Search

Between the sixteenth and nineteenth century, British agriculture underwent a ‘revolutionary’ transformation. Yet despite over a century of research and the recognised centrality of agricultural developments to industrialisation and population growth, the character or chronology of any ‘revolution’ during this period remains contentious. Enquiry has been hampered by the fragmented and locally specific nature of historic accounts and the broad dating of early-modern zooarchaeological assemblages. To address this, we conducted stable isotope analysis on 658 legal documents written on sheepskin parchment; a unique biological resource that records the day, month and year of use (AD 1499 to 1969). We find these provide a high temporal resolution analysis of changing agricultural practices and episodes of disease. Most significantly, they suggest that if an ‘Agricultural Revolution’ occurred in livestock management, it did so from the mid-nineteenth century, in the aftermath of the Napoleonic Wars.

Gigantopithecus blacki was a giant hominid that inhabited densely forested environments of Southeast Asia during the Pleistocene epoch1. Its evolutionary relationships to other great ape species, and the divergence of these species during the Middle and Late Miocene epoch (16-5.3 million years ago), remain unclear2,3. Hypotheses regarding the relationships between Gigantopithecus and extinct and extant hominids are wide ranging but difficult to substantiate because of its highly derived dentognathic morphology, the absence of cranial and post-cranial remains1,3-6, and the lack of independent molecular validation. We retrieved dental enamel proteome sequences from a 1.9-million-year-old G. blacki molar found in Chuifeng Cave, China7,8. The thermal age of these protein sequences is approximately five times greater than that of any previously published mammalian proteome or genome. We demonstrate that Gigantopithecus is a sister clade to orangutans (genus Pongo) with a common ancestor about 12-10 million years ago, implying that the divergence of Gigantopithecus from Pongo forms part of the Miocene radiation of great apes. In addition, we hypothesize that the expression of alpha-2-HS-glycoprotein, which has not been previously observed in enamel proteomes, had a role in the biomineralization of the thick enamel crowns that characterize the large molars in Gigantopithecus9,10. The survival of an Early Pleistocene dental enamel proteome in the subtropics further expands the scope of palaeoproteomic analysis into geographical areas and time periods previously considered incompatible with the preservation of substantial amounts of genetic information. E.C. and F.W. are supported by VILLUM FONDEN (17649) and by the European Commission through a Marie Skłodowska-Curie (MSCA) Individual Fellowship (795569). T.M.-B. is supported by BFU2017-86471-P (MINECO/FEDER, UE), NIHM grant U01 MH106874, Howard Hughes International Early Career grant, Obra Social ‘La Caixa’ and Secretaria d’Universitats i Recerca and CERCA Programme del Departament d’Economia i Coneixement de la Generalitat de Catalunya (GRC 2017 SGR 880). E.C., J.C., J.V.O., D.S. and P.G. are supported by the Marie Skłodowska-Curie European Training Network (ETN) TEMPERA, a project funded by the European Union’s EU Framework Program for Research and Innovation Horizon 2020 under grant agreement no. 722606. M.J.C. and M.M. are supported by the Danish National Research Foundation award PROTEIOS (DNRF128). Work at the Novo Nordisk Foundation Center for Protein Research is funded in part by a donation from the Novo Nordisk Foundation (NNF14CC0001). Research at Chuifeng Cave is made possible by support from the National Natural Science Foundation of China (41572023) and by a grant from the Bagui Scholar of Guangxi. M.K. was supported by a Deutsche Forschungsgemeinschaft (DFG) fellowship (KU 3467/1-1) and the Postdoctoral Junior Leader Fellowship Programme from ‘la Caixa’ Banking Foundation (LCF/BQ/PR19/11700002). M.E.A. is supported by the Independent Research Fund Denmark (7027-00147B). We thank E. Willerslev for critical reading of the manuscript, scientific support and guidance.

Paleoproteomics, the study of ancient proteins, is a rapidly growing field at the intersection of molecular biology, paleontology, archaeology, paleoecology, and history. Paleoproteomics research leverages the longevity and diversity of proteins to explore fundamental questions about the past. While its origins predate the characterization of DNA, it was only with the advent of soft ionization mass spectrometry that the study of ancient proteins became truly feasible. Technological gains over the past 20 years have allowed increasing opportunities to better understand preservation, degradation, and recovery of the rich bioarchive of ancient proteins found in the archaeological and paleontological records. Growing from a handful of studies in the 1990s on individual highly abundant ancient proteins, paleoproteomics today is an expanding field with diverse applications ranging from the taxonomic identification of highly fragmented bones and shells and the phylogenetic resolution of extinct species to the exploration of past cuisines from dental calculus and pottery food crusts and the characterization of past diseases. More broadly, these studies have opened new doors in understanding past human-animal interactions, the reconstruction of past environments and environmental changes, the expansion of the hominin fossil record through large scale screening of nondiagnostic bone fragments, and the phylogenetic resolution of the vertebrate fossil record. Even with these advances, much of the ancient proteomic record still remains unexplored. Here we provide an overview of the history of the field, a summary of the major methods and applications currently in use, and a critical evaluation of current challenges. We conclude by looking to the future, for which innovative solutions and emerging technology will play an important role in enabling us to access the still unexplored "dark" proteome, allowing for a fuller understanding of the role ancient proteins can play in the interpretation of the past.

Mollusc shells represent excellent systems for the preservation and retrieval of genuine biomolecules from archaeological or palaeontological samples. As a consequence, the post-mortem breakdown of intracrystalline mollusc shell proteins has been extensively investigated, particularly with regard to its potential use as a "molecular clock" for geochronological applications. But despite seventy years of ancient protein research, the fundamental aspects of diagenesis-induced changes to protein structures and sequences remain elusive. In this study we investigate the degradation of intracrystalline proteins by performing artificial degradation experiments on the shell of the thorny oyster, Spondylus gaederopus, which is particularly important for archaeological research. We used immunochemistry and tandem mass tag (TMT) quantitative proteomics, a novel analytical approach that allowed us to simultaneously track patterns of structural loss and of peptide bond hydrolysis. Powdered and bleached shell samples were heated in water at four different temperatures (80, 95, 110, 140 °C) for different time durations. The structural loss of carbohydrate and protein groups was investigated by immunochemical techniques (ELLA and ELISA) and peptide bond hydrolysis was studied by tracking the changes in protein/peptide relative abundances over time using TMT quantitative proteomics. We find that heating does not induce instant organic matrix decay, but first facilitates the uncoiling of cross-linked structures, thus improving matrix detection. We calculated apparent activation energies of structural loss: Ea (carbohydrate groups) = 104.7 kJ/mol, Ea (protein epitopes) = 104.4 kJ/mol, which suggests that secondary matrix structure degradation may proceed simultaneously with protein hydrolysis. While prolonged heating at 110 °C (10 days) results in complete loss of the structural signal, surviving peptide sequences were still observed. Eight hydrolysis-prone peptide bonds were identified in the top scoring shell sequence, the uncharacterised protein LOC117318053 from Pecten maximus. Interestingly, these were not the expected "weak" bonds based on published theoretical stabilities calculated for peptides in solution. This further confirms that intracrystalline protein degradation patterns are complex and that the overall microchemical environment plays an active role in protein stability. Our TMT approach represents a major stepping stone towards developing a model for studying protein diagenesis in biomineralised systems.

