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Brain tissue is ubiquitous in the archaeological record. Multiple, independent studies report the finding of black, resinous or shiny brain tissue, and Petrone et al. [2020 “Heat-induced Brain Vitrification from the Vesuvius Eruption in C.E. 79.” N Engl J Med. 382: 383–384; doi:10.1056/NEJMc1909867] raise the intriguing prospect of a role for vitrification in the preservation of ancient biomolecules. However, Petrone et al. (2020) have not made their raw data available, and no detailed laboratory or analytical methodology is offered. Issues of contamination and misinterpretation hampered a decade of research in biomolecular archaeology, such that addressing these sources of bias and facilitating validation of specious findings has become both routine and of paramount importance in the discipline. We argue that the evidence they present does not support their conclusion of heat-induced vitrification of human brain tissue, and that future studies should share palaeoproteomic data in an open access repository to facilitate comparative analysis of the recovery of ancient proteins and patterns of their degradation.

UNLABELLED: Historic legal deeds are one of the most abundant resources in British archives, but also one of the most neglected. Despite the millions that survive, we know remarkably little about their manufacture, including the species of animal on which they were written. Here we present the species identification of 645 sixteenth-twentieth century skins via peptide mass fingerprinting (ZooMS), demonstrating the preferential use of sheepskin parchment. We argue that alongside their abundance and low cost, the use of sheepskins over those of other species was motivated by the increased visibility of fraudulent text erasure and modification afforded by the unique structure of their skin. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s40494-021-00503-6.

Funder: Greek Archaeological Committee UK (GACUK), Funder: Leventis Foundation; Id: http://dx.doi.org/10.13039/501100004117, This study compares histological preservation in archeological bones from different burial types to unravel the histotaphonomy‐to‐funerary practices relationship. Αn intraskeletal approach is also adopted to explore intraindividual (inner ear part of the petrous bone vs. upper/lower limb long bones) and intrabone (proximal vs. distal diaphysis) variability in bone collagen preservation, δ13C, and δ15N. The aim is to (a) target bones that likely retain higher amounts of collagen, (b) better understand the inner ear bone collagen isotopic signature and remodeling, and (c) assess intrabone isotopic and histological homogeneity. For the histological analysis, the data have been collected from 61 specimens (20 individuals) from the medieval/postmedieval cemetery of St. Rombout, Belgium. Thin sections have been studied using optical and scanning electron microscopy. For the collagen and isotopic data, 101 samples have been collected from 21 individuals. Distinct histological patterns are observed only in bones from single coffin burials; however, bone histology can display intraindividual and intrabone variability, which are important to account for interpretations. Collagen wt.%, δ13C, and δ15N show significant intraindividual differences but insignificant intrabone variability. This study also confirms the extraordinary nature of the petrous bone, as the inner ear bone collagen δ13C and δ15N values reflect the dietary input of the first approximately 2–3 years of life.

Next Generation Lab turns large and hitherto unstudied urban assemblages of archaeological leather and bone into a laboratory learning experience for high school students. The students, in turn, provide species identifications and thus increase knowledge on medieval and Renaissance livestock exploitation and material selection by craftsmen.

The rate at which endogenous DNA from differently prepared (butchered, boiled and baked) compact pig bones degrades in five different Danish terrestrial and marine environments over 12 months was investigated. Although > 70% of the estimated endogenous mtDNA is lost after just four weeks of exposure, no cytosine deamination of DNA was recognised. A correlation between the presence of oxygen and the amount of preserved DNA was observed. The results provide valuable information on the interaction between the endogenous DNA and the depositional environment in the early stages of bone diagenesis, which can be a support in the interpretation of the initial diagenetic pathways of archaeological bone.

The degradation of archaeological bones is influenced by many variables. The bone material itself is a composite of both organic and inorganic components, and their degradation depends on processes occurring both before and after burial, and on both intrinsic bone characteristics as well as extrinsic environmental parameters. In this study we attempt to quantify the effect of some of the variables using a novel approach that includes detailed monitoring of the burial environment combined with respirometry studies of bone material from five archaeological sites in West Greenland. First, we compare the state of preservation of excavated bone material with the current burial environment including the soil pH, thawing degree days, soil porosity and soil moisture. Secondly, we investigate oxic degradation of collected bone samples through respirometry and quantify the effects of temperature and moisture on the oxidation rate of individual bones. Finally, we discuss how the oxidation rate is influenced by intrinsic bone parameters. Some of the main conclusions are:1) There is a significant correlation between the current burial environment and the current state of preservation of the bones.2) The oxidation rate measured by respirometry increases on average fourfold as temperature increases by 10 degrees C, and more than hundred-fold when dry bones are soaked in water.3) The oxidation rate of different bones varies over two orders of magnitude due to intrinsic variables such as organic content and state of preservation of the bones.4) The median oxidation rate of wet bone at 15 degrees C corresponds to a yearly loss of 3.8% of their mean organic content, while the median yearly loss for dry bones at 75% RH is 0.02%.5) Respirometry is a promising tool for quantitative degradation studies of bone, but more studies are needed in order to obtain a better understanding of the oxidation processes involved.

We report a simple histological study on skin biopsies from young domestic sheep following each step in transformation from skin to parchment production. During the recreation of historical parchment manufacture, histological analyses were conducted; before and after lime treatment, hair removal, and stretching. Sections were fixed and stained using a variety of histological stains to identify the presence of different molecular classes and the fibrous proteins, collagen and elastin. The results reveal surprisingly few histological changes in most steps in the production process. However, very visible changes in the supramolecular ordering of skin macromolecules (elastin, collagen) occur during the final stage of parchment production when stretched on the frame. Collagen fibres and hair follicles were all strongly re-oriented in the direction of strain. Surprisingly despite the thinness of the lambskin and the exhaustive treatment in lime, not all fats were saponified and even in the final product Oil Red O stained fat bodies were detectable on the hair side of the skin. We believe this study will help compensate for the lack of sources on microscopic changes in parchment during the recreation of its historical manufacture.

