Zooarchaeology By Mass Spectrometry Research Articles

A comparative study of commercially available, minimally invasive, sampling methods on Early Neolithic humeri analysed via palaeoproteomics

Due to methodological advances in the archaeological sciences, an increasing number of archaeological specimens undergo destructive sampling. However, the preservation of cultural heritage is a primary concern. This leads to a dilemma between accessing sample material and obtaining sufficient information for a meaningful analytical outcome. Ideally, sampling a specimen would preserve the object for further macro, micro, and molecular analyses. For palaeoproteomics, a number of minimally invasive sampling approaches have been proposed, representing different benefits and limitations. There have been studies comparing a selection of these protocols, however, these have focused on specimens from a homogenous preservation environment using Zooarchaeology by Mass Spectrometry (ZooMS). Here we expand on earlier work by extending the comparison to specimens from two highly different preservation environments through both ZooMS and liquid-chromatography tandem mass spectrometry (LC-MS/MS). We compare five sampling approaches and seven extraction protocols in total, on 10 Bos sp. humeri from the Early Neolithic site of La Draga, Spain, utilising MALDI-ToF MS and LC-MS/MS to generate proteomic output, while assessing protocol invasiveness using microscopy and 3D imaging. Five humeri originate from Sector A, which is mostly related to dry, terrestrial preservation conditions, while the other five humeri stem from Sector B, which is characterised by its phreatic/aquatic preservation conditions. We show that there is a significant difference in protein recovery and taxonomic specificity between the sampling techniques applied, as well as between burial conditions. Additionally, various surface modifications were observed depending on the specific sampling technique applied. It is therefore essential to assess protein preservation for each sedimentological context within an archaeological site before performing extensive sampling, as protein preservation can be highly inter- and intra-site-specific.

Exploring the provenance of a Byzantine excavated assemblage of textile and leather finds by the application of instrumental analysis

Stereomicroscopy, Scanning Electron Microscopy-Energy Dispersive Spectroscopy (SEM–EDS), Fourier Transform Infrared spectroscopy with Attenuated Total Reflectance probe (FTIR-ATR), High Performance Liquid Chromatography with a Photodiode Array system (HPLC–PDA), X-ray Fluorescence spectroscopy (XRF), Zooarchaeology by Mass Spectrometry (ZooMS), and 14C dating were applied to an assemblage of textile and leather fragments that belong to the collection of the Byzantine & Christian Museum of Athens in Greece and lacked inventory information.The analyses applied, along with bibliographical research, yielded information on the materials, techniques and provenance of the find. The assemblage of finds consists of a gold thread embroidered silk satin, dyed with shellfish-purple; a gold strip-drawn wire embroidered silk tabby; a gold strip-drawn wire and red silk thread tapestry; a gold-thread embroidery on a tabby background; fragments of braided cord; and leather fragments. The material components could have been produced locally, in the area of the Byzantine Empire, though some of them indicate connections with India and/or China. The assemblage, dated between the mid AD 10th and mid eleventh century, was probably found in 1924 by Andreas Xygopoulos during the excavation of a tomb inside the church of Agia Sophia in Thessaloniki, Greece.

Combing through museum collections. A “museomic” application of ZooMS

AbstractThis article presents the application of Zooarchaeology by Mass Spectrometry (ZooMS) to osseous Longobard artifacts from the collection of the “Musei Reali di Torino” (MRT; Torino, Italy). Like most archaeological items made of worked bone/antler in museum collections, the raw material of such specimens is usually attributed to deer, often without accurate taxonomic attribution. Therefore, the main aim of the present investigation was to shed light on taxonomical aspects using biomolecular approaches.We first examined the collagen preservation of the artifacts, then we compared three sampling methods (invasive, eraser‐based, and bag‐based), and we evaluated the quality of the collagen fingerprint obtained. Overall, we found a good, albeit not optimal, biomolecular preservation status, even in heavily restored objects coming from the 19th‐century collections.Out of 37 specimens analyzed through matrix‐assisted laser desorption ionization–time of flight–mass spectrometry (MALDI‐TOF‐MS) and nanoHPLC‐tandem MS, 31 yielded usable data. The results confirmed the widespread use of cervid as the osseous raw material for comb‐making in Longobard times in Piedmont, but we also found that bovine bones (Bos but also other taxa belonging to family Bovidae, such as caprines) and equid bones were exploited—demonstrating opportunistic use of animal resources. As far as the method is concerned, the ZooMS peptide markers useful to distinguish between bovids and cervids (m/z 1580 vsm/z 1550) (Buckley et al., 2009) are more frequently detected when analyzing bone chips, that is, with the invasive sampling method, rather than collagen extracts obtained using non‐invasive techniques. Nonetheless, the eraser method (eZooMS) seems to be a good trade‐off between invasivity and quality of the information obtained: eZooMS sampling does not leave visible marks on the object and therefore can contribute to facilitating the routine application of biomolecular methods in the daily practice of museum conservation laboratories. Indeed, an important outcome of the present study has been the establishment of a close collaboration between museum and biomolecular specialists.Taken together, our results suggest that the Longobards had a preference towards locally available resources, although this work did not highlight a clear association between raw material (deer, cattle, and other bovids) and object typology (in the case of combs) or function, except for buttons. The overall information obtained by this study confirms the potential of biomolecular approaches for reconstructing the biography of museum objects with a long and complex life and demonstrates the value of zooarchaeological study of museum collections.