Ancient protein analysis is a rapidly developing field of research. Proteins ranging in age from the Quaternary to Jurassic are being used to answer questions about phylogeny, evolution, and extinction. However, these analyses are sometimes contentious, and focus primarily on large vertebrates in sedimentary fossilisation environments; there are few studies of protein preservation in fossils in amber. Here we show exceptionally slow racemisation rates during thermal degradation experiments of resin enclosed feathers, relative to previous thermal degradation experiments of ostrich eggshell, coral skeleton, and limpet shell. We also recover amino acids from two specimens of fossil feathers in amber. The amino acid compositions are broadly similar to those of degraded feathers, but concentrations are very low, suggesting that much of the original protein has been degraded and lost. High levels of racemisation in more apolar, slowly racemising amino acids suggest that some of the amino acids were ancient and therefore original. Our findings indicate that the unique fossilisation environment inside amber shows potential for the recovery of ancient amino acids and proteins.

We used palaeoproteomics and peptide mass fingerprinting to obtain secure species identifications of key specimens of early domesticated fauna from South Africa, dating to ca. 2000 BP. It can be difficult to distinguish fragmentary remains of early domesticates (sheep) from similar-sized local wild bovids (grey duiker, grey rhebok, springbok—southern Africa lacks wild sheep) based on morphology alone. Our analysis revealed a Zooarchaeology by Mass Spectrometry (ZooMS) marker (m/z 1532) present in wild bovids and we demonstrate through LC–MS/MS that it is capable of discriminating between wild bovids and caprine domesticates. We confirm that the Spoegrivier specimen dated to 2105 ± 65 BP is indeed a sheep. This is the earliest directly dated evidence of domesticated animals in southern Africa. As well as the traditional method of analysing bone fragments, we show the utility of minimally destructive sampling methods such as PVC eraser and polishing films for successful ZooMS identification. We also show that collagen extracted more than 25 years ago for the purpose of radiocarbon dating can yield successful ZooMS identification. Our study demonstrates the importance of developing appropriate regional frameworks of comparison for future research using ZooMS as a method of biomolecular species identification.

The domestic dog has inhabited the anthropogenic niche for at least 15 000 years, but despite their impact on human strategies, the lives of dogs and their interactions with humans have only recently become a subject of interest to archaeologists. In the Arctic, dogs rely exclusively on humans for food during the winter, and while stable isotope analyses have revealed dietary similarities at some sites, deciphering the details of provisioning strategies have been challenging. In this study, we apply zooarchaeology by mass spectrometry (ZooMS) and liquid chromatography tandem mass spectrometry to dog palaeofaeces to investigate protein preservation in this highly degradable material and obtain information about the diet of domestic dogs at the Nunalleq site, Alaska. We identify a suite of digestive and metabolic proteins from the host species, demonstrating the utility of this material as a novel and viable substrate for the recovery of gastrointestinal proteomes. The recovered proteins revealed that the Nunalleq dogs consumed a range of Pacific salmon species (coho, chum, chinook and sockeye) and that the consumed tissues derived from muscle and bone tissues as well as roe and guts. Overall, the study demonstrated the viability of permafrost-preserved palaeofaeces as a unique source of host and dietary proteomes. 1. Introduction 2. Material and methods (a) Samples (b) Palaeoproteomics (c) Zooarchaeology by mass spectrometry 3. Results and discussion (a) Metaproteomics (b) Host proteins (c) Dietary proteins (d) Challenges and future directions 4. Conclusion

Unidad de excelencia María de Maeztu CEX2019-000940-M The origins, prevalence and nature of dairying have been long debated by archaeologists. Within the last decade, new advances in high-resolution mass spectrometry have allowed for the direct detection of milk proteins from archaeological remains, including ceramic residues, dental calculus, and preserved dairy products. Proteins recovered from archaeological remains are susceptible to post-excavation and laboratory contamination, a particular concern for ancient dairying studies as milk proteins such as beta-lactoglobulin (BLG) and caseins are potential laboratory contaminants. Here, we examine how site-specific rates of deamidation (i.e., deamidation occurring in specific positions in the protein chain) can be used to elucidate patterns of peptide degradation, and authenticate ancient milk proteins. First, we characterize site-specific deamidation patterns in modern milk products and experimental samples, confirming that deamidation occurs primarily at low half-time sites. We then compare this to previously published palaeoproteomic data from six studies reporting ancient milk peptides. We confirm that site-specific deamidation rates, on average, are more advanced in BLG recovered from ancient dental calculus and pottery residues. Nevertheless, deamidation rates displayed a high degree of variability, making it challenging to authenticate samples with relatively few milk peptides. We demonstrate that site-specific deamidation is a useful tool for identifying modern contamination but highlight the need for multiple lines of evidence to authenticate ancient protein data.

Dire wolves are considered to be one of the most common and widespread large carnivores in Pleistocene America1, yet relatively little is known about their evolution or extinction. Here, to reconstruct the evolutionary history of dire wolves, we sequenced five genomes from sub-fossil remains dating from 13,000 to more than 50,000 years ago. Our results indicate that although they were similar morphologically to the extant grey wolf, dire wolves were a highly divergent lineage that split from living canids around 5.7 million years ago. In contrast to numerous examples of hybridization across Canidae2,3, there is no evidence for gene flow between dire wolves and either North American grey wolves or coyotes. This suggests that dire wolves evolved in isolation from the Pleistocene ancestors of these species. Our results also support an early New World origin of dire wolves, while the ancestors of grey wolves, coyotes and dholes evolved in Eurasia and colonized North America only relatively recently. © 2021, The Author(s), under exclusive licence to Springer Nature Limited. Acknowledgements We thank the staff at the Carnegie Museum of Natural History, Cincinnati Museum Center, Danish Zoological Museum, Harrison Zoological Museum, Harvard Museum of Comparative Zoology, Idaho Museum of Natural History, Institute of Archaeology (Russian Academy of Sciences), Institute of Systematics and Animal Ecology (Russian Academy of Sciences), Institute of Zoology (Chinese Academy of Sciences), Instituto de Conservação da Natureza e das Florestas, Kansas Museum of Natural History, La Brea Tar Pits and Museum, Ludwig Maximilian University, McClung Museum, Museum of the Institute of Plant and Animal Ecology (Russian Academy of Sciences), Museum national d’Histoire naturelle, National Museums Scotland, Natural History Museum London, Naturalis Biodiversity Center, Naturhistorisches Museum Bern, Smithsonian National Museum of Natural History, Swedish Naturhistoriska Riksmuseet, SYLVATROP, US Bureau of Reclamation, University of California Museum of Paleontology, University of Texas at El Paso, University of Washington Burke Museum and the Zoological Institute (Russian Academy of Sciences; state assignment no. АААА-А19-119032590102-7) for access to specimens in their care; T. Barnosky, S. Bray, A. Farrell, R. Fischer, A. Harris, J. Harris, A. Henrici, P. Holroyd, R. MacPhee, T. Martin, A. Philpot, J. Saunders, J. Southon, G. Storrs, G. Takeuchi, X. Wang and C. Widga for assistance; and L. DeSantis for comments. A.M. used computational and storage services associated with the Hoffman2 Shared Cluster provided by UCLA Institute for Digital Research and Education’s Research Technology Group. DireGWC was sequenced using the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley, supported by NIH S10 OD018174 Instrumentation Grant. We acknowledge the assistance of the Danish National High-Throughput Sequencing Centre, BGI-Europe, the Garvan Institute of Medical Research and the Australian Cancer Research Foundation (ACRF) Cancer Genomics Facility for assistance in Illumina and BGIseq500 data generation. A.R.P. was supported by a Marie Curie COFUND Junior Research Fellowship (Durham University). A.M. was supported by an NSF grant (award number: 1457106) and the QCB Collaboratory Postdoctoral Fellowship (UCLA). L.A.F.F., J.H., A.H.-B. and G.L. were supported by either European Research Council grant (ERC-2013-StG-337574-UNDEAD and ERC-2019-StG-853272-PALAEOFARM) and/or Natural Environmental Research Council grants (NE/K005243/1 and NE/K003259/1). K.S. was supported by a grant from Barrett, the Honors College at Arizona State University. A.T.O. was supported by the Strategic Initiative Funds, Office of the President, Arizona State University to the Institute of Human Origins DNA and Human Origins at Arizona State University project. L.A.F.F. was supported by a Junior Research Fellowship (Wolfson College, University of Oxford) and L.A.F.F. and A. Carmagnini were supported by the Wellcome Trust (210119/Z/18/Z). S.G. was supported by Carlsbergfondet grant CF14–0995 and Marie Skłodowska-Curie Actions grant 655732-WhereWolf. M.T.P.G. was supported by ERC Consolidator grant 681396-Extinction Genomics. B.S. and J.K. were supported by IMLS MG-30-17-0045-17 and NSF DEB-1754451. A.H.-B. was supported by the Leverhulme Trust (ECF-2017-315). A. Cooper, K.J.M. and H.H. were supported by the Australian Research Council. A.T.S. and G.G. were supported by Australian Government Research Training Program Scholarships. A.T.L. was supported by the Peter Buck Postdoctoral Fellowship from the Smithsonian Institution’s National Museum of Natural History. Y.V.K. was supported by the by State Assignment of the Sobolev Institute of Geology and Mineralogy.