Paleoproteomics and the study of ancient proteins has become an important consideration in bioarchaeological research as we seek to understand the relationship between the physical skeleton and its underlying biochemistry. Osteocalcin is an abundant, non-collagenous protein that is accessible archaeologically due to its affinity for hydroxyapatite in bone. Manufactured by the osteoblast cells during normal bone metabolism, osteocalcin is inherently linked to the process of bone remodelling. Clinical studies have consistently shown the impact of biological (i.e., sex and age) and pathological (i.e., disease) factors on osteocalcin concentrations within the body; however, this connection has yet to be fully explored bioarchaeologically. For this study, 46 individuals from the Danish Sct. Albert’s cemetery (CE 1250–1536) were analyzed with cortical bone samples harvested from the femur. Each sample was demineralized and quantified using established paleoproteomic methods (ELISA) and tested for diagenesis using FTIR-ATR. No significant differences were found in the osteocalcin concentrations between adult males and females. However, males had a wider range of osteocalcin variability perhaps explained in part by their increased prevalence of pathological conditions characterized by bone remodelling. Similarly, non-adults had a wide range of osteocalcin variability but there were no significant differences when compared to the adult mean. No correlations were found between osteocalcin concentrations and diagenesis suggesting that the protein can survive across a range of temporal periods and variable burial environments. While no clear pattern emerged between osteocalcin concentrations and biological and/or pathological factors, this study is significant in that it further explores the use of paleoproteomics in bioarchaeological contexts and how we may unite our macroscopic and biochemical assessments of human skeletal remains.

Pacific salmon (Oncorhynchus spp.) are ecological and cultural keystone species along the Northwest Coast of North America and are ubiquitous in archaeological sites of the region. The inability to morphologically identify salmonid post-cranial remains to species, however, can limit our understanding of the ecological and cultural role different taxa played in the seasonal subsistence practices of Indigenous groups in the past. Here, we present a rapid, cost-effective ZooMS method to distinguish salmonid species based on collagen peptide mass-fingerprinting. Using modern reference material and an assemblage of 28 DNA-identified salmonid bones from the pre-contact Yup'ik site of Nunalleq, Western Alaska, we apply high-resolution mass spectrometry (LC-MS/MS) to identify a series of potential collagen peptide markers to distinguish Pacific salmon. We then confirm these peptide markers with a blind ZooMS analysis (MALDI-TOF-MS) of the archaeological remains. We successfully distinguish five species of anadromous salmon with this ZooMS approach, including one specimen that could not be identified through ancient DNA analysis. Our biomolecular identification of chum (43%), sockeye (21%), chinook (18%), coho (11%) and pink (7%), confirm the exploitation of all five available species of salmonid at Nunalleq.

Avian eggshell survives well in alkaline and neutral soils, but its potential as an archaeological resource remains largely unexplored, mainly due to difficulties in its identification. Here we exploit the release of novel bird genomes and, for the first time on eggshell, use MALDI-ToF (matrix-assisted laser desorption ionisation-time of flight) mass spectrometry in combination with peptide sequencing by LC-MS/MS. The eggshell proteome is revealed as unexpectedly complex, with 5755 proteins identified for a reference collection comprising 23 bird species. We determined 782 m/z markers useful for eggshell identification, 583 of which could be assigned to known eggshell peptide sequences. These were used to identify eggshell fragments recovered from a medieval site at Freeschool Lane, Leicester. We discuss the specificity of the peptide markers and highlight the importance of assessing the level of taxonomic identification achievable for archaeological interpretation.

There has long been debate over the origins of dairy consumption within European populations. Whilst it was previously assumed that lactase persistence (LP) was under positive selection following the advent of agriculture, recent genetic studies of prehistoric human remains have revealed LP may have only emerged in Europe in the last 4000 years. These findings stand in contrast to organic residue analysis of Neolithic pottery indicating the utilisation of dairy products, and zooarchaeological mortality profiles consistent with dairying herds at Neolithic sites. The recent discovery of the milk protein β-lactoglobulin (BLG) within human dental calculus presents a new method via which to explore dairy product consumption in the archaeological past. Here, we apply shotgun proteomic analysis to dental calculus samples from three British Neolithic sites, revealing the earliest identification of BLG in human dental calculus to date. The presence of BLG peptides in individuals who are unlikely to possess LP provides new insight into dairying in the British Neolithic, suggesting the potential processing of milk by Neolithic populations to reduce the lactose content of dairy products.

Biocodicology, the study of the biological information stored in manuscripts, offers the possibility of interrogating manuscripts in novel ways. Exploring the biological data associated to parchment documents will add a deeper level of understanding and interpretation to these invaluable objects, revealing information about book production, livestock economies, handling, conservation and the historic use of the object. As biotechnological methods continue to improve we hope that biocodicology will become a highly relevant discipline in manuscript studies, contributing an additional perspective to the current scholarship. We hope that this review will act as a catalyst enabling further interactions between the heritage science community, manuscript scholars, curators and conservators.

