Ancient metaproteomics: a novel approach for understanding disease and diet in the archaeological record

Proteomics is increasingly being applied to archaeological samples following technological developments in mass spectrometry. This thesis explores how these developments may contribute to the characterisation of disease and diet in the archaeological record. This thesis has a three-fold aim; a) to evaluate the potential of shotgun proteomics as a method for characterising ancient disease, b) to develop the metaproteomic analysis of dental calculus as a tool for understanding both ancient oral health and patterns of individual food consumption and c) to apply these methodological developments to understanding individual lifeways of people enslaved during the 19th century transatlantic slave trade. This thesis demonstrates that ancient metaproteomics can be a powerful tool for identifying microorganisms in the archaeological record, characterising the functional profile of ancient proteomes and accessing individual patterns of food consumption with high taxonomic specificity. In particular, analysis of dental calculus may be an extremely valuable tool for understanding the aetiology of past oral diseases. Results of this study highlight the value of revisiting previous studies with more recent methodological approaches and demonstrate that biomolecular preservation can have a significant impact on the effectiveness of ancient proteins as an archaeological tool for this characterisation. Using the approaches developed in this study we have the opportunity to increase the visibility of past diseases and their aetiology, as well as develop a richer understanding of individual lifeways through the production of molecular life histories

Ancient protein analysis in archaeology

The analysis of ancient proteins from paleontological, archeological, and historic materials is revealing insights into past subsistence practices, patterns of health and disease, evolution and phylogeny, and past environments. This review tracks the development of this field, discusses some of the major methodological strategies used, and synthesizes recent developments in archeological applications of ancient protein analysis. Moreover, this review highlights some of the challenges faced by the field and potential future directions, arguing that the development of minimally invasive or nondestructive techniques, strategies for protein authentication, and the integration of ancient protein analysis with other biomolecular techniques are important research strategies as this field grows

Diet and Health in Middle Bronze Age Italy : a metaproteomic analysis of human dental calculus in two case-studies

Shotgun metaproteomics applied to dental calculus is a tool that is providing unprecedented insights in ancient diet and health reconstruction [1][2]. We apply a proteomic analysis of dental calculus deposits from individuals of two contemporaneous populations, Bovolone (Verona) and Sant’Abbondio (Pompeii), in order to provide more insight into the diet and health of individuals in middle Bronze Age Italy. Here we find that differences in protein preservation among individuals make comparing the two populations challenging. Nevertheless, we detect a number of dietary proteins, including wheats and eggs, which gains insight into food consumption practices during this period of social and economic change

Relief food subsistence revealed by microparticle and proteomic analyses of dental calculus from victims of the Great Irish Famine

This study provides direct evidence of the dependency on relief food in Ireland around the time of the Great Famine (1845 to 1852) through dental calculus analysis of archaeological human remains. The findings show a dominance of corn (maize) and milk from the identified foodstuffs and corroborate the contemporaneous historical accounts of diet and subsistence. It shows that microparticle and proteomic analyses, even when based on small archaeological samples, can provide a valid snapshot of dietary patterns and food consumption. The occurrence of egg protein, generally only included in the diet for the better-off social classes, also highlights how these analytical techniques can provide unanticipated insights into the variability of diet in historical populations.Food and diet were class markers in 19th-century Ireland, which became evident as nearly 1 million people, primarily the poor and destitute, died as a consequence of the notorious Great Famine of 1845 to 1852. Famine took hold after a blight (Phytophthora infestans) destroyed virtually the only means of subsistence—}the potato crop{—}for a significant proportion of the population. This study seeks to elucidate the variability of diet in mid{–}19th-century Ireland through microparticle and proteomic analysis of human dental calculus samples (n = 42) from victims of the famine. The samples derive from remains of people who died between August 1847 and March 1851 while receiving poor relief as inmates in the union workhouse in the city of Kilkenny (52{°}39' N, -7{°}15' W). The results corroborate the historical accounts of food provisions before and during the famine, with evidence of corn (maize), potato, and cereal starch granules from the microparticle analysis and milk protein from the proteomic analysis. Unexpectedly, there is also evidence of egg protein{—}a food source generally reserved only for export and the better-off social classes{—which highlights the variability of the prefamine experience for those who died. Through historical contextualization, this study shows how the notoriously monotonous potato diet of the poor was opportunistically supplemented by other foodstuffs. While the Great Irish Famine was one of the worst subsistence crises in history, it was foremost a social disaster induced by the lack of access to food and not the lack of food availability

Preservation of the metaproteome: variability of protein preservation in ancient dental calculus.

Proteomic analysis of dental calculus is emerging as a powerful tool for disease and dietary characterisation of archaeological populations. To better understand the variability in protein results from dental calculus, we analysed 21 samples from three Roman-period populations to compare: 1) the quantity of extracted protein; 2) the number of mass spectral queries; and 3) the number of peptide spectral matches and protein identifications. We found little correlation between the quantity of calculus analysed and total protein identifications, as well as no systematic trends between site location and protein preservation. We identified a wide range of individual variability, which may be associated with the mechanisms of calculus formation and/or post-depositional contamination, in addition to taphonomic factors. Our results suggest dental calculus is indeed a stable, long-term reservoir of proteins as previously reported, but further systematic studies are needed to identify mechanisms associated with protein entrapment and survival in dental calculus

A unified protocol for simultaneous extraction of DNA and proteins from archaeological dental calculus

Archaeological materials are a finite resource, and efforts should be made to minimize destructive analyses. This can be achieved by using protocols combining extraction of several types of biomolecules or microparticles, which decreases the material needed for analyses while maximizing the information yield. Archaeological dental calculus is a source of several different types of biomolecules, as well as microfossils, and can tell us about the human host, microbiome, diet, and even occupational activities. Here, we present a unified protocol allowing for simultaneous extraction of DNA and proteins from a single sample of archaeological dental calculus. We evaluate the protocol on dental calculus from six individuals from a range of time periods and estimated preservation states, and compare it against previously published DNA-only and protein-only protocols. We find that most aspects of downstream analyses are unaltered by the unified protocol, although minor shifts in the recovered proteome can be detected, such as a slight loss of hydrophilic proteins. Total protein recovery depends on both the amount of starting material and choice of extraction protocol, whereas total DNA recovery is significantly reduced using the unified protocol (mean 43%). Nevertheless, total DNA recovery from dental calculus is generally very high, and we found no differences in DNA fragment characteristics or taxonomic profile between the protocols. In conclusion, the unified protocol allows for simultaneous extraction of two complementary lines of biomolecular evidence from archaeological dental calculus without compromising downstream results, thereby minimizing the need for destructive analysis of this finite resource

New criteria for the molecular identification of cereal grains associated with archaeological artefacts.

The domestication and transmission of cereals is one of the most fundamental components of early farming, but direct evidence of their use in early culinary practices and economies has remained frustratingly elusive. Using analysis of a well-preserved Early Bronze Age wooden container from Switzerland, we propose novel criteria for the identification of cereal residues. Using gas chromatography mass spectrometry (GC-MS), we identified compounds typically associated with plant products, including a series of phenolic lipids (alkylresorcinols) found only at appreciable concentration in wheat and rye bran. The value of these lipids as cereal grain biomarkers were independently corroborated by the presence of macrobotanical remains embedded in the deposit, and wheat and rye endosperm peptides extracted from residue. These findings demonstrate the utility of a lipid-based biomarker for wheat and rye bran and offer a methodological template for future investigations of wider range of archaeological contexts. Alkylresorcinols provide a new tool for residue analysis which can help explore the spread and exploitation of cereal grains, a fundamental component of the advent and spread of farming