The rise of ancient genomics has revolutionised our understanding of human prehistory but this work depends on the availability of suitable samples. Here we present a complete ancient human genome and oral microbiome sequenced from a 5700 year-old piece of chewed birch pitch from Denmark. We sequence the human genome to an average depth of 2.3× and find that the individual who chewed the pitch was female and that she was genetically more closely related to western hunter-gatherers from mainland Europe than hunter-gatherers from central Scandinavia. We also find that she likely had dark skin, dark brown hair and blue eyes. In addition, we identify DNA fragments from several bacterial and viral taxa, including Epstein-Barr virus, as well as animal and plant DNA, which may have derived from a recent meal. The results highlight the potential of chewed birch pitch as a source of ancient DNA., Birch pitch is thought to have been used in prehistoric times as hafting material or antiseptic and tooth imprints suggest that it was chewed. Here, the authors report a 5,700 year-old piece of chewed birch pitch from Denmark from which they successfully recovered a complete ancient human genome and oral microbiome DNA.

Jetsam ambergris, found on beaches worldwide, has always been assumed to originate as a natural product of sperm whales (Physeteroidea). However, only indirect evidence has ever been produced for this, such as the presence of whale prey remains in ambergris. Here, we extracted and analysed DNA sequences from jetsam ambergris from beaches in New Zealand and Sri Lanka, and sequences from ambergris of a sperm whale beached in The Netherlands. The lipid-rich composition of ambergris facilitated high preservation-quality of endogenous DNA, upon which we performed shotgun Illumina sequencing. Alignment of mitochondrial and nuclear genome sequences with open-access reference data for multiple whale species confirms that all three jetsam samples derived originally from sperm whales ( Physeter macrocephalus ). Shotgun sequencing here also provides implications for metagenomic insights into ambergris-preserved DNA. These results demonstrate significant implications for elucidating the origins of jetsam ambergris as a prized natural product, and also for the understanding of sperm whale metabolism and diet, and the ecological mechanisms underlying these coproliths.

Funder: FP7 Ideas: European Research Council; funder-id: http://dx.doi.org/10.13039/100011199; Grant(s): 295729, The recovery and analysis of ancient DNA and protein from archaeological bone is time-consuming and expensive to carry out, while it involves the partial or complete destruction of valuable or rare specimens. The fields of palaeogenetic and palaeoproteomic research would benefit greatly from techniques that can assess the molecular quality prior to sampling. To be relevant, such screening methods should be effective, minimally-destructive, and rapid. This study reports results based on spectroscopic (Fourier-transform infrared spectroscopy in attenuated total reflectance [FTIR-ATR]; n = 266), palaeoproteomic (collagen content; n = 226), and palaeogenetic (endogenous DNA content; n = 88) techniques. We establish thresholds for three different FTIR indices, a) the infrared splitting factor [IRSF] that assesses relative changes in bioapatite crystals’ size and homogeneity; b) the carbonate-to-phosphate [C/P] ratio as a relative measure of carbonate content in bioapatite crystals; and c) the amide-to-phosphate ratio [Am/P] for assessing the relative organic content preserved in bone. These thresholds are both extremely reliable and easy to apply for the successful and rapid distinction between well- and poorly-preserved specimens. This is a milestone for choosing appropriate samples prior to genomic and collagen analyses, with important implications for biomolecular archaeology and palaeontology.

Today, practical, functional and symbolic choices inform the selection of raw materials for worked objects. In cases where we can discern the origin of worked bone, tooth, ivory and antler objects in the past, we assume that similar choices are being made. However, morphological species identification of worked objects is often impossible due to the loss of identifying characteristics during manufacture. Here, we describe a novel non-destructive ZooMS (Zooarchaeology by Mass Spectrometry) method which was applied to bone points from Pre-Contact St. Lawrence Iroquoian village sites in southern Quebec, Canada. The traditional ZooMS technique requires destructive analysis of a sample, which can be problematic when dealing with artefacts. Here we instead extracted proteins from the plastic bags in which the points had been stored. ZooMS analysis revealed hitherto unexpected species, notably black bear (Ursus americanus) and human (Homo sapiens sapiens), used in point manufacture. These surprising results (confirmed through genomic sequencing) highlight the importance of advancing biomolecular research in artefact studies. Furthermore, they unexpectedly and exceptionally allow us to identify and explore the tangible, material traces of the symbolic relationship between bears and humans, central to past and present Iroquoian cosmology and mythology.