The level of glutamine (Gln) deamidation in bone collagen provides information on the diagenetic history of bone but, in order to accurately assess the extent of Gln deamidation, it is important to minimise the conditions that may induce deamidation during the sample preparation. Here we report the results of a preliminary investigation of the variability in glutamine deamidation levels in an archaeological bone due to: a) sampling location within a bone; b) localised diagenesis; and c) sample preparation methods. We then investigate the effects of pre-treatment on three bone samples: one modern, one Medieval and one Pleistocene. The treatment of bone with acidic solutions was found to both induce deamidation and break down the collagen fibril structure. This is particularly evident in the Pleistocene material (∼80,000 years BP) considered in this study. We show that ethylenediaminetetraacetic acid (EDTA), when used as an alternative to hydrochloric acid (HCl) demineralisation, induces minimal levels of deamidation and maintains the collagen fibril structure. Areas of bone exhibiting localised degradation are shown to be correlated with an increase in the levels of Gln deamidation. This indicates that the extent of Gln deamidation could provide a marker for diagenesis but that sampling is important, and that, whenever possible, subsamples should be taken from areas of the bone that are visually representative of the bone as a whole. Although validation of our observations will require analysis of a larger sample set, deamidation measurements could be a valuable screening tool to evaluate the suitability of bone for further destructive collagen analyses such as isotopic or DNA analysis, as well as assessing the overall preservation of bone material at a site. The measure of bone preservation may be useful to help conservators identify bones that may require special long-term storage conditions.

Following the report of Mycobacterium tuberculosis proteins found in archaeological bone by Boros-Major et al. (2011), we attempted to identify M. tuberculosis proteins in mummified lung tissues from which ancient DNA success had already been reported. Using a filter-aided sample preparation protocol modified for ancient samples we applied shotgun proteomics to seven samples of mummified lung, chest and pleura tissues. However, we only identified four peptides with unique matches to the M. tuberculosis complex, none of which were unique to M. tuberculosis, although we did identify a range of human proteins and non-mycobacterial bacterial proteins. In light of these results, we question the validity of the peptide mass fingerprint (PMF) approach presented by Boros-Major et al. (2011), especially because the PMF spectrum presented in Boros-Major et al. (2011) has similarities to that of human collagen, the dominant protein in the tissue under investigation. We explore the challenges of using proteomic approaches to detect M. tuberculosis, and propose that, given the contentious outcomes that have plagued ancient protein research in the past, the susceptibility of ancient material to modern contamination, and the degradation inherent in archaeological samples, caution is needed in the acquisition, analysis and reporting of proteomic data from such material.

Contamination is a potential problem in the study of ancient proteins, either from prior handling of the sample, laboratory consumables, or cross-sample carryover from mass spectrometers. Recently, deamidation of glutamine has been proposed as a measure for assessing the degradation of ancient proteins. Here, we present deamiDATE 1.0, a method for the authentication of ancient proteins using measure of site-specific deamidation rates. We test this approach on shotgun proteomic data derived from bone collagen from modern, archaeological and extinct taxa. We further demonstrate how this method may be used to differentiate between modern contaminants and authentic ancient proteins using a case study from Neolithic dental calculus.

Strontium isotope analysis (87Sr/86Sr) has been employed as a provenancing tool for archaeological wool textiles. To date, the effect of post-depositional (soil burial environment) contamination on keratin samples, which contain ∼ppm concentrations of Sr, has not been rigorously investigated. We compared published methods for removing exogenous Sr from keratinous textiles, using either: (1) compressed N2 gas, (2) HF(aq) solution (with and without a strong oxidising agent to remove dyestuff) or (3) organic solvents. 87Sr/86Sr ratios and Sr contents were determined in undyed and madder-dyed/alum-mordanted moieties of the same wool textile, buried for up to three years in contrasting environments (marine sediment/fenland bog), and two archaeological textiles recovered in Iceland (one typical and one atypical of local manufacture). Undyed experimental samples had low Sr contents (0.07–0.29 ppm) that were increased by both dyeing (0.14–8.92 ppm) and soil burial (0.11–15.01 ppm). The efficacy of Sr removal was: HF(aq) + oxidising agent > organic solvents > HF(aq) > compressed N2. Unburied samples showed little variation in 87Sr/86Sr ratio between cleaning methods (0.00006–0.00035); buried samples showed greater variation (0.00257–0.00713). Archaeological samples showed Sr contents greater than experimental soil burials (1–118 ppm), and 87Sr/86Sr values consistent with Icelandic groundwater (0.70357–0.70540). No cleaning methods retrieved original (unburied and undyed) 87Sr/86Sr ratios except treatment with compressed N2 in undyed samples. Exogenous Sr from the short term soil burial environment is probably mostly present as particulates. We conclude that 87Sr/86Sr ratios of archaeological wool textiles recovered from wet burial environments do not accurately reflect wool provenance even after cleaning with the methods investigated.

This paper presents evidence of the discovery of a new Middle Pleistocene site in central southern England, with undisturbed evidence of hominin occupation well-dated to an interstadial towards the end of Marine Isotope Stage 8, c. 250,000 BP. The site consists of a preserved remnant of a river terrace and its alluvial floodplain overlain by chalk-rich bankside deposits, all abutting a Chalk bedrock riverbank. It preserves an area of occupation with activity focused on the riverbank, complemented by occasional activity on a palaeo-landsurface developed on the surface of the alluvial floodplain. Lithic technology at the site consists almost entirely of handaxe manufacture, allowing attribution to an Acheulian industrial tradition. Mammalian and other palaeo-environmental remains are present and associated with the occupation horizons, including large mammal bones showing signs of hominin interference. Dating was based on OSL determinations on the sediments and amino acid racemisation of molluscan remains, supported by biostratigraphic indications. Besides being a rare instance of an undisturbed Palaeolithic palaeo-landscape covering several hectares, the site contributes to wider Quaternary research concerns over the ability of Middle Pleistocene hominins to tolerate colder climatic episodes in higher latitudes, and over settlement history and changing lithic industrial traditions of northwest Europe in relation to climate change and British peninsularity. It is suggested that the Harnham evidence may represent an insular population that had persisted in southern Britain since MIS 10/9, which became locally extinct during the glacial maximum 8.2 marking the end of MIS 8.