SpecieScan: semi-automated taxonomic identification of bone collagen peptides from MALDI-ToF-MS.

Zooarchaeology by Mass Spectrometry (ZooMS) is a palaeoproteomics method for the taxonomic determination of collagen, which traditionally involves challenging manual spectra analysis with limitations in quantitative results. As the ZooMS reference database expands, a faster and reproducible identification tool is necessary. Here we present SpecieScan, an open-access algorithm for automating taxa identification from raw MALDI-ToF mass spectrometry (MS) data. SpecieScan was developed using R (pre-processing) and Python (automation). The algorithm's output includes identified peptide markers, closest matching taxonomic group (taxon, family, order), correlation scores with the reference databases, and contaminant peaks present in the spectra. Testing on original MS data from bones discovered at Palaeothic archaeological sites, including Denisova Cave in Russia, as well as using publicly-available, externally produced data, we achieved >90% accuracy at the genus-level and ∼92% accuracy at the family-level for mammalian bone collagen previously analysed manually. The SpecieScan algorithm, along with the raw data used in testing, results, reference database, and common contaminants lists are freely available on Github (https://github.com/mesve/SpecieScan).

Use of hare bone for the manufacture of a Clovis bead

A tubular bone bead dating to ~ 12,940 BP was recovered from a hearth-centered activity area at the La Prele Mammoth site in Converse County, Wyoming, USA. This is the oldest known bead from the Western Hemisphere. To determine the taxonomic origin of the bead, we extracted collagen for zooarchaeology by mass spectrometry (ZooMS). We also used micro-CT scanning for morphological analysis to determine likely skeletal elements used for its production. We conclude that the bead was made from a metapodial or proximal phalanx of a hare (Lepus sp.). This find represents the first secure evidence for the use of hares during the Clovis period. While the use of hare bone for the manufacture of beads was a common practice in western North America during the Holocene, its origins can now be traced back to at least the terminal Pleistocene.

Identification and tentative removal of collagen glue in Palaeolithic worked bone objects: implications for ZooMS and radiocarbon dating

Collagen glue has been used for nearly two centuries to consolidate bone material, although its prevalence in museum collections is only now becoming visible. Identifying and removing collagen glue is crucial before the execution of any geochemical or molecular analyses. Palaeolithic bone objects from old excavations intended for radiocarbon dating were first analysed using ZooMS (Zooarchaeology by Mass Spectrometry) to identify the animal species, however peaks characteristic of both cattle and whale were discovered. Two extraction methods for ZooMS were tested to identify the authentic animal species of these objects, which revealed that these were originally whale bone objects that had been consolidated with cattle collagen glue. This is the first time animal collagen glue has been identified in archaeological remains with ZooMS, illustrating again the incredible versatility of this technique. Another technique, Fourier Transform Infrared Spectroscopy in Attenuated Total Reflectance mode (FTIR-ATR), was also tested if it could rapidly identify the presence of collagen glue in archaeological bone material, which was not the case. Two other cleaning methods were tested to remove bone glue contamination prior to radiocarbon dating, along with two modified collagen extraction methods for ZooMS. These methods were applied to bone blank samples (FmC = 0.0031 ± 0.0002, (n = 219), 47 336 ± 277 yr BP) that were experimentally consolidated with collagen glue and to the Palaeolithic bone material (ca. 15 000 and 12 000 yr BP). The experimental bone blanks produced excellent 14C ages, suggesting the cleaning methods were successful, however the 14C ages for some of the Palaeolithic material remained too young considering their contextual age, suggesting that the collagen glue contamination had most likely cross-linked to the authentic collagen molecule. More research is needed in order to gain a deeper understanding of the occurrence and elimination of cross-linked collagen-based glues in material from museum collections.

