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Entangled multiphoton states lie at the heart of quantum information, computing, and communications. In recent years, topology has risen as a new avenue to robustly transport quantum states in the presence of fabrication defects, disorder and other noise sources. Whereas topological protection of single photons and correlated photons has been recently demonstrated experimentally, the observation of topologically protected entangled states has thus far remained elusive. Here, we experimentally demonstrate the topological protection of spatially-entangled biphoton states. We observe robustness in crucial features of the topological biphoton correlation map in the presence of deliberately introduced disorder in the silicon nanophotonic structure, in contrast with the lack of robustness in nontopological structures. The topological protection is shown to ensure the coherent propagation of the entangled topological modes, which may lead to robust propagation of quantum information in disordered systems., Comment: 17 pages, 5 figures

Rationale Although mass spectrometry (MS) is routinely used to determine deamination in peptide mixtures, the effects of the choice of ionisation source have not yet been investigated. In particular, matrix-assisted laser desorption/ionisation (MALDI) has become a popular tool with which to measure levels of glutamine deamidation in ancient proteins. Here we use model synthetic peptides to rigorously compare MALDI and electrospray ionisation (ESI). Methods We used two synthetic peptides, with glutamine (Q) in one substituted for glutamic acid (E) in the other, to investigate the suitability of MALDI and ESI sources for the assessment of deamidation in peptides using MS. We also compared measurements of the same Q- and E-containing peptide mixtures using two different mass analysers (time-of-flight (TOF) and Fourier transform ion cyclotron resonance (FT-ICR)). Results When standard mixtures of the Q- and E-containing peptides were analysed using MALDI, under-representation of the E-containing peptide was observed. This observation was consistent between analyses carried out using either TOF or FT-ICR-MS. When the same mixtures were analysed using ESI FT-ICR-MS, no ionisation bias was observed. Conclusions MALDI may not be a suitable ionisation method for the determination of deamidation in peptide mixtures. However, ESI was successfully used to determine the ratio in known mixtures of Q- and E-containing peptides. These preliminary observations warrant further investigation into ionisation bias when measuring deamidation in other peptide sequences.

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.

Fourier transform infrared (FTIR) spectroscopy using attenuated total reflection (ATR) is commonly used for the examination of bone. During sample preparation bone is commonly ground, changing the particle size distribution. Although previous studies have examined changes in crystallinity caused by the intensity of grinding using FTIR, the effect of sample preparation (i.e. particle size and bone tissue type) on the FTIR data is still unknown. This study reports on the bone powder particle size effects on mid-IR spectra and within sample variation (i.e. periosteal, mesosteal, trabecular) using FTIR-ATR. Twenty-four archaeological human and faunal bone samples (5 heated and 19 unheated) of different chronological age (Neolithic to post-Medieval) and origin (Belgium, Britain, Denmark, Greece) were ground using either (1) a ball-mill grinder, or (2) an agate pestle and mortar, and split into grain fractions (>500 μm, 250–500 μm, 125–250 μm, 63–125 μm, and 20–63 μm). Bone powder particle size has a strong but predictable effect on the infrared splitting factor (IRSF), carbonate/phosphate (C/P) ratio, and amide/phosphate (Am/P) values. The absorbance and positions of the main peaks, the 2nd derivative components of the phosphate and carbonate bands, as well as the full width at half maximum (FWHM) of the 1010 cm−1 phosphate peak are particle size dependent. This is likely to be because of the impact of the particle size on the short- and long-range crystal order, as well as the contact between the sample and the prism, and hence the penetration depth of the IR light. Variations can be also observed between periosteal, cortical and trabecular areas of bone. We therefore propose a standard preparation method for bone powder for FTIR-ATR analysis that significantly improves accuracy, consistency, reliability, replicability and comparability of the data, enabling systematic evaluation of bone in archaeological, anthropological, paleontological, forensic and biomedical studies.