Peptide mass fingerprinting of bone collagen (ZooMS) has previously been proposed as a method to calculate the extent of the non-enzymatic degradation of glutamine into glutamic acid (deamidation). Temporal and spatial variation of glutamine deamidation at a single site, however, has not been investigated. Here we apply ZooMS screening of Châtelperronian and Early Holocene bone specimens from Quinçay, France, to explore temporal and spatial variation in glutamine deamidation. Our results indicate that chronological resolution is low, while spatial variation is high. Nevertheless, our analysis allows the identification of bone specimens that have undergone diagenetic histories remarkably different (either in length or in type) from spatially related bone specimens. Therefore, ZooMS ammonium-bicarbonate screening is capable of testing bone assemblage homogeneity, which could guide subsequent analysis and interpretation.

Fibre products, such as textiles and animal pelts, are often recovered in the corrosion crust of archaeological metal artefacts. Because clothed burials are an important resource for the study of past societies, accurate fibre identification is important. However, extreme mineralisation of animal fibres can render microscopic visualisation difficult for species identification. Peptide mass fingerprinting (PMF) has been successfully used to identify the species origin in both collagen and keratin-made archaeological artefacts. The approach requires little material but the state of degradation (protein hydrolysis) is a limiting factor as it might impact on the identification of key markers. In this study we analysed pelt and textile fragments found in association with copper-alloy objects with different degrees of mineralisation; samples were obtained from a Viking-Age (10th c.) grave in Britain and from a burial in Mongolia (3rd c. BC to 2nd c. AD). Species identification was possible in all but one sample, revealing PMF can be applied to corrosion products, thereby further expanding the value of these objects for textile research.

Eggshell is a potentially common archaeological resource, but it tends to be ignored. The recent development of ZooMS (zooarchaeology by mass spectrometry) as a rapid and robust system for taxonomic identification of preserved eggshell fragments has facilitated new insights into patterns of egg use in the past. This paper presents a case study of egg use at two sites in Anglo-Scandinavian York (Hungate and Coppergate). The results described below suggest that the relative prevalence of goose eggshell may become a useful indicator of status, consistent with other characteristics of the two sites, and also demonstrate an apparent lack of exploitation of eggs of wild birds in York during the Anglo-Scandinavian period. These results highlight the interpretative potential of eggshell, which can now begin to be more fully explored. Copyright © 2013 John Wiley & Sons, Ltd.

Analysis of ancient cattle husbandry is compromised by the need for conventional analysis of sex and age at death to use different elements of the skeleton, usually from different individuals. We demonstrate a technique using aDNA acquired from cattle mandibles and combined with age-at-death estimation based on dental eruption in the same specimens. Application to small sample groups from two sites in Na h-Eileanan Siar (the Scottish Western Isles) show a high success rate, and demonstrates the potential for a more nuanced interpretation of ancient cattle husbandry.

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.

The last few years have seen an enormous proliferation of ancient biomolecules research, especially in the field of ancient DNA. Ancient DNA studies have been transformed by the advent of next generation sequencing, with the first Pleistocene sample being analysed in 2005, and several complete and draft genomes that have been compiled from ancient DNA to date. At the same time, although less conspicuous, research on ancient proteins has also seen advances, with the time limit for research on ancient biomolecules now extending to over 1 million years. Here we review which effects these developments have on research in Quaternary science. We identify several lines of research that have the potential to profit substantially from these recent developments in ancient biomolecules research. First, the identification of taxa can be made using ancient biomolecules, and in the case of ancient DNA, specimens can even be assigned to specific populations within a species. Second, increasingly large DNA data sets from Pleistocene animals allow the elucidation of ever more precise pictures of the population dynamic processes whereby organisms respond to climate and environmental change. With the accompanying better understanding of process in the Quaternary, past ecologies can also more realistically be interpreted from proxy data sets. The dominant message from this research so far is that the Quaternary saw a great deal more dynamism in populations than had been forecast by conventional palaeoecology. This suggests that reconstructions of past environmental conditions need to be done with caution. Third, ancient DNA can also now be obtained directly from sediments to elucidate the presence of both plant and animal species in an area even in the absence of identifiable fossils, be it macro- or micro-fossils. Finally, the analysis of proteins enables the identification of bone remains to genus and sometimes species level far beyond the survival time of DNA, at least in temperate regions, illustrating that precise data is now forthcoming from seemingly unlikely sources. Together, these approaches allow the study of environmental dynamics throughout a substantial part, and perhaps even the entire Quaternary (the last 2.6 million years).

Identifying animals to species from relict proteins is a powerful new archaeological tool. Here the authors apply the method to answer questions relating to the Salish of west coast North America. Did they weave their blankets out of dog hair? The proteomic analysis shows that they did, interweaving it with goat, and that the woolly dog was increasingly superseded by sheep in the later nineteenth century.

It is generally a challenge to interpret incomplete and degraded skeletal assemblages found during archaeological excavations. Several events after the death of animals and humans, before and during burial, will have influenced the situation as seen upon excavation. This postmortem sequence of events can be called the taphonomic history of bones. Taphonomic signatures as detected using histology can provide additional data on deposition/burial and the evolution of the burial environment. This article reports on the results of a histological characterisation of diagenetic alterations in a bone assemblage from the Roman period burial and settlement site of Castricum, located in the province of Noord-Holland in the Netherlands. The aim is to assess the relationship between bone histology and known taphonomic events. Both transmitted light microscopy and scanning electron microscopy were used to investigate histological changes. In 1995, the excavation at Castricum uncovered the skeletal material of humans and a variety of animal species that had received different burial treatments. The humans, as well as some dogs and cattle, were buried as complete inhumations. The skeletal remains of horses provided evidence of surface exposure before deposition. In addition, both environmental changes (e.g. variations in groundwater levels) and disturbances by later human activity are known to have occurred since the internment of the bones. Together with the animal refuse bones from the site, this assemblage provides an excellent opportunity to investigate the relationship between early taphonomy, burial conditions and histological appearance. The complex taphonomic history of the Castricum skeletal material was found to be reflected in several characteristic alterations of the bone microstructure such as the extent of bioerosion, cracking, staining and inclusions of framboidal pyrite crystals. This allowed for the reconstruction of a postmortem sequence of events. Copyright © 2011 John Wiley & Sons, Ltd.