Using ZooMS to assess archaeozoological insights and unravel human subsistence behaviour at La Viña rock shelter (northern Iberia)

The highly fragmented nature of Palaeolithic faunal assemblages is a regular limitation in archaeozoological analyses as it prevents a precise taxonomic identification following comparative anatomy criteria. In this paper, we integrate Zooarchaeology by Mass Spectrometry (ZooMS) analysis of unidentifiable bone fragments within archaeozoological and taphonomic data to overcome this limitation and disentangle the subsistence strategies of Solutrean and Magdalenian human groups of the La Viña rock shelter (northern Iberia). The ZooMS results are consistent with the archaeozoological data showing preferential exploitation of red deer complemented by horses and caprids, whereas large bovids and reindeer are a minor component. This study also reveals significant species variability in classifying unidentifiable bones to mammal-size classes. The combination of biomolecular methods and traditional archaeozoology and taphonomy provides a higher resolution of faunal diversity in Palaeolithic assemblages and hominin subsistence behaviour related to carcass transport and seasonality.

Peptide mass fingerprinting as a tool to assess micromammal biodiversity in Pleistocene South Africa: The case of Klipdrift Shelter

Remains of small mammals from archaeological sites are often used as palaeoenvironmental proxies in the reconstruction of past environments. Yet, identification of micromammals to species-level based on morphological traits is often difficult due to fragmentation of diagnostic skeletal elements. Here we test the potential of Zooarchaeology by Mass Spectrometry (ZooMS) as a tool for the taxonomic identification of micromammal remains from Middle Stone Age (MSA) sequences in South Africa. ZooMS peptide markers are first established for 14 extant micromammal species present in the region. These novel peptide markers are then used to identify micromammal bone remains from the MSA levels of Klipdrift Shelter (c. 72–51 ka), De Hoop Nature Reserve, South Africa. Our study shows that collagen preservation in micromammal bones from MSA contexts is sufficient for successful ZooMS analysis. To our knowledge, these results represent the oldest material successfully analysed with ZooMS from an African context. The peptide markers developed as part of this study can be used to characterize a larger number of micromammal assemblages. This holds significant promise for the future application of ZooMS to prehistoric material in South Africa and elsewhere in the continent.

Large-scale application of palaeoproteomics (Zooarchaeology by Mass Spectrometry; ZooMS) in two Palaeolithic faunal assemblages from China.

The application of Zooarchaeology by Mass Spectrometry (ZooMS) on Pleistocene sites in Europe and northern Asia has resulted in the discovery of important new hominin fossils and has expanded the range of identified fauna. However, no systematic, large-scale application of ZooMS on Palaeolithic sites in East Asia has been attempted thus far. Here, we analyse 866 morphologically non-diagnostic bones from Jinsitai Cave in northeast China and Yumidong Cave in South China, from archaeological horizons dating to 150-10 ka BP. Bones from both sites revealed a high degree of collagen preservation and potentially time-related deamidation patterns, despite being located in very distinct environmental settings. At Jinsitai, we identified 31 camel bones, five of which were radiocarbon dated to 37-20 ka BP. All dated specimens correspond to colder periods of Marine Isotope Stages 3 and 2. We regard the presence of camels at Jinsitai as evidence of wild camels being a megafauna taxon targeted, most likely by early modern humans, during their expansion across northeast Asia. This large-scale application of ZooMS in China highlights the potential of the method for furthering our knowledge of the palaeoanthropological and zooarchaeological records of East Asia.

Tryps-IN: A streamlined palaeoproteomics workflow enables ZooMS analysis of 10,000-year-old petrous bones from Jordan rift-valley

Poor preservation of collagen in dry and/or arid environments has hindered the application of Zooarchaeology by mass spectrometry (ZooMS) analysis in many regions of the world. As a result, many zooarchaeological investigations have relied exclusively on morphological assessment of fragmentary remains due to the inadequate preservation of biomolecules. The climatic conditions of Southwest Asia include extreme temperature fluctuations unconducive to preservation of proteins and DNA. We performed zooarchaeological analysis of remains from the 10,000-year-old site of Shkārat Msaied in Jordan and sub-sampled twenty-eight petrous bones, the hardest bone in the mammalian skeleton, for species identification by ZooMS. Using an unconventional and simplified extraction protocol we call Tryps-IN, in which digestion was performed without removal of the demineralising EDTA, we taxonomically identified several fragments, outperforming an established ZooMS workflow. A subset of identifications was subsequently confirmed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) protein sequencing. The new methodology presented here opens the possibility of further bioarchaeological investigation of other fragmentary faunal assemblages within this region of archaeological significance.

