Zandra Fagernäs

Ancient human dental calculus is one of the richest archives of archaeological biomolecular information, providing direct evidence of diet, oral health, and the oral microbiome. Proteomic analyses of this biological matrix have so far focused mainly on oral microbes and dietary proteins, with milk proteins such as beta-lactoglobulin (BLG) providing the largest corpus of proteomic evidence. Despite the close relation between the various stages of dental calculus formation and mineralization with the dental enamel surface, proteins from the dental enamel matrix have not previously been reported outside of dental enamel tissue. Here we reanalysed 498 ancient dental calculus proteomes from 14 published studies (n=434 individuals) reporting the presence of BLG, spanning from the Neolithic to the Victorian Era and applying different protein extraction protocols (FASP, GASP, SP3 and in-solution digestion). Dental enamel matrix proteins were identified in ten studies (n=37 individuals), with amelogenin being the most frequently detected. Enamel peptides occurred more often in studies that applied SP3, although amelogenin was successfully identified through both SP3 and FASP. Structural proteins, including enamelin, ameloblastin, and MMP20, were also identified. The detection of AMELX and AMELY peptide sequences provided new insights into cases where the sex was previously undetermined. These findings establish dental enamel proteins as a new category of biomolecules detected in dental calculus, broadening its application beyond diet and microbiome studies to possible sex estimation.

Ancient human dental calculus is one of the richest archives of archaeological biomolecular information, providing direct evidence of diet, oral health, and the oral microbiome. Proteomic analyses of this biological matrix have so far focused mainly on oral microbes and dietary proteins, with milk proteins such as beta-lactoglobulin (BLG) providing the largest corpus of proteomic evidence. Despite the close relation between the various stages of dental calculus formation and mineralization with the dental enamel surface, proteins from the dental enamel matrix have not previously been reported outside of dental enamel tissue. Here we reanalysed 498 ancient dental calculus proteomes from 14 published studies (n=434 individuals) reporting the presence of BLG, spanning from the Neolithic to the Victorian Era and applying different protein extraction protocols (FASP, GASP, SP3 and in-solution digestion). Dental enamel matrix proteins were identified in ten studies (n=37 individuals), with amelogenin being the most frequently detected. Enamel peptides occurred more often in studies that applied SP3, although amelogenin was successfully identified through both SP3 and FASP. Structural proteins, including enamelin, ameloblastin, and MMP20, were also identified. The detection of AMELX and AMELY peptide sequences provided new insights into cases where the sex was previously undetermined. These findings establish dental enamel proteins as a new category of biomolecules detected in dental calculus, broadening its application beyond diet and microbiome studies to possible sex estimation. Highlights: Dental calculus entraps oral microbes along with endogenous and exogenous particles during formation and mineralization We conduct reanalysis of 14 published ancient dental calculus studies (n = 434 individuals) spanning the Neolithic to Victorian Era Dental enamel proteins AMELX, AMELY, AMBN, COL17A1, ENAM and MMP20 are identified in ancient human dental calculus Amelogenin was the most frequently detected enamel protein We expand dental calculus palaeoproteomics beyond diet and oral microbiome to potentially include sex estimation

The integration of biomolecular studies of past organisms with geoarchaeological studies can significantly improve our understanding of the relative chronology and context of archaeologically (in)visible behaviors. However, the complexity and sedimentological heterogeneity of archaeological deposits at a microscopic scale is often not taken into consideration in biomolecular studies. Here, we investigate the preservation and retrieval of paleoproteomic data from bone fragments embedded in Pleistocene resin-impregnated sediment blocks. We show that resin impregnation has minimal effect on skeletal protein taxonomic identifications in modern skeletal material but observe an increase in oxidation of methionines. We then successfully retrieve proteins from resin-impregnated blocks from the Paleolithic sites of Bacho Kiro Cave, La Ferrassie, and Quinçay. The taxonomic identifications of minute bones encased in resin are in line with previous analyses of the faunal communities of these sites, with a diversity of taxa (Bos sp./Bison sp., Equus sp., Ursus sp., and Caprinae) observed at a microscale in Bacho Kiro. This differs from results from La Ferrassie where most of the samples are identified as a single taxon (Bos sp./Bison sp.) across different areas of the site. The block from Quinçay only provided taxonomic identification of two out of eleven bone-derived samples, likely due to diagenesis. Our work indicates that paleoproteomes can be retrieved from bone fragments at a microstratigraphic resolution, enabling the detailed study of faunal community composition at a scale that more closely matches that of past human occupations.

Metagenomic investigation of archaeological dental calculus has provided insights into the changing oral health, disease, and diet of past human societies, but little is known about the oral microbiota of exceptionally high-status individuals, whose diet and lifestyle sharply differed from the general population. Here we analyze the dental calculus metagenome of King Richard III of England (1452-1485) and compare it to new and previously published dental calculus metagenomes from predominantly non-elite contexts in England, Ireland, the Netherlands, and Germany spanning the Neolithic to the present. Deep sequencing and de novo assembly enabled the investigation of metagenome-assembled genomes (MAGs) from periopathogens within the genus Tannerella . DNA preservation within the dental calculus of King Richard III was found to be exceptional, yielding an extraordinarily well-preserved oral microbiota. Oral microbiome species diversity fell within the range previously observed among other northern European populations over the past 7,000 years, suggesting that a royal lifestyle and a rich diet did not substantially shift his oral microbiome composition. Reconstructed Tannerella genomes contained many virulence factors found today among periopathogens, including Tannerella forsythia . Insufficient plant and animal DNA was recovered to investigate diet, suggesting that dental calculus may not be a sufficient source of dietary DNA for dietary reconstruction, even when well-preserved. The dental calculus of King Richard III has produced one of the richest and best preserved ancient oral metagenomes studied to date and contributes to understanding the ecology and evolution of the human oral microbiome.