Fish are a large, highly diverse, and anthropologically important group of vertebrates. However, fish bones are underrepresented in the archaeological literature because they are less stable than those of other taxa and identification of bone to species is often difficult or impossible. We explore a new identification system, ZooMS (Zooarchaeology by Mass Spectrometry), which is based upon protein barcoding. As proteins can be cleaved enzymatically and analyzed by mass spectrometry in a repeatable way, protein barcoding is used widely in microbiological contexts for quick and inexpensive protein identification; mass spectra reflect the differences in protein sequence and can therefore be reproducibly linked to a particular protein or protein fragment. ZooMS uses peptide fingerprinting of bone collagen as a method for rapid identification of archaeological bone. This has involved the identification of masses related to peptides of known sequence. For mammals, sufficient sequence information is available for this approach but for groups, such the teleost fish, species are highly diverse and there are few available collagen sequences. Here we report a preliminary investigation into the identification of fish species by peptide mass fingerprinting that does not require sequence information. Collagen mass spectra are used to identify eight species of bony fishes (Osteichthyes) from four orders: Clupeiformes, Salmoniformes, Gadiformes, and Perciformes. The method is applied to both modern and archaeological fish remains and offers the capacity to identify traditionally unidentifiable fish fragments, thus increasing the Number of Identified Specimens (NISP) and providing invaluable information in specialized contexts.

Bone collagen is found throughout most of the archaeological record. Under experimental conditions, collagen is apparently preserved as an intact molecule, with amino acid compositions and isotopic profiles only changing when almost all of the protein is lost. The ubiquity of collagen in archaeological bone has lead to the development of the use of collagen peptide mass fingerprints for the identification of bone fragments—Zooarchaeology by Mass Spectrometry (ZooMS). We report a novel, but a simple method for the partial extraction of collagen for ZooMS that uses ammonium bicarbonate buffer but avoids demineralisation. We compared conventional acid demineralisation with ammonium bicarbonate buffer extraction to test ZooMS in a range of modern and archaeological bone samples. The sensitivity of the current generation of mass spectrometers is high enough for the non-destructive buffer method to extract sufficient collagen for ZooMS. We envisage that a particular advantage of this method is that it leaves worked bone artefacts effectively undamaged post-treatment, suitable for subsequent analysis or museum storage or display. Furthermore, it may have potential as a screening tool to aid curators in the selection of material for more advanced molecular analysis—such as DNA sequencing.

As part of an ongoing project at the World Heritage Site of Bukhara, we investigated the glue used in the construction of the Khoja Zaynuddin mosque. Analysis by a range of techniques confirmed that it consisted of a collagen-based glue. However, the glue contained many non-protein constituents. The presence of lipid material suggests that the glue was produced by a relatively unsophisticated process. More surprisingly, various marker compounds of urine were found. Study of the viscosity of a mixture of a modern collagen-based glue and urea showed that the presence of urea at a concentration typical of human urine reduced the viscosity of the glue, thus improving its spreading characteristics and enabling its use at a lower temperature. Whilst the advantages of adding urea to glue were known to craftsmen in Europe and America in the 19th century, it would appear that the builders of Bukhara were aware of the technique, using less pure components, at a significantly earlier date.

Archaeological work in advance of construction at a site on the edge of York, UK, yielded human remains of prehistoric to Romano-British date. Amongst these was a mandible and cranium, the intra-cranial space of which contained shrunken but macroscopically recognizable remains of a brain. Although the distinctive surface morphology of the organ is preserved, little recognizable brain histology survives. Though rare, the survival of brain tissue in otherwise skeletalised human remains from wet burial environments is not unique. A survey of the literature shows that similar brain masses have been previously reported in diverse circumstances. We argue for a greater awareness of these brain masses and for more attention to be paid to their detection and identification in order to improve the reporting rate and to allow a more comprehensive study of this rare archaeological survival.

During the late Pleistocene, a large number of species colonized North America from Eurasia through the Bering Land Bridge. At least four different genera within the tribe Ovibovini are known from this time, the holarctically distributed Ovibo s moschatus (the extant musk ox) and Praeovibos , and the nearctically distributed Bootherium and Euceratherium . Questions about the taxonomic relationships of the four North American musk oxen arose soon after their description and continue to be unresolved at the present. In this study we present the first DNA and protein analyses, of the four mentioned genera. Phylogenetic analysis at the mitochondrial level indicates that Praeovibos falls within the diversity of modern Ovibos , which might indicate that Praeovibos is simply an earlier morphotype of the extant species O. moschatus . In contrast Euceratherium and Bootherium are clearly distinct genera, in agreement with the findings of previous morphological studies.

Image analysis techniques have been used to investigate the likelihood of being able to classify and assign a probability regarding the plant origin of individual starch granules in a collection of granules. Quantifiable variables were used to characterize the granules, and the assignments and probabilities were calculated objectively. We consider the classification of images containing granules of a single species and of mixed species and the possibility of assigning a class to granules of unknown species in an image of a slide obtained from the dental calculus of chimpanzees.

