Jessica Hendy

Direct evidence of milk consumption from ancient human dental calculus

Milk is a major food of global economic importance, and its consumption is regarded as a classic example of gene-culture evolution. Humans have exploited animal milk as a food resource for at least 8500 years, but the origins, spread, and scale of dairying remain poorly understood. Indirect lines of evidence, such as lipid isotopic ratios of pottery residues, faunal mortality profiles, and lactase persistence allele frequencies, provide a partial picture of this process; however, in order to understand how, where, and when humans consumed milk products, it is necessary to link evidence of consumption directly to individuals and their dairy livestock. Here we report the first direct evidence of milk consumption, the whey protein β-lactoglobulin (BLG), preserved in human dental calculus from the Bronze Age (ca. 3000 BCE) to the present day. Using protein tandem mass spectrometry, we demonstrate that BLG is a species-specific biomarker of dairy consumption, and we identify individuals consuming cattle, sheep, and goat milk products in the archaeological record. We then apply this method to human dental calculus from Greenlands medieval Norse colonies, and report a decline of this biomarker leading up to the abandonment of the Norse Greenland colonies in the 15th century CE.

Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification

To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341–534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions.

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.

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

ABSTRACT 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.

Poor preservation of antibodies in archaeological human bone and dentine

Abstract The growth of proteomics-based methods in archaeology prompted an investigation of the survival of non-collagenous proteins, specifically immunoglobulin G (IgG), in archaeological human bone and dentine. Over a decade ago reports were published on extracted, immunoreactive archaeological IgG, and the variable yields of IgG molecules detected by Western blots of 1D and 2D SDS-PAGE gels. If IgG can indeed be recovered from archaeological skeletal material, it offers remarkable opportunities for exploring the history of disease - for example in applying functional anti-malarial IgGs to study past patterns of malaria. More recently, the field has seen a move away from immunological approaches and towards the use of shotgun proteomics via mass spectrometry. Using previously published techniques, this study attempted to extract and characterize archaeological IgG proteins. In only one extraction method were immunoglobulin derived peptides identified, and these displayed extensive evidence of degradation. The failure to extract immunoglobulins by all but one method, along with observed patterns of protein degradation, suggests that IgG may be an unsuitable target for detecting disease-associated antigens. This research highlights the importance of revisiting previously ‘successful’ biomolecular methodologies using emerging technologies.

A guide to ancient protein studies

Palaeoproteomics is an emerging neologism used to describe the application of mass spectrometry-based approaches to the study of ancient proteomes. As with palaeogenomics (the study of ancient DNA), it intersects evolutionary biology, archaeology and anthropology, with applications ranging from the phylogenetic reconstruction of extinct species to the investigation of past human diets and ancient diseases. However, there is no explicit consensus at present regarding standards for data reporting, data validation measures or the use of suitable contamination controls in ancient protein studies. Additionally, in contrast to the ancient DNA community, no consolidated guidelines have been proposed by which researchers, reviewers and editors can evaluate palaeoproteomics data, in part due to the novelty of the field. Here we present a series of precautions and standards for ancient protein research that can be implemented at each stage of analysis, from sample selection to data interpretation. These guidelines are not intended to impose a narrow or rigid list of authentication criteria, but rather to support good practices in the field and to ensure the generation of robust, reproducible results. As the field grows and methodologies change, so too will best practices. It is therefore essential that researchers continue to provide necessary details on how data were generated and authenticated so that the results can be independently and effectively evaluated. We hope that these proposed standards of practice will help to provide a firm foundation for the establishment of palaeoproteomics as a viable and powerful tool for archaeologists, anthropologists and evolutionary biologists.Palaeoproteomics is an emerging field at the intersection of evolutionary biology, archaeology and anthropology. This Perspective provides a best practice primer for researchers, reviewers and editors.

Proteomic evidence of dietary sources in ancient dental calculus

Archaeological dental calculus has emerged as a rich source of ancient biomolecules, including proteins. Previous analyses of proteins extracted from ancient dental calculus revealed the presence of the dietary milk protein β-lactoglobulin, providing direct evidence of dairy consumption in the archaeological record. However, the potential for calculus to preserve other food-related proteins has not yet been systematically explored. Here we analyse shotgun metaproteomic data from 100 archaeological dental calculus samples ranging from the Iron Age to the post-medieval period (eighth century BC to nineteenth century AD) in England, as well as 14 dental calculus samples from contemporary dental patients and recently deceased individuals, to characterize the range and extent of dietary proteins preserved in dental calculus. In addition to milk proteins, we detect proteomic evidence of foodstuffs such as cereals and plant products, as well as the digestive enzyme salivary amylase. We discuss the importance of optimized protein extraction methods, data analysis approaches and authentication strategies in the identification of dietary proteins from archaeological dental calculus. This study demonstrates that proteomic approaches can robustly identify foodstuffs in the archaeological record that are typically under-represented due to their poor macroscopic preservation.

Author Correction: A guide to ancient protein studies

In the version of this Perspective originally published, in the third paragraph of the section ‘Selection and sampling’, the sentence beginning ‘Pyrolysis–gas chromatography’ should have also referred to high-performance liquid chromatography; the sentence has now been amended to read ‘Pyrolysis–gas chromatography/mass spectrometry (py–GC/MS) and high-performance liquid chromatography (HPLC) can be used to detect the presence of amino acids62 in any putative proteinaceous sample.’

Ancient proteins from ceramic vessels at Çatalhöyük West reveal the hidden cuisine of early farmers

The analysis of lipids (fats, oils and waxes) absorbed within archaeological pottery has revolutionized the study of past diets and culinary practices. However, this technique can lack taxonomic and tissue specificity and is often unable to disentangle signatures resulting from the mixing of different food products. Here, we extract ancient proteins from ceramic vessels from the West Mound of the key early farming site of Çatalhöyük in Anatolia, revealing that this community processed mixes of cereals, pulses, dairy and meat products, and that particular vessels may have been reserved for specialized foods (e.g., cow milk and milk whey). Moreover, we demonstrate that dietary proteins can persist on archaeological artefacts for at least 8000 years, and that this approach can reveal past culinary practices with more taxonomic and tissue-specific clarity than has been possible with previous biomolecular techniques.Ancient diets have been reconstructed from archaeological pottery based on lipid remains, but these can lack specificity. Here, Hendy and colleagues analyze ancient proteins from ceramic vessels up to 8000 years old to produce a more nuanced understanding of ancient food processing and diet.

Evidence of milk consumption in Northern and Southern Middle Bronze Age Italy through a metaproteomic analysis of human dental calculus

• Detecting any difference in the animal species raised Shotgun metaproteomics consists of the proteolytic digestion of proteins belonging to a mixture extremely increased in complexity. We applied a GASP (Gel-Aided Sample Preparation) protocol, modified for ancient mineralized samples [7]. Extractions were performed at BioArCh, Department of Archaeology at the University of York. Extracted peptides were analysed using liquid chromatography tandem mass spectrometry (QExactive) at the Mass Spectrometry laboratory of the Target Discovery Institute at the University of Oxford. Spectra were searched using Mascot (Matrix ScienceTM) against Uniprot database. Dental calculus is used as a powerful tool in bioarchaeology to detect direct evidence of the health status, the diet and possibly the occupation of an individual and/or of a population[4][5][6]. We performed a shotgun metaproteomic analysis of 9 samples of dental calculus; 6 samples from Bovolone, and 3 from Sant’Abbondio. Only supragingival calculus has been sampled, predominately from premolars and molars, and collected in 2.0 mL tubes. We used between 1.9 and 26.7 mg of dental calculus for protein extraction.