Motivation Classification of archaeological animal samples is commonly achieved via manual examination of matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) spectra. This is a time-consuming process which requires significant training and which does not produce a measure of confidence in the classification. We present a new, automated method for arriving at a classification of a MALDI-ToF sample, provided the collagen sequences for each candidate species are available. The approach derives a set of peptide masses from the sequence data for comparison with the sample data, which is carried out by cross-correlation. A novel way of combining evidence from multiple marker peptides is used to interpret the raw alignments and arrive at a classification with an associated confidence measure. Results To illustrate the efficacy of the approach, we tested the new method with a previously published classification of parchment folia from a copy of the Gospel of Luke, produced around 1120 C.E. by scribes at St Augustine’s Abbey in Canterbury, UK. In total, 80 of the 81 samples were given identical classifications by both methods. In addition, the new method gives a quantifiable level of confidence in each classification. Availability and implementation The software can be found at https://github.com/bioarch-sjh/bacollite, and can be installed in R using devtools. Supplementary information Supplementary data are available at Bioinformatics online.

The living tree sloths Choloepus and Bradypus are the only remaining members of Folivora, a major xenarthran radiation that occupied a wide range of habitats in many parts of the western hemisphere during the Cenozoic, including both continents and the West Indies. Ancient DNA evidence has played only a minor role in folivoran systematics, as most sloths lived in places not conducive to genomic preservation. Here we utilize collagen sequence information, both separately and in combination with published mitochondrial DNA evidence, to assess the relationships of tree sloths and their extinct relatives. Results from phylogenetic analysis of these datasets differ substantially from morphology-based concepts: Choloepus groups with Mylodontidae, not Megalonychidae; Bradypus and Megalonyx pair together as megatherioids, while monophyletic Antillean sloths may be sister to all other folivorans. Divergence estimates are consistent with fossil evidence for mid-Cenozoic presence of sloths in the West Indies and an early Miocene radiation in South America., Facultad de Ciencias Naturales y Museo

Cattle were domesticated ∼10,000 years ago, but analysis of modern breeds has not elucidated their origins. Verdugo et al. performed genome-wide analysis of 67 ancient Near Eastern Bos taurus DNA samples. Several populations of ancient aurochs were progenitors of domestic cows. These genetic lineages mixed ∼4000 years ago in a region around the Indus Valley. Interestingly, mitochondrial analysis indicated that genetic material likely derived from arid-adapted Bos indicus (zebu) bulls was introduced by introgression.Science, this issue p. 173Genome-wide analysis of 67 ancient Near Eastern cattle, Bos taurus, remains reveals regional variation that has since been obscured by admixture in modern populations. Comparisons of genomes of early domestic cattle to their aurochs progenitors identify diverse origins with separate introgressions of wild stock. A later region-wide Bronze Age shift indicates rapid and widespread introgression of zebu, Bos indicus, from the Indus Valley. This process was likely stimulated at the onset of the current geological age, ~4.2 thousand years ago, by a widespread multicentury drought. In contrast to genome-wide admixture, mitochondrial DNA stasis supports that this introgression was male-driven, suggesting that selection of arid-adapted zebu bulls enhanced herd survival. This human-mediated migration of zebu-derived genetics has continued through millennia, altering tropical herding on each continent.

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.

Zooarchaeology by Mass Spectrometry (ZooMS) is rapidly becoming a staple in archaeological and cultural heritage science. Developed a decade ago, this peptide mass fingerprinting technique is expanding from a small group of researchers mainly involved in method development to a broader group of scientists using it as another tool in their toolboxes. With new researchers beginning to use the method, it is imperative that a user-friendly, standardized approach be established. A major barrier has been the often haphazard and changing nomenclature used to label peptide markers necessary for taxonomic identification. Consistent, reliable, and easy-to-understand nomenclature is key to the growth of ZooMS, particularly as the reference library continues to expand. We propose a new set of standardized guidelines for peptide markers based on their position in the type I collagen sequence from the beginning of the highly conserved, helical region. Since this region has no insertions or deletions over a wide range of taxonomic groups, the proposed nomenclature system can be used reliably and consistently across all vertebrate taxa. We propose to label ZooMS peptide markers with the gene, followed by the position of the first and last amino acid of the marker from the start of the helical region. Significance statement We propose a standardized nomenclature system for ZooMS peptide markers that provides consistent labels across multiple, broad taxonomic groups. This system unambiguously locates the marker peptides in the type I collagen sequence, avoids duplication of marker names, and facilitates the creation of large ZooMS databases which can include all vertebrates.

Bacteria play an important role in the degradation of bone material. However, much remains to be learnt about the structure of their communities in degrading bone, and how the depositional environment influences their diversity throughout the exposure period. We genetically profiled the bacterial community in an experimental series of pig bone fragments (femur and humeri) deposited at different well-defined environments in Denmark. The bacterial community in the bone fragments and surrounding depositional environment were studied over one year, and correlated with the bioerosion damage patterns observed microscopically in the bones. We observed that the bacterial communities within the bones were heavily influenced by the local microbial community, and that the general bone microbial diversity increases with time after exposure. We found the presence of several known collagenase producing bacterial groups, and also observed increases in the relative abundance of several of these in bones with tunneling. We anticipate that future analyses using shotgun metagenomics on this and similar datasets will be able to provide insights into mechanisms of microbiome driven bone degradation. © 2020 Eriksen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

How humans got their goatsLittle is known regarding the location and mode of the early domestication of animals such as goats for husbandry. To investigate the history of the goat, Dalyet al.sequenced mitochondrial and nuclear sequences from ancient specimens ranging from hundreds to thousands of years in age. Multiple wild populations contributed to the origin of modern goats during the Neolithic. Over time, one mitochondrial type spread and became dominant worldwide. However, at the whole-genome level, modern goat populations are a mix of goats from different sources and provide evidence for a multilocus process of domestication in the Near East. Furthermore, the patterns described support the idea of multiple dispersal routes out of the Fertile Crescent region by domesticated animals and their human counterparts.Science, this issue p.85

In Western Europe, the Middle to Upper Paleolithic transition is associated with the disappearance of Neandertals and the spread of anatomically modern humans (AMHs). Current chronological, behavioral, and biological models of this transitional period hinge on the Châtelperronian technocomplex. At the site of the Grotte du Renne, Arcy-sur-Cure, morphological Neandertal specimens are not directly dated but are contextually associated with the Châtelperronian, which contains bone points and beads. The association between Neandertals and this "transitional" assemblage has been controversial because of the lack either of a direct hominin radiocarbon date or of molecular confirmation of the Neandertal affiliation. Here we provide further evidence for a Neandertal-Châtelperronian association at the Grotte du Renne through biomolecular and chronological analysis. We identified 28 additional hominin specimens through zooarchaeology by mass spectrometry (ZooMS) screening of morphologically uninformative bone specimens from Châtelperronian layers at the Grotte du Renne. Next, we obtain an ancient hominin bone proteome through liquid chromatography-MS/MS analysis and error-tolerant amino acid sequence analysis. Analysis of this palaeoproteome allows us to provide phylogenetic and physiological information on these ancient hominin specimens. We distinguish Late Pleistocene clades within the genus Homo based on ancient protein evidence through the identification of an archaic-derived amino acid sequence for the collagen type X, alpha-1 (COL10α1) protein. We support this by obtaining ancient mtDNA sequences, which indicate a Neandertal ancestry for these specimens. Direct accelerator mass spectometry radiocarbon dating and Bayesian modeling confirm that the hominin specimens date to the Châtelperronian at the Grotte du Renne.