Grouping groupers in the Mediterranean: Ecological baselines revealed by ancient proteins.

Marine historical ecology provides a means to establish baselines to inform current fisheries management. Groupers (Epinephelidae) are key species for fisheries in the Mediterranean, which have been heavily overfished. Species abundance and distribution prior to the 20th century in the Mediterranean remains poorly known. To reconstruct the past biogeography of Mediterranean groupers, we investigated whether Zooarchaeology by Mass Spectrometry (ZooMS) can be used for identifying intra-genus grouper bones to species level. We discovered 22 novel, species-specific ZooMS biomarkers for groupers. Applying these biomarkers to Kinet Höyük, a Mediterranean archaeological site, demonstrated 4000 years of regional Epinephelus aeneus dominance and resiliency through millennia of fishing pressures, habitat degradation and climatic changes. Combining ZooMS identifications with catch size reconstructions revealed the Epinephelus aeneus capacity for growing 30 cm larger than hitherto documented, revising the maximum Total Length from 120 to 150 cm. Our results provide ecological baselines for a key Mediterranean fishery which could be leveraged to define and assess conservation targets.

Late Pleistocene Environments of East Kazakhstan (Based on Ushbulak Site Materials)

We reconstruct environmental conditions at various stages of the Late Pleistocene and Early Holocene of East Kazakhstan. The reconstructions are based on materials from the stratified Ushbulak site in the Shilikta Valley, spanning a period from the Early Upper Paleolithic to the Bronze Age. Climatic changes were evaluated using natural science methods-mineralogical, ZooArchaeology by Mass Spectrometry (ZooMS), OSL- and AMS-dating, etc. Sevaral stages, relating to environmental changes, are evaluated. The fi rst period (~52–37 ka BP) was period of moderately warm and relatively humid climate, with predominantly forest-steppe, meadow-steppe, and semidesert landscapes. The second period (~25–21 ka BP) coincided with a transition from a moderately warm to a very cold and more arid climate dominated by steppes. The third period (~18–16 ka BP) was transitional from the glacial maximum to the postglacial interstadial, with a relatively cool and arid climate and mostly steppe and forest-steppe landscapes. The fourth period (~15–14 ka BP) was characterized by the warmest climate in the Late Pleistocene; steppe and forest-steppe vegetation dominated. During the latest, Early Holocene period, the climate was warm and humid, with savanna-like landscapes. The analysis of natural-climatic conditions allows us to conclude that the early stage of the site’s functioning, characterized by the highest intensity of settlement, was optimal for ancient man.

Archaeology demonstrates sustainable ancestral Coast Salish salmon stewardship over thousands of years.

Salmon are an essential component of the ecosystem in Tsleil-Waututh Nation's traditional, ancestral, and contemporary unceded territory, centred on present-day Burrard Inlet, BC, Canada, where Tsleil-Waututh people have been harvesting salmon, along with a wide variety of other fishes, for millennia. Tsleil-Waututh Nation is a Coast Salish community that has called the Inlet home since time immemorial. This research assesses the continuity and sustainability of the salmon fishery at təmtəmíxʷtən, an ancestral Tsleil-Waututh settlement in the Inlet, over thousands of years before European contact (1792 CE). We apply Zooarchaeology by Mass Spectrometry (ZooMS) analysis to 245 archaeological salmon vertebrae to identify the species that were harvested by the ancestral Tsleil-Waututh community that lived at təmtəmíxʷtən. The results demonstrate that Tsleil-Waututh communities consistently and preferentially fished for chum salmon (Oncorhynchus keta) over the period of almost 3,000 years. The consistent abundance indicates a sustainable chum salmon fishery over that time, and a strong salmon-to-people relationship through perhaps 100 generations. This research supports Tsleil-Waututh Nation's stewardship obligations under their ancestral legal principles to maintain conditions that uphold the Nation's way of life.