The dodo Raphus cucullatus Linnaeus, 1758, an extinct and flightless, giant pigeon endemic to Mauritius, hasfascinated people since its discovery, yet has remained surprisingly poorly known. Until the mid-19th century, almost all thatwas known about the dodo was based on illustrations and written accounts by 17th century mariners, often of questionableaccuracy. Furthermore, only a few fragmentary remains of dodos collected prior to the bird’s extinction exist. Ourunderstanding of the dodo’s anatomy was substantially enhanced by the discovery in 1865 of subfossil bones in a marsh calledthe Mare aux Songes, situated in southeastern Mauritius. However, no contextual information was recorded during earlyexcavation efforts, and the majority of excavated material comprised larger dodo bones, almost all of which wereunassociated. Here we present a modern interdisciplinary analysis of the Mare aux Songes, a 4200-year-old multitaxicvertebrate concentration Lagerst€atte. Our analysis of the deposits at this site provides the first detailed overview of theecosystem inhabited by the dodo. The interplay of climatic and geological conditions led to the exceptional preservation ofthe animal and associated plant remains at the Mare aux Songes and provides a window into the past ecosystem of Mauritius.This interdisciplinary research approach provides an ecological framework for the dodo, complementing insights on itsanatomy derived from the only associated dodo skeletons known, both of which were collected by Etienne Thirioux and arethe primary subject of this memoir.

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

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.

In this paper, we describe palaeoproteomic evidence obtained from a stained medieval birth girdle using a previously developed dry non-invasive sampling technique. The parchment birth girdle studied (Wellcome Collection Western MS. 632) was made in England in the late fifteenth century and was thought to be used by pregnant women while giving birth. We were able to extract both human and non-human peptides from the manuscript, including evidence for the use of honey, cereals, ovicaprine milk and legumes. In addition, a large number of human peptides were detected on the birth roll, many of which are found in cervico-vaginal fluid. This suggests that the birth roll was actively used during childbirth. This study is, to our knowledge, the first to extract and analyse non-collagenous peptides from a birth girdle using this sampling method and demonstrates the potential of this type of analysis for stained manuscripts, providing direct biomolecular evidence for active use.

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

In this paper we describe a dry non-invasive extraction method to detect palaeoproteomic evidence from stained manuscripts. The manuscript analysed in this study is a medieval parchment birth girdle (Wellcome Collection Western MS. 632) made in England and thought to be used by pregnant women while giving birth. Using a dry non-invasive sampling method we were able to extract both human and non-human peptides from the stains, including evidence for the use of honey, cereals, ovicaprine milk and legumes. In addition, a large number of human peptides were detected on the birth roll, many of which are found in cervico-vaginal fluid. This suggests that the birth roll was actively used during childbirth. This study is the first to extract and analyse non-collagenous peptides from a parchment document using a dry non-invasive sampling method and demonstrates the potential of this type of analysis for stained manuscripts, providing direct biomolecular evidence for active use.

The extensive peat bogs of Southern Scandinavia have yielded rich Mesolithic archaeological assemblages, with one of the most iconic artefacts being the bone point. Although great in number they remain understudied. Here we present a combined investigation of the typology, protein-based species composition, and absolute chronology of Maglemosian bone points. The majority of the bone points are made from cervids and bovines. However, changes both in species composition and barb morphology can be directly linked to a paucity of finds lasting nearly 600 years in Southern Scandinavia around 10,300 cal BP. We hypothesize that this hiatus was climate-driven and forced hunter-gatherers to abandon the lakes. Furthermore, the marked change in bone points coincides with a change in lithic technology. We, therefore, propose that the Maglemose culture in Southern Scandinavia is fundamentally divided into an Early Complex and a Late Complex. Results - Bone points - Radiocarbon dating - Protein analysis Discussion Methods - Materials - Radiocarbon dating - Proteomics

We report demonstrations of both quadrature squeezed vacuum and photon number difference squeezing generated in an integrated nanophotonic device. Squeezed light is generated via strongly driven spontaneous four-wave mixing below threshold in silicon nitride microring resonators. The generated light is characterized with both homodyne detection and direct measurements of photon statistics using photon number-resolving transition edge sensors. We measure $1.0(1)$~dB of broadband quadrature squeezing (${\sim}4$~dB inferred on-chip) and $1.5(3)$~dB of photon number difference squeezing (${\sim}7$~dB inferred on-chip). Nearly-single temporal mode operation is achieved, with measured raw unheralded second-order correlations $g^{(2)}$ as high as $1.95(1)$. Multi-photon events of over 10 photons are directly detected with rates exceeding any previous quantum optical demonstration using integrated nanophotonics. These results will have an enabling impact on scaling continuous variable quantum technology., Comment: Minor revisions, and Fig. 5 corrected; Now published in Science Advances