Deamidation of glutamine (Q) and asparagine (N) has been recognized as a marker of degradation and aging in ancient proteins. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to study deamidation in wool textiles, we identified eight peptides from α-keratin proteins in sheep wool that could potentially be used to assess the level of degradation. For each chosen peptide, the extent of deamidation was determined by comparing the calculated theoretical distribution with the measured distribution using a genetic algorithm that gives the best fit to the measured distribution. Variations in the levels of deamidation were observed between peptides and in modern wool samples buried for up to 8 years in which deamidation levels were relatively low under short-term burial. In contrast, deamidation was higher in archeological textile fragments from medieval sites ranging from the 9th to 13th century in York (United Kingdom) and Newcastle (United Kingdom) and from the 13th to 16th century in Reykholt (Iceland). Major differences were observed between the British and the Icelandic samples, showing a negative correlation between age of samples and levels of deamidation, but highlighting the effect of local environment. In addition, nanoscale liquid chromatography-electrospray ionization tandem mass spectrometry (nanoLC-ESI-MS/MS) data indicated that deamidation in wool's α-keratin was influenced by primary and higher-order structures. Predominance of deamidation on glutamine rather than asparagine in the archeological samples was attributed to a higher abundance of Q in the α-helical core domain of keratins, neighboring residues and steric hindrance preventing deamidation of N.

The facile racemization of protein-bound amino acids plays an important role in the aging and pathologies of living tissues, and it can be exploited for protein geochronological studies in subfossil biominerals. However, the in-chain degradation pathways of amino acids are complex and difficult to elucidate. Serine has proven to be particularly elusive, and its ability to racemize as a peptide-bound residue (like asparagine and aspartic acid) has not been demonstrated. This study investigates the patterns of degradation of a model peptide (WNSVWAW) at elevated temperatures, quantifying the extent of racemization and peptide bond hydrolysis using reverse-phase high-performance liquid chromatography (RP-HPLC) and tracking the presence of degradation products by MALDI-MS. We provide direct evidence that, under these experimental conditions, both serine and asparagine are able to undergo racemization as internally bound residues, which shows their potential for initiating protein breakdown and provides an explanation for the presence of d-enantiomers in living mammalian tissues.

We report the characterisation of meat and bone meal (MBM) standards (Set B-EFPRA) derived from cattle, sheep, pig and chicken, each rendered at four different temperatures (133, 137, 141 and 145 °C). The standards, prepared for an EU programme STRATFEED (to develop new methodologies for the detection and quantification of illegal addition of mammalian tissues in feeding stuffs), have been widely circulated and used to assess a range of methods for identification of the species composition of MBM. The overall state of mineral alteration and protein preservation as a function of temperature was monitored using small angle X-ray diffraction (SAXS), amino acid composition and racemization analyses. Progressive increases in protein damage and mineral alteration in chicken and cattle standards was observed. In the case of sheep and pig, there was greater damage to the proteins and alteration of the minerals at the lowest treatment temperature (133 °C), suggesting that the thermal treatments must have been compromised in some way. This problem has probably impacted upon the numerous studies which tested methods against these heat treatments. We use protein mass spectrometric methods to explore if thermostable proteins could be used to identify rendered MBM. In more thermally altered samples, so-called 'thermostable' proteins such as osteocalcin which has been proposed as a ideal target to speciate MBM were no longer detectable, but the structural protein type I collagen could be used to differentiate all four species, even in the most thermally altered samples.

We investigate the origin of archaeological wool textiles preserved by anoxic waterlogging from seven medieval archaeological deposits in north-western Europe (c. 700–1600 AD), using geospatial patterning in carbon (δ13C), nitrogen (δ15N) and non-exchangeable hydrogen (δ2H) composition of modern and ancient sheep proteins. δ13C, δ15N and δ2H values from archaeological wool keratin (n = 83) and bone collagen (n = 59) from four sites were interpreted with reference to the composition of modern sheep wool from the same regions. The isotopic composition of wool and bone collagen samples clustered strongly by settlement; inter-regional relationships were largely parallel in modern and ancient samples, though landscape change was also significant. Degradation in archaeological wool samples, examined by elemental and amino acid composition, was greater in samples from Iceland (Reykholt) than in samples from north-east England (York, Newcastle) or northern Germany (Hessens). A nominal assignment approach was used to classify textiles into local/non-local at each site, based on maximal estimates of isotopic variability in modern sheep wool. Light element stable isotope analysis provided new insights into the origins of wool textiles, and demonstrates that isotopic provenancing of keratin preserved in anoxic waterlogged contexts is feasible. We also demonstrate the utility of δ2H analysis to understand the location of origin of archaeological protein samples.

Shamanic belief systems represent the first form of religious practice visible within the global archaeological record. Here we report on the earliest known evidence of shamanic costume: modified red deer crania headdresses from the Early Holocene site of Star Carr (c. 11 kya). More than 90% of the examples from prehistoric Europe come from this one site, establishing it as a place of outstanding shamanistic/cosmological significance. Our work, involving a programme of experimental replication, analysis of macroscopic traces, organic residue analysis and 3D image acquisition, metrology and visualisation, represents the first attempt to understand the manufacturing processes used to create these artefacts. The results produced were unexpected-rather than being carefully crafted objects, elements of their production can only be described as expedient.

East African elephants have been hunted for their ivory for millennia but the nineteenth century witnessed strongly escalating demand from Europe and North America. It has been suggested that one consequence was that by the 1880s elephant herds along the coast had become scarce, and to meet demand, trade caravans trekked farther into interior regions of East Africa, extending the extraction frontier. The steady decimation of elephant populations coupled with the extension of trade networks have also been claimed to have triggered significant ecological and socio-economic changes that left lasting legacies across the region. To explore the feasibility of using an isotopic approach to uncover a ‘moving frontier’ of elephant extraction, we constructed a baseline isotope data set (δ13C, δ15N, δ18O and 87Sr/86Sr) for historic East African elephants known to have come from three distinct regions (coastal, Rift Valley, and inland Lakes). Using the isotope results with other climate data and geographical mapping tools, it was possible to characterise elephants from different habitats across the region. This baseline data set was then used to provenance elephant ivory of unknown geographical provenance that was exported from East Africa during the late nineteenth and early twentieth centuries to determine its likely origin. This produced a better understanding of historic elephant geography in the region, and the data have the potential to be used to provenance older archaeological ivories, and to inform contemporary elephant conservation strategies.

International audience; One contribution of 16 to a theme issue 'From DNA barcodes to biomes'. Over the last few centuries, many cetacean species have witnessed dramatic global declines due to industrial overharvesting and other anthropogenic influences, and thus are key targets for conservation. Whale bones recovered from archaeological and palaeontological contexts can provide essential baseline information on the past geographical distribution and abundance of species required for developing informed conservation policies. Here we review the challenges with identifying whale bones through traditional anatomical methods, as well as the opportunities provided by new molecular analyses. Through a case study focused on the North Sea, we demonstrate how the utility of this (pre)historic data is currently limited by a lack of accurate taxonomic information for the majority of ancient cetacean remains. We then discuss current opportunities presented by molecular identification methods such as DNA barcoding and collagen peptide mass fingerprinting (zooarchaeology by mass spectrometry), and highlight the importance of molecular identifications in assessing ancient species' distributions through a case study focused on the Mediterranean. We conclude by considering high-throughput molecular approaches such as hybridization capture followed by next-generation sequencing as cost-effective approaches for enhancing the ecological informativeness of these ancient sample sets. This article is part of the themed issue 'From DNA barcodes to biomes'.