Is it possible to identify cooked, rather than burnt, bone? Mild heating (≤100 °C,1 h) – typical of cooking – does not lead to detectable changes in any biochemical parameter of bone yet measured. If it is only possible to detect charred bone, how is it possible to detect cooking in the archaeological record? In a previous paper (Koon et al., 2003, J. Arch. Sci.), we used a Transmission Electron Microscopy (TEM) based approach to investigate changes in the organization of the bone protein, collagen, as it is heated, using bone from heating experiments and short term burials. The work revealed that mineralized collagen, despite requiring aggressive treatment to gelatinise the protein (e.g. 90 °C, 240+ h), readily accumulates minor damage. We believe that the presence of mineral matrix stabilises the collagen enabling the damage to accumulate, but preventing it from causing immediate gelatinisation. Once the mineral is removed, the damage can be observed using appropriate visualization methods. In this paper the visualization technique was tested in a blind study of bovine bone from the Anglo-Scandinavian site of Coppergate, York. The purpose of the study was to determine if the method could discriminate between bones thought likely, on the basis of zoo-archaeological and spatial evidence, to have been cooked (high meat yield bones from a domestic context) and those which were butchered but unlikely to have been cooked (low yield bones from a butchery site). The results of the TEM analysis identified two clear groups of bones, one set more damaged than the other. This finding was consistent with archaeozoological interpretation, with the exception of one bone from the domestic context, which was not identified as having been cooked.

We describe a method of isolating and analyzing a single collagen peptide able to distinguish between sheep and goat bone collagen. The 33 amino acid peptide from both sheep and goat collagen was sequenced and shown to differ between the two species at two positions. Analysis of a range of caprines indicated that the sequence changes occurred between the divergence of the Himalayan tahr (Hemitragus jemlahicus) and the ibex (Capra ibex) and that the proposed goat marker is diagnostic of all Capra species and breeds. The survival of these markers in archaeological bones was tested using a set of 26 ovicaprid specimens from Domuztepe, a Neolithic site in south central Turkey. These markers were used to test the osteological determination of 24 of the Domuztepe bones, and determine the species for two immature specimens. The collagen-peptide method has advantages over other non-morphological methods of sheep/goat distinction because of the long-term survival of collagen over other biomolecules such as ancient DNA. The results also highlighted the problems in relying upon one morphological criterion, in this case on the distal radius, to distinguish between sheep and goat bones.

The detection and identification of protein residues from archaeological artefacts has been the subject of considerable controversy over the past two decades, this controversy spurring the application of a range of different analytical methods. Part of the debate surrounds the ability of the methods to uniquely identify original proteins. Another is the apparent contradiction between the ease of extraction in the laboratory of residues that have managed to survive in sediment over millennia; some studies use simple solvents including water, whilst others claim that efficient extraction requires the destruction of the artefact. Desorption electrospray ionisation mass spectrometry (DESI-MS) offers the potential to directly analyse proteins bound to the surface of artefacts. The method permits desorption of analytes under ambient conditions directly from a wide range of surfaces with little or no sample preparation. This has already led to its application in a range of areas, including forensic analysis of trace levels of narcotic drugs. It does not require destruction of the artefact (e.g. by drilling or dissolution) and can work in concert with other methods (for instance it could be used as a screening tool prior to lipid extraction). Here we report the construction of a DESI-MS interface and consider the potential of the method to contribute to the debate surrounding the preservation of proteins on artefacts. The DESI-MS interface was fitted to a conventional ion trap mass spectrometer and tested using peptides and proteins artificially applied to the surface of flint flakes and potsherd samples. A range of intact proteins was analysed; the DESI interface identified test proteins up to a maximum molecular weight of approximately 20 kDa. Peptides generated from these and larger proteins could be detected using DESI-MS following tryptic digestion in situ. The collected data allowed identification of the originating proteins. The potential applications to archaeological science of this new analytical tool are discussed.

DNA appears to decay by random chain scission resulting in a predictable range of fragment lengths. Collagen decay has also been modelled in this same way, although it has become increasingly evident that collagen decay does not follow this same pattern. Radiocarbon and stable isotope analysis now use ultra-filtration to isolate large fragments (>30% of original polymer length) even in Pleistocene bone. How then does collagen decay? This study contrasts experimentally degraded samples with collagen extracted from forensic, archaeological and fossil bone. In experimentally degraded bone, values for amino acid and elemental (C:N) composition, bulk δ 13C, δ 15N, and aspartic acid racemisation (AAR) changed very little until 99% of the collagen was lost, suggesting that the collagen triple helix and polypeptide chains remained remarkably intact. This suggestion was demonstrated directly by examining the integrity of individual polypeptide chains using cyanogen bromide (CNBr) cleavage followed by SDS-PAGE electrophoresis. In ancient samples, AAR values remain remarkably stable and the pattern of CNBr-cleavage was only replaced with a smear of smaller polypeptides in the oldest (Pleistocene) bones investigated. Smearing may reflect both modification of the methionine resides (the sites of CNBr-cleavage) and/or partial hydrolysis of the collagen molecule. The findings reveal why it is not usually necessary to worry about collagen diagenesis; it is mostly intact. However, evidence of partial deterioration of the oldest bone samples suggests that alternative purification strategies may increase yields in some samples.

Recent work in various parts of the world has suggested the possibility of ancient starch granules surviving and adhering to archaeological artefacts. Often this information is used to infer aspects of diet. One additional source for recovery of archaeological starch granules is dental calculus. The presence of plant food debris in dental calculus is well known but has not been not widely investigated using archaeological material. The extraction of starch granules from dental calculus represents a direct link to the consumption of starchy food by humans or animals. Using dental calculus also sidesteps many other questions still inherent in using starch granules to reconstruct aspects of ancient diet, such as the effects of diagenesis on their morphology; as the starches are trapped inside a concreted matrix they are less likely to alter over time. We used amylase digestion by a starch-specific enzyme to confirm the material as starch.