Rates of peptide bond hydrolysis and other diagenetic reactions are not favourable for Mesozoic protein survival. Proteins hydrolyse into peptide fragments and free amino acids that, in open systems such as bone, can leach from the specimen and be further degraded. However, closed systems are more likely to retain degradation products derived from endogenous proteins. Amino acid racemisation data in experimental and subfossil material suggests that mollusc shell and avian eggshell calcite crystals can demonstrate closed system behaviour, retaining endogenous amino acids. Here, high-performance liquid chromatography reveals that the intra-crystalline fraction of Late Cretaceous (estimated ~80 Ma) titanosaur sauropod eggshell is enriched in some of the most stable amino acids (Glx, Gly, Ala, and possibly Val) and those that racemise are fully racemic, despite being some of the slowest racemising amino acids. These results are consistent with degradation trends deduced from modern, thermally matured, sub-fossil, and ~3.8 Ma avian eggshell, as well as ~30 Ma calcitic mollusc opercula. Selective preservation of certain fully racemic amino acids, which do not racemise in-chain, along with similar concentrations of free versus total hydrolysable amino acids, likely suggests complete hydrolysis of original peptides. Liquid chromatography-tandem mass spectrometry supports this hypothesis by failing to detect any non-contamination peptide sequences from the Mesozoic eggshell. Pyrolysis-gas chromatography-mass spectrometry reveals pyrolysates consistent with amino acids as well as aliphatic hydrocarbon homologues that are not present in modern eggshell, suggestive of kerogen formation deriving from eggshell lipids. Raman spectroscopy yields bands consistent with various organic molecules, possibly including N-bearing molecules or geopolymers. These closed-system amino acids are possibly the most thoroughly supported non-avian dinosaur endogenous protein-derived constituents, at least those that have not undergone oxidative condensation with other classes of biomolecules. Biocrystal matrices can help preserve mobile organic molecules by trapping them (perhaps with the assistance of resistant organic polymers), but trapped organics are nevertheless prone to diagenetic degradation even if such reactions might be slowed in exceptional circumstances. The evidence for complete hydrolysis and degradation of most amino acids in the eggshell raises concern about the validity of reported polypeptide sequences from open-system non-avian dinosaur bone and other Mesozoic fossils.

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

Ancient proteomics is being applied to samples dating further and further back in time, with many palaeontological specimens providing protein sequence data for phylogenetic analysis as well as protein degradation studies. However, fossils are a precious material and proteomic analysis is destructive and costly. In this paper we consider three different techniques (ATR-FTIR, MALDI-ToF MS and chiral AA analysis) to screen fossil material for potential protein preservation, aiming to maximise the proteomic information recovered and saving costly time consuming analyses which may produce low quality results. It was found that splitting factor and C/P indices from ATR-FTIR were not a reliable indicator of protein survival as they are confounded by secondary mineralisation of the fossil material. Both MALDI-ToF MS and chiral AA analysis results were able to successfully identify samples with surviving proteins, and it is suggested that one or both of these analyses be used for screening palaeontological specimens.Significance: This study has shown both chiral amino acid analysis and MALDI-ToF MS are reliable screening methods for predicting protein survival in fossils. Both these methods are quick, cheap, minimally destructive (1 mg and 15 mg respectively) and can provide crucial additional information about the endogeneity of the surviving proteins. It is hoped that the use of these screening methods will encourage the examination of a wide range of palaeontological specimens for potential proteomic analysis. This in turn will give us a better under standing of protein survival far back in time and under different environmental conditions.

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.

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

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

We report our experimental results on topologically protected path-entangled photonic states using dimer chains in silicon photonics. These results highlight the potential of the lattice topology to protect photonic quantum information.