The authors acknowledge Science Foundation Ireland European Research Council (ERC) Support Award 12/ERC/B2227, Valeria Mattiangeli, and the Trinity Genome Sequencing Laboratory (TrinSeq) for MiSeq support. This work was supported by the Marie Curie International Fellowship PALIMPSEST FP7-PEOPLE-2011-IEF 299101, a University of Manchester Research Institute seedcorn grant, British Academy Postdoctoral Fellowship funding, and ERC Investigator Grant 295729-CodeX. Tissue-thin parchment made it possible to produce the first pocket Bibles: Thousands were made in the 13th century. The source of this parchment, often called “uterine vellum,” has been a long-standing controversy in codicology. Use of the Latin term abortivum in many sources has led some scholars to suggest that the skin of fetal calves or sheep was used. Others have argued that it would not be possible to sustain herds if so many pocket Bibles were produced from fetal skins, arguing instead for unexpected alternatives, such as rabbit. Here, we report a simple and objective technique using standard conservation treatments to identify the animal origin of parchment. The noninvasive method is a variant on zooarchaeology by mass spectrometry (ZooMS) peptide mass fingerprinting but extracts protein from the parchment surface by using an electrostatic charge generated by gentle rubbing of a PVC eraser on the membrane surface. Using this method, we analyzed 72 pocket Bibles originating in France, England, and Italy and 293 additional parchment samples that bracket this period. We found no evidence for the use of unexpected animals; however, we did identify the use of more than one mammal species in a single manuscript, consistent with the local availability of hides. These results suggest that ultrafine vellum does not necessarily derive from the use of abortive or newborn animals with ultrathin hides, but could equally well reflect a production process that allowed the skins of maturing animals of several species to be rendered into vellum of equal quality and fineness. Postprint

We used high-sensitivity, high-resolution tandem mass spectrometry to shotgun sequence ancient protein remains extracted from a 43 000 year old woolly mammoth ( Mammuthus primigenius ) bone preserved in the Siberian permafrost. For the first time, 126 unique protein accessions, mostly low-abundance extracellular matrix and plasma proteins, were confidently identified by solid molecular evidence. Among the best characterized was the carrier protein serum albumin, presenting two single amino acid substitutions compared to extant African ( Loxodonta africana ) and Indian ( Elephas maximus ) elephants. Strong evidence was observed of amino acid modifications due to post-mortem hydrolytic and oxidative damage. A consistent subset of this permafrost bone proteome was also identified in more recent Columbian mammoth ( Mammuthus columbi ) samples from temperate latitudes, extending the potential of the approach described beyond subpolar environments. Mass spectrometry-based ancient protein sequencing offers new perspectives for future molecular phylogenetic inference and physiological studies on samples not amenable to ancient DNA investigation. This approach therefore represents a further step into the ongoing integration of different high-throughput technologies for identification of ancient biomolecules, unleashing the field of paleoproteomics.

We present a novel method for the isolation and analysis of the bone collagen (I) α2 chain carboxytelopeptide as a species biomarker. Conventional methods for the analysis and sequencing of mixtures of proteins and peptides commonly involve using the protease trypsin to cleave proteins present in the sample. However, in the study of collagen, these methods result in very complex mixtures of peptides that are difficult to analyze and the acquired results are not reproducible. Here we use bacterial collagenase (from Clostridium histolyticum) for its ability to cleave the highly unusual Gly-Xaa-Yaa repeating sequence of collagen, where Xaa usually is Pro and Yaa often is Hyp. Followed by a simple isolation step using a reverse phase solid phase extraction cartridge, the α2 (I) chain carboxytelopeptide can be readily analyzed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and the results can be used to distinguish between different species of origin. © 2007 Elsevier Inc. All rights reserved.

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.

Evolutionary biologists are increasingly relying on ancient DNA from archaeological animal bones to study processes such as domestication and population dispersals. As many animal bones found on archaeological sites are likely to have been cooked, the potential for DNA preservation must be carefully considered to maximise the chance of amplification success. Here, we assess the preservation of mitochondrial DNA in a medieval cattle bone assemblage from Coppergate, York, UK. These bones have variable degrees of thermal alterations to bone collagen fibrils, indicative of cooking. Our results show that DNA preservation is not reliant on the presence of intact collagen fibrils. In fact, a greater number of template molecules could be extracted from bones with damaged collagen. We conclude that moderate heating of bone may enhance the retention of DNA fragments. Our results also indicate that ancient DNA preservation is highly variable, even within a relatively recent assemblage from contexts conducive to organic preservation, and that diagenetic parameters based on protein diagenesis are not always useful for predicting ancient DNA survival.

Although the application of sequencing-by-synthesis techniques to DNA extracted from bones has revolutionized the study of ancient DNA, it has been plagued by large fractions of contaminating environmental DNA. The genetic analyses of hair shafts could be a solution: We present 10 previously unexamined Siberian mammoth ( Mammuthus primigenius ) mitochondrial genomes, sequenced with up to 48-fold coverage. The observed levels of damage-derived sequencing errors were lower than those observed in previously published frozen bone samples, even though one of the specimens was >50,000 14 C years old and another had been stored for 200 years at room temperature. The method therefore sets the stage for molecular-genetic analysis of museum collections.

Xiang et al. (1) assert that chickens were domesticated on the North China plain 10,000 y ago. Although a great deal remains unknown about the temporal and geographic origins of poultry husbandry, this claim is extraordinary. We welcome the increasing application of modern bioarcheological techniques to questions pertaining to animal domestication in China, but we are skeptical about these conclusions for several reasons.

Parchment represents an invaluable cultural reservoir. Retrieving an additional layer of information from these abundant, dated livestock-skins via the use of ancient DNA (aDNA) sequencing has been mooted by a number of researchers. However, prior PCR-based work has indicated that this may be challenged by cross-individual and cross-species contamination, perhaps from the bulk parchment preparation process. Here we apply next generation sequencing to two parchments of seventeenth and eighteenth century northern English provenance. Following alignment to the published sheep, goat, cow and human genomes, it is clear that the only genome displaying substantial unique homology is sheep and this species identification is confirmed by collagen peptide mass spectrometry. Only 4% of sequence reads align preferentially to a different species indicating low contamination across species. Moreover, mitochondrial DNA sequences suggest an upper bound of contamination at 5%. Over 45% of reads aligned to the sheep genome, and even this limited sequencing exercise yield 9 and 7% of each sampled sheep genome post filtering, allowing the mapping of genetic affinity to modern British sheep breeds. We conclude that parchment represents an excellent substrate for genomic analyses of historical livestock.