Testing osteometric species determination on zooarchaeological dolphin remains from Late Neolithic and Early Bronze Age sites in Ash-Sharqiyyah, Sultanate of Oman

Different Late Neolithic and Early Bronze Age sites located in the Ras al-Hadd cape and Ras al-Jinz Bay area (Ash-Sharqiyyah South Governorate, Sultanate of Oman) have provided thousands of zooarchaeological dolphin remains suggesting a strong reliance on the exploitation of these animals. Dolphins are hard to identify to the species level due to a highly comparable interspecies osteological morphology as well as a general lack of extensive osteological reference collections. As a result, such remains are frequently identified as “dolphin”, without any further species identification being undertaken. In this study, we assess whether an osteometric method for distinguishing the nine dolphin species that are present in Omani waters can be used to identify the zooarchaeological specimens. Zooarchaeology by Mass-Spectrometry (ZooMS) was also undertaken on a subset of the specimens but proved ineffective due to the poor preservation of the material in an arid climate. This evidence strengthens the need for effective species identification methods based on traditional zooarchaeological methods. This research is based on our ongoing analysis of the thousands of dolphin remains from the Omani zooarchaeological assemblages.

Subsistence strategies during the Gravettian in the rock shelter of La Viña (Asturias, N Spain)

The rock shelter of La Viña, located on the northern Atlantic coast of the Iberian Peninsula, contains a rich archaeological sequence dated from the Mousterian to the Magdalenian. This site preserves an extended Gravettian occupation where a diversified assemblage of fauna, lithic and bone tools has been attested. In this paper, we undertake the archaeozoological and taphonomic study of the macromammals found in the Gravettian levels X and IX of the Western Sector, recovered during the campaigns directed by Fortea between 1987 and 1992. The taphonomy indicates a high presence of fire-altered bone elements and butchery marks related to ungulate skinning and disarticulation activities, with a low presence of carnivore modifications, being Cervus elaphus, followed by Rupicapra rupicapra and Capra pyrenaica the most common taxa, therefore confirming the anthropogenic origin of the bone assemblage. Given the high fragmentation of the infantile remains identified, a biomolecular study by Zooarchaeology by Mass Spectrometry (ZooMS) was carried out to increase the taxonomic data and unravel the hunting strategies and site seasonality. Proteomic results revealed a high presence of newborn red deer and ibex, thus suggesting females herd captures during late spring/early summer.

Using bone technology and ZooMS to understand indigenous use of marine mammals at Iita, Northwest Greenland

The site of Iita in northwestern Greenland had near-continual occupation from 1000 CE to the mid-twentieth century, which presents an unparalleled opportunity to examine diachronic changes in the use of marine mammals for food and raw material resources by Pre-Inuit (Late Dorset) and Inuit–Inughuit—two culturally distinct Indigenous groups. Limited wood in High Arctic environments necessitated the use of antler, bone, and ivory for tool making. Choices in the selection and processing of osseous material reflect changes in the way these two groups used the same resources. However, modification of osseous material during the toolmaking process is often to such an extent that few diagnostic features remain for species identification. Here, we include Zooarchaeology by Mass Spectrometry (ZooMS) to identify typically indeterminate pieces of manufacturing debris from preserved collagen. The minimally destructive extraction technique using polishing films had a 99% success rate. The results suggest that selection of raw materials from certain marine mammals, particularly narwhal, by toolmakers at Iita was more frequent than previously understood through traditional faunal analysis alone. There is a paucity of narwhal from the Pre-Inuit (Late Dorset) assemblage, which contrasts starkly with the high frequency of narwhal in the Inuit–Inughuit assemblage. We demonstrate the importance of combining the analysis of bone-tool manufacturing debris with traditional dietary faunal remains to improve our understanding of resource use in coastal environments.

Machine Learning for collagen peptide biomarker determination in the taxonomic identification of archaeological fish remains

Species identification of archaeofaunal remains can be informative of changing local and global ecosystems, as well as how we interacted with them in the past. However, with the vast majority of assemblages being dominated by morphologically indeterminate specimens, methods of biomolecular species identification are becoming more popular, such as the protein fingerprint-based identification approach known as ZooMS (Zooarchaeology by Mass Spectrometry). As larger datasets are being produced, Machine Learning techniques such as those using Random Forest algorithms have been considered to expedite the identification process. However, it has proven difficult to extract meaningful biomarkers from these processes alone and so far only tackled for mammals. Here, we introduce a novel approach based on the principles of the ID3 algorithm for biomarker identification from ZooMS spectral databases, focussing on archaeological fish remains from Europe and the Caribbean. We show that the tool is highly effective at generating comprehensible lists of family- and genus-level biomarkers. At family-level identification, the average sensitivity across five families of fish was ∼0.9, where a specificity of 1 would indicate a greatly effective algorithm that outperforms both traditional Random Forest and Naïve Bayes approaches; at the genus level the mean sensitivity reduced to ∼0.8 across the nine genera tested. However, some anomalous matches were produced, with accuracy dropping when distinguishing between genera of the same family, such as Epinephelus and Mycteroperca belonging to the Serranidae. Therefore, while this tool has value in rapidly producing lists of biomarkers and can efficaciously identify new ZooMS fingerprints based on those markers, it indicates that manual intervention remains a requirement at finer taxonomic resolutions.