Ancient protein analysis is providing new insights into the evolutionary relationships between hominin fossils across the Pleistocene. Protein identification commonly relies on the proteolysis of a protein extract using a single protease, trypsin. As with modern proteome studies, alternative or additional proteases have the potential to increase both proteome size and protein sequence recovery. This could enhance the recovery of phylogenetic information from ancient proteomes. Here we identify 18 novel hominin bone specimens from the Kleine Feldhofer Grotte using MALDI-TOF MS peptide mass fingerprinting of collagen type I. Next, we use one of these hominin bone specimens and three Late Pleistocene Equidae specimens identified in a similar manner and present a comparison of the bone proteome size and protein sequence recovery obtained after using nanoLC-MS/MS and parallel proteolysis using six different proteases, including trypsin. We observe that the majority of the preserved bone proteome is inaccessible to trypsin. We also observe that for proteins recovered consistently across several proteases, protein sequence coverage can be increased significantly by combining peptide identifications from two or more proteases. Our results thereby demonstrate that the proteolysis of Pleistocene proteomes by several proteases has clear advantages when addressing evolutionary questions in palaeoproteomics.SignificanceMaximizing proteome and protein sequence recovery of ancient skeletal proteomes is important when analyzing unique hominin fossils. As with modern proteome studies, palaeoproteomic analysis of Pleistocene bone and dentine samples has almost exclusively used trypsin as its only protease, despite the demonstrated advantages of alternative proteases to increase proteome recovery in modern proteome studies. We demonstrate that Pleistocene bone proteomes can be significantly expanded by using additional proteases beside trypsin, and that this also improves sequence coverage of individual proteins. The use of several alternative proteases beside trypsin therefore has major benefits to maximize the phylogenetic information retrieved from ancient skeletal proteomes.

Ancient starch research illuminates aspects of human ecology and economic botany that drove human evolution and cultural complexity over time, with a special emphasis on past technology, diet, health, and adaptation to changing environments and socio-economic systems. However, lapses in prevailing starch research demonstrate the exaggerated expectations for the field that have been generated over the last few decades. This includes an absence of explanation for the millennial-scale survivability of a biochemically degradable polymer, and difficulties in establishing authenticity and taxonomic identification. This paper outlines new taphonomic and authenticity criteria to guide future work toward designing research programs that fully exploit the potential of ancient starch while considering growing demands from readers, editors, and reviewers that look for objective compositional identification of putatively ancient starch granules.

Defect modes in two-dimensional periodic photonic structures have found use in diverse optical devices. For example, photonic crystal cavities confine optical modes to subwavelength volumes and can be used for enhancement of nonlinearity, lasing and cavity quantum electrodynamics. Defect-core photonic crystal fibres allow for supercontinuum generation and endlessly single-mode fibres with large cores. However, these modes are notoriously fragile: small structural change leads to significant detuning of resonance frequency and mode volume. Here, we show that photonic topological crystalline insulator structures can be used to topologically protect the mode frequency at mid-gap and minimize the volume of a photonic defect mode. We experimentally demonstrate this in a femtosecond-laser-written waveguide array by observing the presence of a topological zero mode confined to the corner of the array. The robustness of this mode is guaranteed by a topological invariant that protects zero-dimensional states embedded in a two-dimensional environment—a novel form of topological protection that has not been previously demonstrated. Eigenmodes of photonic crystal defects have now been topologically protected in an experimental demonstration that also shows how to minimize the mode volume.

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

Chemotaxis provides a mechanism for directing the transport of molecules along chemical gradients. Here, we show the chemotactic migration of dye molecules in response to the gradients of several different neutral polymers. The magnitude of chemotactic response depends on the structure of the monomer, polymer molecular weight and concentration, and the nature of the solvent. The mechanism involves cross-diffusion up the polymer gradient, driven by favorable dye-polymer interaction. Modeling allows us to quantitatively evaluate the strength of the interaction and the effect of the various parameters that govern chemotaxis.