Gelatine is a component of a wide range of foods. It is manufactured as a by-product of the meat industry from bone and hide, mainly from bovine and porcine sources. Accurate food labelling enables consumers to make informed decisions about the food they buy. Since labelling currently relies heavily on due diligence involving a paper trail, there could be benefits in developing a reliable test method for the consumer industries in terms of the species origin of gelatine. We present a method to determine the species origin of gelatines by peptide mass spectrometry methods. An evaluative comparison is also made with ELISA and PCR technologies. Commercial gelatines were found to contain undeclared species. Furthermore, undeclared bovine peptides were observed in commercial injection matrices. This analytical method could therefore support the food industry in terms of determining the species authenticity of gelatine in foods.

Aspartic acid racemization (AAR) represents one of the major types of non-enzymatic covalent modification that leads to an age-dependent accumulation of abnormal protein in numerous human tissues. In vivo racemization is an autonomic process during the "natural" ageing of proteins, and correlates with the age of long-lived proteins. Consequently AAR can be used as molecular indicator of protein ageing as well as for the identification of permanent proteins that age with the human organism. Although long-living, structural proteins are mainly affected, AAR may be significant on a time scale also relevant to enzymes and signaling proteins. It may result in a loss of protein function due to proteolysis or due to changes in the molecular structure. In vivo racemization may also increase in pathological conditions. AAR has already been discussed as a relevant pathophysiological factor in the pathogenesis of diseases of old age such as atherosclerosis, lung emphysema, presbyopia, cataract, degenerative diseases of cartilage and cerebral age-related dysfunctions. Although the details of the biological consequences of AAR have to be further elucidated, it is evident that AAR plays a role in the molecular biology of ageing.

No large group of recently extinct placental mammals remains as evolutionarily cryptic as the approximately 280 genera grouped as 'South American native ungulates'. To Charles Darwin, who first collected their remains, they included perhaps the 'strangest animal[s] ever discovered'. Today, much like 180 years ago, it is no clearer whether they had one origin or several, arose before or after the Cretaceous/Palaeogene transition 66.2 million years ago, or are more likely to belong with the elephants and sirenians of superorder Afrotheria than with the euungulates (cattle, horses, and allies) of superorder Laurasiatheria. Morphology-based analyses have proved unconvincing because convergences are pervasive among unrelated ungulate-like placentals. Approaches using ancient DNA have also been unsuccessful, probably because of rapid DNA degradation in semitropical and temperate deposits. Here we apply proteomic analysis to screen bone samples of the Late Quaternary South American native ungulate taxa Toxodon (Notoungulata) and Macrauchenia (Litopterna) for phylogenetically informative protein sequences. For each ungulate, we obtain approximately 90% direct sequence coverage of type I collagen α1- and α2-chains, representing approximately 900 of 1,140 amino-acid residues for each subunit. A phylogeny is estimated from an alignment of these fossil sequences with collagen (I) gene transcripts from available mammalian genomes or mass spectrometrically derived sequence data obtained for this study. The resulting consensus tree agrees well with recent higher-level mammalian phylogenies. Toxodon and Macrauchenia form a monophyletic group whose sister taxon is not Afrotheria or any of its constituent clades as recently claimed, but instead crown Perissodactyla (horses, tapirs, and rhinoceroses). These results are consistent with the origin of at least some South American native ungulates from 'condylarths', a paraphyletic assembly of archaic placentals. With ongoing improvements in instrumentation and analytical procedures, proteomics may produce a revolution in systematics such as that achieved by genomics, but with the possibility of reaching much further back in time., La lista completa de autores puede consultarse en el documento o en la página web de la revista., Facultad de Ciencias Naturales y Museo

Human colonization of the New World is generally believed to have entailed migrations from Siberia across the Bering isthmus. However, the limited archaeological record of these migrations means that details of the timing, cause and rate remain cryptic. Here, we have used a combination of ancient DNA, 14C dating, hydrogen and oxygen isotopes, and collagen sequencing to explore the colonization history of one of the few other large mammals to have successfully migrated into the Americas at this time: the North American elk ( Cervus elaphus canadensis ), also known as wapiti. We identify a long-term occupation of northeast Siberia, far beyond the species’s current Old World distribution. Migration into North America occurred at the end of the last glaciation, while the northeast Siberian source population became extinct only within the last 500 years. This finding is congruent with a similar proposed delay in human colonization, inferred from modern human mitochondrial DNA, and suggestions that the Bering isthmus was not traversable during parts of the Late Pleistocene. Our data imply a fundamental constraint in crossing Beringia, placing limits on the age and mode of human settlement in the Americas, and further establish the utility of ancient DNA in palaeontological investigations of species histories.

Immunological detection of proteins adsorbed to mineral and ceramic surfaces has proved not only difficult but controversial. Unlike the immunological detection of proteins associated with carbonate or phosphate minerals (e.g. shells and bones) proteins adsorbed to siliceous minerals cannot readily be removed by dissolution of the mineral phase. We have previously examined alternative extraction methodologies which claim to bring the protein into solution, but found none of these to be effective. Here we report a novel strategy for immuno-detection of proteins adsorbed to siliceous minerals, the Digestion and Capture Immunoassay (DACIA). The method involves the use of cold, concentrated (4M) hydrofluoric acid (HF) with the simultaneous capture of liberated protein onto a solid phase. The combination of low temperatures and surface stabilisation enables us to detect epitopes from even partially degraded proteins. The method may have a wide application in forensic, archaeological, soil and earth sciences.

In ancient and/or damaged artefacts containing keratinous materials, the species of origin of the materials can be difficult to identify through visual examination; therefore, a minimally destructive methodology for species identification is required. While hair fibres from some species have seen substantial characterisation, others such as horn or baleen have received little or no attention, or lack protein sequences allowing formal identification using proteomics techniques.We used the PMF method (Peptide Mass Fingerprinting with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS)) to catalogue and identify diagnostic peptide markers up to the genus level. Sequences were checked using nanoflow liquid chromatography/electrospray ionisation tandem mass spectrometry (nanoLC/ESI-MS/MS) and unidentified peptides were searched against a theoretical database generated by substituting amino acids in keratin sequences.Specific peptides were identified by m/z and sequences characterised whenever possible for a range of species belonging to Bovidae and Camelidae, and for tissues such as baleen and horn. The theoretical database allowed an increase in the number of peptides of up to 10% in species with little genetic information.A proteomics approach can successfully identify specific markers for the identification of materials to the genus level, and should be considered when identification by other means is not possible. Identification by PMF is fast, reliable and inexpensive.

Claims of extreme survival of DNA have emphasized the need for reliable models of DNA degradation through time. By analysing mitochondrial DNA (mtDNA) from 158 radiocarbon-dated bones of the extinct New Zealand moa, we confirm empirically a long-hypothesized exponential decay relationship. The average DNA half-life within this geographically constrained fossil assemblage was estimated to be 521 years for a 242 bp mtDNA sequence, corresponding to a per nucleotide fragmentation rate ( k ) of 5.50 × 10 –6 per year. With an effective burial temperature of 13.1°C, the rate is almost 400 times slower than predicted from published kinetic data of in vitro DNA depurination at pH 5. Although best described by an exponential model ( R 2 = 0.39), considerable sample-to-sample variance in DNA preservation could not be accounted for by geologic age. This variation likely derives from differences in taphonomy and bone diagenesis, which have confounded previous, less spatially constrained attempts to study DNA decay kinetics. Lastly, by calculating DNA fragmentation rates on Illumina HiSeq data, we show that nuclear DNA has degraded at least twice as fast as mtDNA. These results provide a baseline for predicting long-term DNA survival in bone.