Although the recent history of human colonisation and impact on Mauritius is well documented, virtually no records of the pre-human native ecosystem exist, making it difficult to assess the magnitude of the changes brought about by human settlement. Here, we describe a 4000-year-old fossil bed at Mare aux Songes (MAS) in south-eastern Mauritius that contains both macrofossils (vertebrate fauna, gastropods, insects and flora) and microfossils (diatoms, pollen, spores and phytoliths). With >250 bone fragments/m(2) and comprising 50% of all known extinct and extant vertebrate species (n(s)=44) of Mauritius, MAS may constitute the first Holocene vertebrate bone Concentration-Lagerstatte identified on an oceanic volcanic island. Fossil remains are dominated by extinct giant tortoises Cylindraspis spp. (63%), passerines (similar to 10%), small bats (7.8%) and dodo Raphus cucullatus (7.1%). Twelve radiocarbon ages [four of them duplicates] from bones and other material suggest that accumulation of fossils took place within several centuries. An exceptional combination of abiotic conditions led to preservation of bones, bone collagen, plant tissue and microfossils. Although bone collagen is well preserved, DNA from dodo and other Mauritian vertebrates has proved difficult. Our analysis suggests that from ca 4000 years ago (4 ka), rising sea levels created a freshwater lake at MAS, generating an oasis in an otherwise dry environment which attracted a diverse vertebrate fauna. Subsequent aridification in the south-west Indian Ocean region may have increased carcass accumulation during droughts, contributing to the exceptionally high fossil concentration. The abundance of floral and faunal remains in this Lagerstatte offers a unique opportunity to reconstruct a pre-human ecosystem on an oceanic island, providing a key foundation for assessing the vulnerability of island ecosystems to human impact. (C) 2008 Elsevier Ltd. All rights reserved.

We have applied cluster analysis to mercury intrusion porosimetry data from 219 archaeological bones (121 human and 98 animal) and soil chemistry data from 219 accompanying soil samples (1 per bone sample), to investigate the influence of soil chemistry on bone preservation. The samples chosen for the study were obtained from sites ranging in time from the pre-modern to the Mesolithic and were representative of burial environments across Europe (from the Baltic to the Mediterranean). These results represent the single largest database for archaeological bone preservation in the European Holocene to date and demonstrate the potential for large-scale diagenetic studies to help develop long term preservation strategies for our European heritage. Despite the variety of sites and environments, bones could be categorised into only four main diagenetic types. Furthermore, soil chemistry appears to significantly affect only one type of preservation, the pathway characterised by loss of mineral. In neutral to basic soils, taphonomy and in particular the differences between the treatment of human and animal remains, becomes the dominating factor in determining preservation. Using these results, strategies for heritage management of archaeological sites can be suggested; grouping sites into those requiring immediate excavation and those where in situ preservation is viable. © 2006 Elsevier Ltd. All rights reserved.

In order to assess the distribution of African Palaeolithic sites and hominin remains, we need an understanding of the factors that may affect the preservation of archaeological material in the fossil record. One of the main factors that is thought to affect seriously the global distribution of fossil remains is taphonomy, but it would seem that taphonomic processes may themselves be overprinted by collagen degradation. The rate of collagen loss is highly temperature dependent, and it has been shown that bone is more likely to be preserved in areas of lower temperature and so lower collagen loss. Hence, it would be expected that fossil fauna remains would be distributed in areas of low collagen loss, i.e. low temperature. Conversely, lithics will not be subject to a temperature bias in their preservation. We have tested this hypothesis through the use of archaeological material. Our results show that the distributions of both lithic and faunal assemblages are non-random across the African continent, with all archaeological sites being found in areas of relatively low collagen degradation. This implies that bone collagen degradation is not the only factor affecting the distribution of African Palaeolithic sites and hominin remains. We suggest that the site distribution is not a reflection of bias in excavation history, but is a real phenomenon reflecting hominin habitat choice.

The majority of ancient DNA studies on human specimens have utilised teeth and bone as a source of genetic material. In this study the levels of endogenous contamination (i.e. present within the sample prior to sampling for the DNA analysis) are assessed within human bone and teeth specimens sampled from the cemetery of Santa Lucia alle Malve, Matera, Italy. This site is of exceptional interest, because the samples have been assayed for 18 measures of biochemical and physical preservation, and it is the only one identified in a study of more than 107 animal and 154 human bones from 43 sites across Europe, where a significant number of human bones was well preserved. The findings demonstrate several important issues: (a) although teeth are more resilient to contamination than bone, both are readily contaminated (presumably through handling or washing), and (b) once contaminated in this way, both are difficult (if not impossible) to decontaminate. Furthermore, although assessed on bone samples, several of the specific biochemical and physical characteristics that describe overall sample preservation, levels of microbial attack and related increases in sample porosity directly correlate with the presence of observable contamination in both bone and teeth samples from individual samples. While we can only speculate on the cause of this relationship, we posit that they provide useful guides for the assessment of whether samples are likely to be contaminated or not.

The antiquity of dairying in regions considered to be marginal, such as the Western Isles of Scotland, has generated considerable debate. Complementary biomolecular methods are now available for identifying milk residues on ceramic vessels, which provides direct evidence for this practice in the past. A range of late Bronze Age and Iron Age ceramic cooking vessels were selected from two sites on South Uist, an island in the Outer Hebrides. The presence of milk proteins and lipids on a high proportion of potsherds confirms that these vessels were originally used to process dairy products. These data were integrated with evidence from the faunal remains and ethnographic accounts, in order to examine the wider significance and implications of dairy production in the Western Isles of Scotland during the first millennium BC. Further evidence from the pottery typologies and their depositional contexts were considered in order to comment on preparation and consumption practices.

It has been demonstrated that the protein osteocalcin can survive in bone in the archaeological record, and postulated that it has the potential to survive over geological time periods. The precise mechanism for this longevity of survival is not yet fully understood, and has not been extensively studied in comparison to other diagenetic aspects of archaeological bone. We report a comparison between osteocalcin survival and the state of preservation of more than 60 bones from 14 archaeological sites. The amount of osteocalcin, assayed immunologically, was compared with diagenetic parameters that measure: the amount of ‘collagen’ in the bone, the mineral changes, the porosity, and the histological preservation of the material. The findings indicate that microbial taphonomy and mineral alteration of bone have a profoundly damaging effect on the preservation of osteocalcin.

