Alkylresorcinol detection and identification in archaeological pottery using ultra-high-performance liquid chromatography-quadrupole/Orbitrap mass spectrometry.

RATIONALE: Alkylresorcinols (AR) are cereal-specific biomarkers and have recently been found in archaeological pots. However, their low concentrations and high susceptibility to degradation make them difficult to detect using conventional gas chromatography mass spectrometry (GC/MS). Here we describe the development of a more sensitive liquid chromatography mass spectrometry (LC/MS) method to detect these compounds. METHOD: A method based on the use of ultra-high-performance liquid chromatography (UHPLC) coupled to an Orbitrap mass analyser was established and validated for the detection of low-concentration ARs in pottery. During the preliminary experiments, UHPLC-Q/Orbitrap MS (ultra-high-performance liquid chromatography-quadrupole/Orbitrap mass spectrometry) was demonstrated to be more sensitive, and a wide range of AR homologues in cereal extracts were detected, unlike UHPLC-QTOFMS (ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry) and GC/MS. The developed method was utilised to profile AR homologue distribution in modern cereal samples and reanalyse AR-containing pots from the archaeological site of Must Farm. RESULTS: A highly sensitive LC/MS method with a limit of detection (LOD) of 0.02 µg/g and a limit of quantification (LOQ) of 0.06 µg/g was used to profile ARs in five modern cereal grains. The obtained LOD is 250 times lower than that obtained using the conventional GC/MS approach. AR 21:0 was the most abundant homologue in all four Triticum spp.-einkorn, emmer, Khorasan wheat and common wheat. Meanwhile, AR 25:0 was the predominant homologue in barley, potentially enabling differentiation between wheat and barley. The developed LC/MS-based method was successfully used to analyse ARs extracted from Must Farm potsherds and identified the cereal species most likely processed in the pots-emmer wheat. CONCLUSION: The described method offers an alternative and more sensitive approach for detecting and identifying ARs in ancient pottery. It has been successfully utilised to detect AR homologues in archaeological samples and discriminate which cereal species-wheat and barley-were processed in the pots.

Optimising a method for aragonite precipitation in simulated biogenic calcification media.

Resolving how factors such as temperature, pH, biomolecules and mineral growth rate influence the geochemistry and structure of biogenic CaCO3, is essential to the effective development of palaeoproxies. Here we optimise a method to precipitate the CaCO3 polymorph aragonite from seawater, under tightly controlled conditions that simulate the saturation state (Ω) of coral calcification fluids. We then use the method to explore the influence of aspartic acid (one of the most abundant amino acids in coral skeletons) on aragonite structure and morphology. Using ≥200 mg of aragonite seed (surface area 0.84 m2), to provide a surface for mineral growth, in a 330 mL seawater volume, generates reproducible estimates of precipitation rate over Ωaragonite = 6.9-19.2. However, unseeded precipitations are highly variable in duration and do not provide consistent estimates of precipitation rate. Low concentrations of aspartic acid (1-10 µM) promote aragonite formation, but high concentrations (≥ 1 mM) inhibit precipitation. The Raman spectra of aragonite precipitated in vitro can be separated from the signature of the starting seed by ensuring that at least 60% of the analysed aragonite is precipitated in vitro (equivalent to using a seed of 200 mg and precipitating 300 mg aragonite in vitro). Aspartic acid concentrations ≥ 1mM caused a significant increase in the full width half maxima of the Raman aragonite v1 peak, reflective of increased rotational disorder in the aragonite structure. Changes in the organic content of coral skeletons can drive variations in the FWHM of the Raman aragonite ν1 peak, and if not accounted for, may confuse the interpretation of calcification fluid saturation state from this parameter.

Dating the Paleolithic: Trapped charge methods and amino acid geochronology.

Despite the vast array of different geochronological tools available, dating the Paleolithic remains one of the discipline's greatest challenges. This review focuses on two different dating approaches: trapped charge and amino acid geochronology. While differing in their fundamental principles, both exploit time-dependent changes in signals found within crystals to generate a chronology for the material dated and hence, the associated deposits. Within each method, there is a diverse range of signals that can be analyzed, each covering different time ranges, applicable to different materials and suitable for different paleoenvironmental and archaeological contexts. This multiplicity of signals can at first sight appear confusing, but it is a fundamental strength of the techniques, allowing internal checks for consistency and providing more information than simply a chronology. For each technique, we present an overview of the basis for the time-dependent signals and the types of material that can be analyzed, with examples of their archaeological application, as well as their future potential.

