for biochemical characterisation of the damage ancient protein residues accumulate over millennia, providing, a rigorous approach to understand and quantify molecular damage affecting this category of biomolecules in cultural heritage material.

Members of the TEMPERA consortium are amongst the pioneers who first applied MS-based ancient protein sequencing in art and archaeology . Ancient protein sequencing was used (UoL) to identify food residues in archaeological pottery , or to characterise paint binders and the chemical damage affecting them (UoN). More recently, paleoproteomics methods allowed characterisation of proteins in mortars and identification of the species of origin of zooarchaeological remains, prehistoric skin garments (UCPH), or medieval parchment ( UoY). In the last year, UCPH demonstrated the feasibility of identification of protein-based wood adhesives. UoY pioneered the ZooMS technique for identification of animal remains.

Investigation of ancient samples presents specific challenges and requirements. All experimental phases require adjustments specific for cultural heritage material. In particular, only a close interaction and collaboration among museum curators, restorers and analytical scientists can lead to the development of innovative sampling procedures compatible with both analytical and conservation requirements. Most of the MS proteomics methods so far used are not initially designed for proteins heavily affected by extended fragmentation, contamination by environmental factors, and biomolecular damage accumulated over millennia.

The involvement of industrial partners in the TEMPERA network will allow access to their industrial expertise. Private-sector collaborators: Thermo and DEVRO, will benchmark their most advanced technological developments against this category of extremely demanding samples.

Research themes

The TEMPERA network will allow development of dedicated methodological solutions for paleoproteomics analysis of different categories of cultural heritage materials. It will also allow mutual sharing of expertise and experience among leading paleoproteomics laboratories in Europe, aiming at defining common standard analytical protocols, for uniformed treatment of European cultural heritage material. Ultimately, the TEMPERA network will train the right specialists to operate globally at top level in the field. ESRs’ research projects will cover the most relevant topics concerning application of paleoproteomics to cultural heritage.

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Paint binders in figurative arts

Supervisor: Caroline Tokarski @ UoL

PhD Candidate: Francesca Galluzzi - see detailed project description

The study of paintings consists of identifying pigments and binders to highlight the techniques used by the artists but also to propose the most appropriate conservation conditions and restoration treatments. Throughout the past centuries, artists have used a wide range of natural components to find the best painting recipes and formulation. The interaction between the different compounds (pigment particles and organic molecules) ensures the cohesion of the paint layer and its adhesion to the support, but often it is also associated to degradation mechanisms resulting from natural ageing or inappropriate conservation/restoration conditions of artworks. Among binders used for artworks, proteins represent a key molecule and proteomics emerged recently as the most informative technique to study proteins in figurative art. In this thematic section, both bottom up and top down proteomics will be used to study protein-based materials starting with their accurate identifications and identification of their biological origins, to the identification of their modifications and breakdown patterns. This information is crucial for any restoration or conservation procedure.

Paint binders in polychromy

Supervisor: Enrico Cappellini @ UCPH

PhD Candidate: Fabiana Di Gianvincenzo- see detailed project description

The study of the polychromy of ancient artefacts over the past decades has completely changed our perception of the ancient world and its aesthetics. Ancient art and architecture was very far from the pure white we usually see in museum exhibitions, but rather vibrant with colour and it is now acknowledged that colour was an integral part of shape. The choice of the binding medium was of crucial importance to the final appearance and effect of the polychromy. The perception of a painted object is the final result of how the complex and highly heterogeneous structure of the paint interacts with light. Understanding the composition of paint binders has paramount relevance in leading to a better understanding and perception of the original appearance of painted objects. In this thematic section of the project, MS-based ancient protein sequencing will be used to confidently identify the biological species of origin and the raw materials used as binders in the polychromy decorating ancient sculptures and architecture. This knowledge will be fundamental to plan long-term conservation and restoration, to define optimal conservation and display conditions, as well as to prevent or reduce decay processes.