Diagenetic alteration to archaeological bone can cause significant disruption to both the biogenic mineral structure and the preservation of biomolecular resources such as protein and DNA over archaeological time. We report here the use of a technique, small-angle X-ray scattering, which makes it possible to examine the alteration to the mineral surface due to diagenesis. This method has previously been applied to archaeological bone thin sections, but has been modified in this case for use on bone powder as a high-throughput screening technique for bone preservation. Our results show that mineral structural change is not necessarily reflected in the currently used methods of measuring lattice perfection, and that the preservation of archaeological biomolecules may be linked to structural alteration as much as to crystallinity. © 2004 Published by Elsevier Ltd.

Unlike burnt bone, cooked bone leaves no obvious trace. The ability to identify cooked bone has far reaching implications within the fields of palaeo-anthropology, archaeology and forensic science. There is, however, only limited literature on low temperature heated bone. This is not due to a lack of significance, but from an absence of any means of identifying such heating at temperatures insufficient to cause charring. The situation is further complicated by the fact that diagenetic alteration to bone may mimic heat induced changes. A previous Transmission Electron Microscopy (TEM) study showed that heat induced morphological changes to the collagen fibrils occur after low temperature heating of fish bone. This paper investigates whether these findings could be replicated on mammal bone. A series of experiments were carried out using sheep humeri. These results were compared with cooked and uncooked bones recovered from experimental burials, representing a variety of different environments (moorland, woodland and garden soil). Morphological changes to the fibrils were seen following only very mild heating events, such as short-term roasting of fleshed bone. However, similar changes were observed in unheated bone which had been buried in a low pH (3.5–4.5) soil for 7 years. Within a given burial cooked and uncooked bone was easily distinguishable. The technique, therefore, has direct application in forensic studies and may be of value in distinguishing heated from unheated bone within a given archaeological assemblage.

Ceramics were subjected to organic residue analysis from two collections: a series of middle Copper Age (Bodrogkeresztúr) vessels hitherto known as ‘milk jugs’, curated in the Magyar Nemzeti Múzeum, Budapest, and a collection of early Baden (Boleráz) vessels from the recently discovered settlement of Gyo”r-Szabadrét-domb, in western Hungary. The aim of the analyses was to establish whether or not these vessels, often associated with milk based on typological criteria, were actually used to process, store or serve dairy products. The results of the analyses revealed that no dairy products could be securely identified in the so-called ‘milk jugs’. Nevertheless dairy products were identified in other vessel types.

Although there has been great interest and controversy concerning the detection of proteins in archaeological residues, very few studies have considered the effect of different extraction methods. Using well-defined control materials, this study compares the extraction efficiency of a number of different extraction reagents. Immunological and spectrophotometric assays are used to assess the amount of protein removed by each extraction method and the effectiveness is reported with reference to previous work. None of the previously reported methods were found to be effective at removing the protein from the mineral surface. We conclude that proteins are very strongly bound to the surface probably via short-range bonds. Although this may be encouraging for the long-term preservation of proteins in this context, extraction methods will have to be re-evaluated in order to achieve efficient recovery.

If bone is considered as a composite of collagen (protein) and bioapatite (mineral), then three pathways of diagenesis are identified: (1) chemical deterioration of the organic phase; (2) chemical deterioration of the mineral phase; and (3) (micro) biological attack of the composite. The first of these three pathways is relatively unusual and will only occur in environments that are geochemically stable for bone mineral. However, because rates of biomolecular deterioration in the burial environment are slow, such bones would yield useful biomolecular information. In most environments, bones are not in thermodynamic equilibrium with the soil solution, and undergo chemical deterioration (path 2). Dissolution of the mineral exposes collagen to biodeterioration, and in most cases the initial phase of dissolution will be followed by microbial attack (path 3). Biological attack (3) also proceeds by initial demineralization; therefore paths 2 and 3 are functionally equivalent. However,in a bone that follows path 3 the damage is more localized than in path 2, and regions equivalent to path 1 may therefore exist outside these zones of destruction. Other biomolecules, such as blood proteins, cellular lipids and DNA, exist within the physiological spaces within bone.For these biomolecules, death history may be particularly important for their survival.

International audience; This paper presents the first study that combines the use of ancient crop and animal stable isotopes (carbon and nitrogen) and Zooarchaeology Mass Spectrometry species identication (ZooMS) for reconstructing early farming practices at Kouphovouno, a MiddleeLate Neolithic village in southern Greece (c. 5950e4500 cal. BC). Debate surrounding the nature of early farming predominantly revolves around the intensity of crop cultivation: did early farmers move around the landscape while practicing temporary farming methods such as slash and burn agriculture or did they create more permanent elds by investing high labor inputs into smaller pieces of land that produced higher crop yields? The need to address these questions using a direct assessment of the intensity and scale of cultivation is apparent, and an integrated stable isotope approach provides such an opportunity. The results of this study support the model of small-scale mixed farming, where crop cultivation and animal husbandry are closely integrated. The farmers directed their intensive management towards crops grown for human consumption (free-threshing wheat), while growing fodder crop (hulled barley) more extensively. Pulses were cultivated under a high-manuring/high-watering regime, likely in garden plots in rotation with free-threshing wheat. The diets of the livestock enable us to investigate which parts of the landscape were used for browsing and grazing and indicate that animal management changed in the Late Neolithic. The sheep and goats were now kept in smaller numbers and grazed together and new pasture grasses may have been sought for the grazing of cattle. This study demonstrates that beyond its applicability for palaeodietary reconstruction, analysis of stable isotopes of archaeological crop and animal remains has important implications for understanding the relationship between humans, plants and animals in an archaeological context.