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.

Palaeoproteomic Profiling of Conservation Layers on a 14th Century Italian Wall Painting

Meaghan Mackie, Patrick Rüther, Diana Samodova, Fabiana Di Gianvincenzo, Clara Granzotto, David Lyon, David A. Peggie, Helen Howard, Lynne Harrison, Lars Juhl Jensen, Jesper V. Olsen, Enrico Cappellini

Ahead of display, a non‐original layer was observed on the surface of a fragment of a wall painting by Ambrogio Lorenzetti (active 1319, died 1348/9). FTIR analysis suggested proteinaceous content. Mass spectrometry was used to better characterise this layer and revealed two protein components: sheep and cow glue and chicken and duck egg white. Analysis of post‐translational modifications detected several photo‐oxidation products, which suggest that the egg experienced prolonged exposure to UV light and was likely applied long before the glue layer. Additionally, glycation products detected may indicate naturally occurring glycoprotein degradation or reaction with a carbohydrate material such as starch, identified by ATR‐FTIR in a cross‐section of a sample taken from the painting. Palaeoproteomics is shown to provide detailed characterisation of organic layers associated with mural paintings and therefore aids reconstruction of the conservation history of these objects.

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Ancient Biomolecules and Evolutionary Inference

Enrico Cappellini, Ana Prohaska, Fernando Racimo, Frido Welker, Mikkel Winther Pedersen, Morten E. Allentoft, Peter de Barros Damgaard, Petra Gutenbrunner, Julie Dunne, Simon Hammann, Mélanie Roffet-Salque, Melissa Ilardo, J. Víctor Moreno-Mayar, Yucheng Wang, Martin Sikora, Lasse Vinner, Jürgen Cox, Richard P. Evershed, and Eske Willerslev

Over the past three decades, studies of ancient biomolecules—particularly ancient DNA, proteins, and lipids—have revolutionized our understanding of evolutionary history. Though initially fraught with many challenges, today the field stands on firm foundations. Researchers now successfully retrieve nucleotide and amino acid sequences, as well as lipid signatures, from progressively older samples, originating from geographic areas and depositional environments that, until recently, were regarded as hostile to long-term preservation of biomolecules. Sampling frequencies and the spatial and temporal scope of studies have also increased markedly, and with them the size and quality of the data sets generated. This progress has been made possible by continuous technical innovations in analytical methods, enhanced criteria for the selection of ancient samples, integrated experimental methods, and advanced computational approaches. Here, we discuss the history and current state of ancient biomolecule research, its applications to evolutionary inference, and future directions for this young and exciting field.

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Early Pleistocene enamel proteome sequences from Dmanisi resolve Stephanorhinus phylogeny

Enrico Cappellini, Frido Welker, Luca Pandolfi, Jazmin Ramos Madrigal, Anna Fotakis, David Lyon, Victor L. Moreno Mayar, Maia Bukhsianidze, Rosa Rakownikow Jersie-Christensen, Meaghan Mackie, Aurelien Ginolhac, Reid Ferring, Martha Tappen, Eleftheria Palkopoulou, Diana Samodova, Patrick L. Ruther, Marc R. Dickinson, Tom Stafford, Yvonne L. Chan, Anders Gotherstrom, Senthivel KSS Nathan, Peter D. Heintzman, Joshua D. Kapp, Irina Kirillova, Yoshan Moodley, Jordi Agusti, Ralf-Dietrich Kahlke, Gocha Kiladze, Bienvenido Martinez-Navarro, Shanlin Liu, Marcela Sandoval Velasco, Mikkel-Holger S. Sinding, Christian D. Kelstrup, Morten E. Allentoft, Anders Krogh, Ludovic Orlando, Kirsty Penkman, Beth Shapiro, Lorenzo Rook, Love Dalen, M. Thomas P. Gilbert, Jesper V. Olsen, David Lordkipanidze, Eske Willerslev

Ancient DNA (aDNA) sequencing has enabled unprecedented reconstruction of speciation, migration, and admixture events for extinct taxa. Outside the permafrost, however, irreversible aDNA post-mortem degradation has so far limited aDNA recovery within the ~0.5 million years (Ma) time range. Tandem mass spectrometry (MS)-based collagen type I (COL1) sequencing provides direct access to older genetic information, though with limited phylogenetic use. In the absence of molecular evidence, the speciation of several Early and Middle Pleistocene extinct species remain contentious. In this study, we address the phylogenetic relationships of the Eurasian Pleistocene Rhinocerotidae using ~1.77 million years (Ma) old dental enamel proteome sequences of a Stephanorhinus specimen from the Dmanisi archaeological site in Georgia (South Caucasus). Molecular phylogenetic analyses place the Dmanisi Stephanorhinus as a sister group to the woolly (Coelodonta antiquitatis) and Merck's rhinoceros (S. kirchbergensis) clade. We show that Coelodonta evolved from an early Stephanorhinus lineage and that this genus includes at least two distinct evolutionary lines. As such, the genus Stephanorhinus is currently paraphyletic and its systematic revision is therefore needed. We demonstrate that Early Pleistocene dental enamel proteome sequencing overcomes the limits of ancient collagen- and aDNA-based phylogenetic inference, and also provides additional information about the sex and the taxonomic assignment of the specimens analysed. Dental enamel, the hardest tissue in vertebrates, is highly abundant in the fossil record. Our findings reveal that palaeoproteomic investigation of this material can push biomolecular investigation further back into the Early Pleistocene.

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