Paper vs. parchment

Supervisors: Matthew Collins @ UoY and Gordon Paul @ DEVRO

PhD Candidate @ UoY (paper project): Gopaiah Talari - see detailed project description

PhD Candidate @ DEVRO (parchment project): Carla Soto - see detailed project description

The shortage of parchment in the Late Medieval period was almost endemic in Europe, but in the 12t h century a new kind of writing material appeared in Italy, introduced by merchants trading with Arabs: paper. In the first four or five centuries of its manufacturing in Europe, paper was neither cheap nor available in unlimited quantities. The raw material - rags - were a strategic commodity. Italians discovered that if the paper is dipped into a warm dilute gelatin solution, pressed and allowed to dry, it can be written on without fear of the ink bleeding. This gelatin sized paper was both tougher and stronger and therefore more reminiscent of parchment. Some three centuries after the introduction of paper, a second innovation: printing, transformed the Medieval world. Initially paper was neither sufficiently cheap nor abundant to be the only printed medium, and both media were used. During the earliest years printing was done on both vellum and paper. Vernacular texts were more commonly written on paper, whereas religious texts and legal documents were transcribed on the more expensive parchment. As part of this thematic section two project will explore the effect of production methods on parchment and gelatin, and direct evidence for these in early printed books.

Ancient bone protein degradation

Supervisor: Leila Birolo @ UoN

PhD Candidate: Georgia Ntasi - see detailed project description

A challenging, as well as intriguing peculiarity of ancient proteins is their signature pattern of deterioration and aging represented by covalent modifications. Distinction between modifications occurred in vivo and diagenetically derived alterations will provide important information on the physiology of ancient organisms, and on the aging and deterioration of artworks. Rigorous identification of protein degradation products and the reconstruction of their formation pathways, through spontaneous reactions with endogenous or exogenous compounds, will also be associated with specific environmental factors, leading to develop a more efficient preservation of ancient samples. Much work still needs to be done to understand and characterize the whole range of modifications occurring during proteins aging in samples exposed to a wide spectrum of different environmental conditions. In this thematic section, characterisation of modifications known to accumulate in ancient samples over millennia, such as deamidation and oxidation, as well as blind search for other, less common, spontaneous modifications will be used to identify environmental changes, aging and diagenetic factors.

Mass spectrometry for paleoproteomics

Supervisors: Jesper V. Olsen and Christian Kelstrup @ UCPH

PhD Candidate (Top-Down with JVO): Patrick Rüther - see detailed project description

PhD Candidate (Hardware with CK): Diana Samodova - see detailed project description

The latest generation of high-resolution tandem mass spectrometers can reliably and rapidly detect proteins in complex mixtures, even at the very limited quantities typical of ancient samples. Despite these developments, ancient protein analysis is still in its infancy. Protein extraction and preparation for MS analysis is the first critical phase affecting recovery success. Simplification of sample preparation by integration of different chemical treatments: reduction, alkylation and digestion, into single-steps already proved to improve recovery in the most recalcitrant ancient samples. The recent availability of new high-resolution mass spectrometric instruments offers increased sensitivity in the low attomoles range. The possibility to combine complementary fragmentation methods (HCD + ETD) maximises the possibility to improve peptide identification and analyze ancient protein modifications with unprecedented depth and accuracy. Confident use of these technologies is still limited to very few specialized research units, with direct connections with instrument suppliers.

Computational paleoproteomics

Supervisor: Jürgen Cox @ MPI

PhD Candidate: Petra Gutenbrunner - see detailed project description

Investigation of archaeological samples, when the target is species identification, can prove to be particularly challenging, as the genome of many non-domestic species used to produce everyday use objects, such as elk and deer for bone combs, or mink for elaborate fur garments, is still not available yet. Similarly, the streamlined identification of pathological variants, reported in publicly available protein databases, but commonly ignored in ordinary peptide-spectrum matching processes, could favour identifications of pathologies in ancient samples. In these cases reliable options for homology search, identification of sequence variants and amino acid substitutions, or de novo sequencing, integrated within the main search-engine would improve a lot the identification of cryptic, potentially species-diagnostic, peptides. Finally, improved algorithms for blind identification of spontaneous post-translational modifications will allow a better characterisation of the damage pattern affecting each sample, with direct relevance in conservation.

DISCLAIMER: The information reported on this website reflects only the TEMPERA consortium view. The European Research Agency is not responsible for any use that may be made of the information it contains.

🇪🇺 This project has received funding from the European Union's EU Framework Programme for Research and Innovation Horizon 2020 under Grant Agreement No. 722606. 🇪🇺