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

The potential information to be obtained from the large corpus of codices and documents written on parchment is immense. Aside from the obvious textual information there is a wealth of biomolecular information trapped in the fibres of the parchment itself. We have developed a non-invasive method for sampling parchment, using conventional conservation erasers, enabling us to objectively identify the species origin of the membranes through the use of peptide mass fingerprinting (PMF) and from this begin to shed light on the manufacturing processes. By performing a widespread and systematic analysis of species origin of parchment documents we can begin to address questions about local animal economies, craft production and book production, demonstrating the importance of parchment objects., Fiddyment Sarah, Collins Matthew. From field to frame. The contribution of bioarchaeological methods to understanding parchment production. In: Gazette du livre médiéval, n°63.2017. pp. 55-63.

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

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

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

The cellular cytoplasm is crowded with macromolecules and other species that occupy up to 40% of the available volume. Previous studies have reported that for high crowder molecule concentrations, colloidal tracer particles have a dampened diffusion due to the higher solution viscosity. However, these studies employed uniform distributions of crowder molecules. We report a scenario, previously unexplored experimentally, of increased tracer transport driven by a non-uniform concentration of crowder macromolecules. In gradients of polymeric crowder, tracer particles undergo transport several times higher than that of their bulk diffusion rate. The direction of the transport is toward regions of lower crowder concentration. Mechanistically, hard-sphere interactions and the resulting volume exclusion between the tracer and crowder increases the effective diffusion by inducing a convective motion of tracers. Strikingly, soft deformable particles show even greater enhancement in transport in crowder gradients compared to similarly sized hard particles. We propose a model that supports the data and quantifies a “diffusiophoretic buoyancy force” when a tracer is in a gradient of crowder concentration. Overall, this demonstration of enhanced transport in non-uniform distributions of crowder is anticipated to clarify aspects of multi-component intracellular transport.

This work was supported by the Max Planck Society, the Leverhulme Trust (through a Leverhulme prize to C.S.), the Mäxi Foundation Zurich (to F.R.), and the National Science Foundation (BCS-1516633 to C.W.). During the European Middle Ages, the opening of long-distance Asian trade routes introduced exotic goods, including ultramarine, a brilliant blue pigment produced from lapis lazuli stone mined only in Afghanistan. Rare and as expensive as gold, this pigment transformed the European color palette, but little is known about its early trade or use. Here, we report the discovery of lapis lazuli pigment preserved in the dental calculus of a religious woman in Germany radiocarbon-dated to the 11th or early 12th century. The early use of this pigment by a religious woman challenges widespread assumptions about its limited availability in medieval Europe and the gendered production of illuminated texts. Publisher PDF

© 2019 Elsevier B.V. The discovery of petrous bone as an excellent repository for ancient biomolecules has been a turning point in biomolecular archaeology, especially in aDNA research, but excessive and uncontrolled sampling could result in loss of this valuable resource for future research. This study reports on the histological (optical microscopy), physical (FTIR-ATR), elemental (CHN) and biochemical (collagen and DNA analysis) preservation of 15 human petrous bones spanning from the c. 2100 BCE to 1850 CE. Through the combined application of a number of diagenetic parameters (general histological index; infrared splitting factor; carbonate/phosphate ratio; amide/phosphate ratio; col wt%; % C, % N and C/N of whole bone and collagen; % endogenous DNA), we provide new insights into petrous bone micromorphological characteristics and diagenesis, and new evidence to enhance screening practices for aDNA and collagen analysis.

We report experiments showing robustness of correlated and entangled biphoton states generated and guided in silicon quantum circuits with non-trivial topology. These results open new avenues to develop robust quantum qubits for quantum information systems.

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.

We present a complete ancient human genome and oral microbiome sequenced from a piece of resinous "chewing gum" recovered from a Stone Age site on the island of Lolland, Denmark, and directly dated to 5,858-5,661 cal. BP (GrM-13305; 5,007+/-11). We sequenced the genome to an average depth-of-coverage of 2.3x and find that the individual who chewed the resin was female and genetically more closely related to western hunter-gatherers from mainland Europe, than hunter-gatherers from central Scandinavia. We use imputed genotypes to predict physical characteristics and find that she had dark skin and hair, and blue eyes. Lastly, we also recovered microbial DNA that is characteristic of an oral microbiome and faunal reads that likely associate with diet. The results highlight the potential for this type of sample material as a new source of ancient human and microbial DNA.

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.

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.