Palaeoproteomics focuses on studying the past by analysing proteins preserved in historical, archaeological, and palaeontological materials. Over the past fifteen years, the number of publications and research questions in this field has grown substantially, as has the number of researchers and research groups utilising palaeoproteomic methods. PAASTA (Palaeoproteomics And Archaeology, Society for Techniques and Advances) is an international early career researcher-driven community, which aims to bring together researchers from all subdisciplines of palaeoproteomics by providing a platform for open communication. Here, we discuss ways in which this fast-growing research field can benefit from adopting principles of open science and increased communication and collaboration between researchers. We approach this question from all stages of a typical research project, from experimental design and sample selection, to data analysis and raw data sharing. Although we acknowledge that each of these guidelines and recommendations may not be applicable to every research project, we aim to provide a foundation which, when applied collectively, will strengthen the future of palaeoproteomics.

Analysis of archaeological and palaeontological dental enamel allows for the study of taxonomy, phylogenetic relationships, and genetic sex of Pleistocene fauna and hominin remains. However, high-quality data is required for the study of degraded, chemically modified ancient proteomes. Protein contamination may hinder acquisition of such data, due to the abundance and better preservation of modern contaminating proteins. Here, we artificially contaminate a Pleistocene woolly rhinoceros tooth in order to investigate decontamination of archaeological dental enamel. We find that although the contaminating proteins are not identified, likely due to a lack of denaturation and digestion steps in archaeological dental enamel protein extraction protocols, contamination leads to significant loss of endogenous proteomic information. We thereafter compare five published decontamination methods, and find that a simple water or bleach wash prior to demineralization is most efficient at removing protein contamination. The water wash does not affect the endogenous proteome, while a bleach wash may lead to a loss of shorter intercrystalline peptides. We therefore conclude that decontamination is necessary in the study of ancient dental enamel, to ensure maximal retrieval of endogenous proteomic information, and can easily be achieved by incorporating a water or bleach wash step into the protein extraction protocol.

Denisovans are an extinct hominin group defined by ancient genomes of Middle to Late Pleistocene fossils from southern Siberia. Although genomic evidence suggests their widespread distribution throughout eastern Asia and possibly Oceania, so far only a few fossils from the Altai and Tibet are confidently identified molecularly as Denisovan. We identified a hominin mandible (Penghu 1) from Taiwan (10,000 to 70,000 years ago or 130,000 to 190,000 years ago) as belonging to a male Denisovan by applying ancient protein analysis. We retrieved 4241 amino acid residues and identified two Denisovan-specific variants. The increased fossil sample of Denisovans demonstrates their wider distribution, including warm and humid regions, as well as their shared distinct robust dentognathic traits that markedly contrast with their sister group, Neanderthals.

The integration of biomolecular studies of past organisms with geoarchaeological studies can significantly improve our understanding of the relative chronology and context of archaeologically (in)visible behaviours. However, the complexity and sedimentological heterogeneity of archaeological deposits at a microscopic scale is often not taken into consideration in biomolecular studies. Here, we investigate the preservation and retrieval of palaeoproteomic data from bone fragments embedded in Pleistocene resin-impregnated sediment blocks. We show that resin impregnation has minimal effect on skeletal protein taxonomic identifications in modern skeletal material, but observe an increase in oxidation-related post-translational modifications. We then successfully retrieve proteins from resin-impregnated blocks from the Palaeolithic sites of Bacho Kiro Cave, La Ferrassie and Quinçay. The taxonomic identifications of minute bones encased in resin are in line with previous analyses of the faunal communities of these sites, with a diversity of taxa ( Bos sp./ Bison sp., Equus sp., Ursus sp., and Caprinae) observed at a microscale in Bacho Kiro. This differs from results from La Ferrassie where most of the samples are identified as a single taxon ( Bos sp./ Bison sp.) across different areas of the site. The block from Quinçay only provided taxonomic identification of two out of eleven bone-derived samples, likely due to diagenesis. Our work indicates that palaeoproteomes can be retrieved from bone fragments at a microstratigraphic resolution, enabling the detailed study of faunal community composition at a scale that more closely matches that of past human occupations. Significance Statement: Resin-embedded sediment blocks are widely used in archaeology and soil sciences to reconstruct past environments and human behavior, but their potential for biomolecular analysis is underexplored. Here, we demonstrate that ancient proteins can be successfully retrieved from bone fragments embedded in resin-impregnated sediment blocks from Pleistocene archaeological sites. Our findings show that resin impregnation has a minimal impact on protein recovery and that palaeoproteomics enables taxonomic identification of Pleistocene bone fragments at a microstratigraphic scale. This approach allows for reconstructing past faunal communities with unprecedented detail, improving our understanding of ancient ecosystems and the environmental contexts of early hominin occupations.