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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.

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.

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.

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.

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.

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.

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.

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.

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.

The diagenesis of ancient bone collagen appears bewildering in its variety. We have simulated collagen diagenesis using a simple conceptual model in which the degradation is controlled by two key processes: (i) depolymerization of collagen by chemical hydrolysis of peptide bonds; and (ii) dissolution (i.e. melting free) of those polypeptide fragments retained by fewer than a critical number of hydrogen bonds, biodeterioration being ignored. The model predicts that the curve describing weight loss is approximately sigmoidal in form, a pattern in accord with the sparse experimental data for non-mineralized collagen. The conclusion from studies of mineralized collagen is that the weight loss curve is exponential (i.e. a first-order reaction), but we argue that the experimental design of those studies was flawed. Other predictions of the model relating to the changing size distribution of residual fragments, N-termini, physical properties and significance of cross-linking are reported. Cross-linking appears to be a particularly significant phenomenon, which causes a marked tailing of the weight loss curve and thus the enhanced preservation of cross-linked, low collagen bone. The validity of applying this simple conceptual model to archaeological collagen is discussed.