Ivory from early Anglo-Saxon burials in Lincolnshire – A biomolecular study

Ivory bag rings have been found in more than 70 cemeteries across southern, central, and eastern England dating to between the late-5th and 7th centuries AD. These rings are most frequently found in richly furnished female graves, and would have served as the framework for bags that hung at the waist. Debate over the source of this ivory has prevailed since the 19th century, with walrus and mammoth ivory considered as possible contenders to elephantid ivory. Recent excavations at an early Anglo-Saxon cemetery in Scremby, Lincolnshire revealed a number of elaborate female burials containing such bag rings. Using radiocarbon dating this study aimed to establish whether the rings were contemporary with the burials before seeking to identify the species of ivory through Zooarchaeology by mass spectrometry (ZooMS). Strontium analysis was also used to identify the place of residence of the elephantids at the time of tusk formation. Through a multi-methodological approach, we have established that the ivory used for the Scremby bag rings came from elephants living in an area of young volcanic rocks in Africa at some point during the 5th and 6th centuries AD. This preliminary evidence allows us to consider the networks and socio-economic factors that facilitated the distribution of ivory from Africa to the British Isles at this time.

Neanderthal subsistence, taphonomy and chronology at Salzgitter‐Lebenstedt (Germany): a multifaceted analysis of morphologically unidentifiable bone

ABSTRACTPleistocene faunal assemblages are often highly fragmented, hindering taxonomic identifications and interpretive potentials. In this paper, we apply four different methodologies to morphologically unidentifiable bone fragments from the Neanderthal open‐air site of Salzgitter‐Lebenstedt (Germany). First, we recorded zooarchaeological attributes for all 1362 unidentifiable bones recovered in 1977. Second, we applied zooarchaeology by mass spectrometry (ZooMS) to 761 fragments, and calculated glutamine deamidation values. Third, we assessed the collagen preservation of 30 fragments by near‐infrared spectroscopy (NIR) and, finally, we pretreated 10 bones with high predicted collagen values for radiocarbon dating. All returned dates at, or beyond, the limit of radiocarbon dating, indicating an age of older than 51 000 years ago. The ZooMS faunal spectrum confirms a cold environment, dominated by reindeer, alongside mammoth, horse and bison. The low occurrence of carnivore modifications (1%) contrasts with an abundance of human modifications (23%). Cut marks and marrow fractures were observed across reindeer, horse and bison. The mammoth remains are less well preserved and show a lower degree of human modifications, indicating, perhaps, a different taphonomic history. Overall, this study illustrates the importance of retaining, studying and incorporating the unidentifiable bone fraction to optimize interpretations of site formation and subsistence behaviour at Palaeolithic sites.

Solving the two-decades-old murder case through joint application of ZooMS and ancient DNA approaches

Bones are one of the most common biological types of evidence in forensic cases. Discriminating human bones from irrelevant species is important for the identification of victims; however, the highly degraded bones could be undiagnostic morphologically and difficult to analyze with standard DNA profiling approaches. The same challenge also exists in archaeological studies. Here, we present an initial study of an analytical strategy that involves zooarchaeology by mass spectrometry (ZooMS) and ancient DNA methods. Through the combined strategy, we managed to identify the only biological evidence of a two-decades-old murder case — a small piece of human bone out of 19 bone fragments — and confirmed the kinship between the victim and the putative parents through joint application of next-generation sequencing (NGS) and Sanger sequencing methods. ZooMS effectively screened out the target human bone while ancient DNA methods improve the DNA yields. The combined strategy in this case outperforms the standard DNA profiling approach with shorter time, less cost, as well as higher reliability for the genetic identification results.Highlights• The first application of zooarchaeology by mass spectrometry technique in the forensic case for screening out human bones from bone fragment mixtures.• Application of ancient DNA technique to recover the highly degraded DNA sequence from the challenging sample that failed standard DNA profiling approaches.• A fast, sensitive, and low-cost strategy that combines the strengths of protein analysis and DNA analysis for kinship identification in forensic research.