Non-enzymatic deamidation accumulates in aging tissues in vivo and has been proposed to be potentially useful as a molecular clock. The process continues post mortem, and here we explore the increase in levels of deamidation in archaeological collagen, as measured during Zooarchaeology by Mass Spectrometry (ZooMS) analysis.With the high sensitivity of current generation mass spectrometers, ZooMS provides a non-destructive and highly cost-effective method to characterise collagen peptides. Deamidation can be detected by mass spectrometry as a +0.984 Da mass shift; therefore, aside from its original purpose, peptide mass-fingerprinting for bone identification, ZooMS concurrently yields a 'thermal indicator' of the samples.By analysis of conventional ZooMS spectra, we determined the deamidation rate for glutamine residues in 911 bone collagen samples from 50 sites, with ages varying from medieval to Palaeolithic. The degree of deamidation was compared to diagenetic parameters and nearby sequence properties.The extent of deamidation was found to be influenced more by burial conditions and thermal age than, for example, chronological age, the extent of bioerosion or crystallinity. The method lends itself mostly to screening heterogenic deposits of bone to identify outliers.

Hardy, Karen et al., Neanderthals disappeared sometime between 30,000 and 24,000 years ago. Until recently, Neanderthals were understood to have been predominantly meat-eaters; however, a growing body of evidence suggests their diet also included plants. We present the results of a study, in which sequential thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS) and pyrolysis-gas chromatographymass spectrometry (Py-GC-MS) were combined with morphological analysis of plant microfossils, to identify material entrapped in dental calculus from five Neanderthal individuals from the north Spanish site of El Sidrón. Our results provide the first molecular evidence for inhalation of wood-fire smoke and bitumen or oil shale and ingestion of a range of cooked plant foods. We also offer the first evidence for the use of medicinal plants by a Neanderthal individual. The varied use of plants that we have identified suggests that the Neanderthal occupants of El Sidrón had a sophisticated knowledge of their natural surroundings which included the ability to select and use certain plants. © Springer-Verlag 2012., Field work was supported by Consejería de Cultura del Principado de Asturias, and some technical aspects by Ministerio de Ciencia e Innovación, Spain, grant CGL2006-02131.

The British Quaternary, spanning roughly the past 2.6 million years, is unmatched for the biodiversity and abundance of its fossil localities and its record of climatic contrasts. However, with all but the most recent deposits beyond radiocarbon range, and with no readily datable volcanic rocks, it has been difficult to get accurate dates for many British Pleistocene deposits. Penkman et al. have developed new analytical methods based on the intra-crystalline proteins of stable biominerals (in the 'opercula' shell closure of the freshwater gastropod Bithynia), common in Quaternary deposits. They obtain confident assignments for various strata to global marine isotope stages, securely placing Britain's rich record of faunal and archaeological change into a broader context. Marine and ice-core records show that the Earth has experienced a succession of glacials and interglacials during the Quaternary (last ∼2.6 million years), although it is often difficult to correlate fragmentary terrestrial records with specific cycles. Aminostratigraphy is a method potentially able to link terrestrial sequences to the marine isotope stages (MIS) of the deep-sea record1,2. We have used new methods of extraction and analysis of amino acids, preserved within the calcitic opercula of the freshwater gastropod Bithynia, to provide the most comprehensive data set for the British Pleistocene based on a single dating technique. A total of 470 opercula from 74 sites spanning the entire Quaternary are ranked in order of relative age based on the extent of protein degradation, using aspartic acid/asparagine (Asx), glutamic acid/glutamine (Glx), serine (Ser), alanine (Ala) and valine (Val). This new aminostratigraphy is consistent with the stratigraphical relationships of stratotypes, sites with independent geochronology, biostratigraphy and terrace stratigraphy3,4,5,6. The method corroborates the existence of four interglacial stages between the Anglian (MIS 12) and the Holocene in the terrestrial succession. It establishes human occupation of Britain in most interglacial stages after MIS 15, but supports the notion of human absence during the Last Interglacial (MIS 5e)7. Suspicions that the treeless ‘optimum of the Upton Warren interstadial’ at Isleworth pre-dates MIS 3 are confirmed. This new aminostratigraphy provides a robust framework against which climatic, biostratigraphical and archaeological models can be tested.

Biomolecules can experience aging processes that limit their long-term functionality in organisms. Typical markers of protein aging are spontaneous chemical modifications, such as AAR (amino acid racemization) and AAI (amino acid isomerization), mainly involving aspartate and asparagine residues. Since these modifications may affect folding and turnover, they reduce protein functionality over time and may be linked to pathological conditions. The present mini-review describes evidence of AAR and AAI involvement in the misfolding and brain accumulation of Aβ (amyloid β-peptide), a central event in AD (Alzheimer's disease) synaptic dysfunctions. Structural alterations introduced by site-specific modifications linked to protein aging may affect Aβ production, polymerization and clearance, and therefore play a pivotal role in the pathogenesis of sporadic and genetic forms of AD. Early changes associated with molecular aging also have significant long-term consequences for Aβ folding and turnover. New fast, reproducible and accurate methods for the screening of protein aging markers in biological samples may contribute to improve diagnostic and therapeutic approaches in AD.

The minerals involved in the formation of metazoan skeletons principally comprise glassy silica, calcium phosphate or carbonate. Because of their ancient heritage, glass sponges (Hexactinellida) may shed light on fundamental questions such as molecular evolution, the unique chemistry and formation of the first skeletal silica-based structures, and the origin of multicellular animals. We have studied anchoring spicules from the metre-long stalk of the glass rope sponge (Hyalonema sieboldi; Porifera, Class Hexactinellida), which are remarkable for their size, durability, flexibility and optical properties. Using slow-alkali etching of biosilica, we isolated the organic fraction, which was revealed to be dominated by a hydroxylated fibrillar collagen that contains an unusual [Gly-3Hyp-4Hyp] motif. We speculate that this motif is predisposed for silica precipitation, and provides a novel template for biosilicification in nature.

Many rare and valuable ancient specimens now carry the scars of ancient DNA research, as questions of population genetics and phylogeography require larger sample sets. This fuels the demand for reliable techniques to screen for DNA preservation prior to destructive sampling. Only one such technique has been widely adopted: the extent of aspartic acid racemization (AAR). The kinetics of AAR are believed to be similar to the rate of DNA depurination and therefore a good measure of the likelihood of DNA survival. Moreover, AAR analysis is only minimally destructive. We report the first comprehensive test of AAR using 91 bone and teeth samples from temperate and high-latitude sites that were analysed for DNA. While the AAR range of all specimens was low (0.02–0.17), no correlation was found between the extent of AAR and DNA amplification success. Additional heating experiments and surveys of the literature indicated that d / l Asx is low in bones until almost all the collagen is lost. This is because aspartic acid is retained in the bone within the constrained environment of the collagen triple helix, where it cannot racemize for steric reasons. Only if the helix denatures to soluble gelatin can Asx racemize readily, but this soluble gelatine is readily lost in most burial environments. We conclude that Asx d / l is not a useful screening technique for ancient DNA from bone.