Estimating the dwarfing rate of an extinct Sicilian elephant.

Evolution on islands, together with the often extreme phenotypic changes associated with it, has attracted much interest from evolutionary biologists. However, measuring the rate of change of phenotypic traits of extinct animals can be challenging, in part due to the incompleteness of the fossil record. Here, we use combined molecular and fossil evidence to define the minimum and maximum rate of dwarfing in an extinct Mediterranean dwarf elephant from Puntali Cave (Sicily).1 Despite the challenges associated with recovering ancient DNA from warm climates,2 we successfully retrieved a mitogenome from a sample with an estimated age between 175,500 and 50,000 years. Our results suggest that this specific Sicilian elephant lineage evolved from one of the largest terrestrial mammals that ever lived3 to an island species weighing less than 20% of its original mass with an estimated mass reduction between 0.74 and 200.95 kg and height reduction between 0.15 and 41.49 mm per generation. We show that combining ancient DNA with paleontological and geochronological evidence can constrain the timing of phenotypic changes with greater accuracy than could be achieved using any source of evidence in isolation.

Assessment of different screening methods for selecting palaeontological bone samples for peptide sequencing.

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 understanding of protein survival far back in time and under different environmental conditions.

The role of aspartic acid in reducing coral calcification under ocean acidification conditions.

Biomolecules play key roles in regulating the precipitation of CaCO3 biominerals but their response to ocean acidification is poorly understood. We analysed the skeletal intracrystalline amino acids of massive, tropical Porites spp. corals cultured over different seawater pCO2. We find that concentrations of total amino acids, aspartic acid/asparagine (Asx), glutamic acid/glutamine and alanine are positively correlated with seawater pCO2 and inversely correlated with seawater pH. Almost all variance in calcification rates between corals can be explained by changes in the skeletal total amino acid, Asx, serine and alanine concentrations combined with the calcification media pH (a likely indicator of the dissolved inorganic carbon available to support calcification). We show that aspartic acid inhibits aragonite precipitation from seawater in vitro, at the pH, saturation state and approximate aspartic acid concentrations inferred to occur at the coral calcification site. Reducing seawater saturation state and increasing [aspartic acid], as occurs in some corals at high pCO2, both serve to increase the degree of inhibition, indicating that biomolecules may contribute to reduced coral calcification rates under ocean acidification.

Author Correction: The dental proteome of Homo antecessor.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Screening archaeological bone for palaeogenetic and palaeoproteomic studies.

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.

The dental proteome of Homo antecessor.

The phylogenetic relationships between hominins of the Early Pleistocene epoch in Eurasia, such as Homo antecessor, and hominins that appear later in the fossil record during the Middle Pleistocene epoch, such as Homo sapiens, are highly debated1-5. For the oldest remains, the molecular study of these relationships is hindered by the degradation of ancient DNA. However, recent research has demonstrated that the analysis of ancient proteins can address this challenge6-8. Here we present the dental enamel proteomes of H. antecessor from Atapuerca (Spain)9,10 and Homo erectus from Dmanisi (Georgia)1, two key fossil assemblages that have a central role in models of Pleistocene hominin morphology, dispersal and divergence. We provide evidence that H. antecessor is a close sister lineage to subsequent Middle and Late Pleistocene hominins, including modern humans, Neanderthals and Denisovans. This placement implies that the modern-like face of H. antecessor-that is, similar to that of modern humans-may have a considerably deep ancestry in the genus Homo, and that the cranial morphology of Neanderthals represents a derived form. By recovering AMELY-specific peptide sequences, we also conclude that the H. antecessor molar fragment from Atapuerca that we analysed belonged to a male individual. Finally, these H. antecessor and H. erectus fossils preserve evidence of enamel proteome phosphorylation and proteolytic digestion that occurred in vivo during tooth formation. Our results provide important insights into the evolutionary relationships between H. antecessor and other hominin groups, and pave the way for future studies using enamel proteomes to investigate hominin biology across the existence of the genus Homo.

Early Pleistocene enamel proteome from Dmanisi resolves Stephanorhinus phylogeny.

The sequencing of ancient DNA has enabled the reconstruction of speciation, migration and admixture events for extinct taxa1. However, the irreversible post-mortem degradation2 of ancient DNA has so far limited its recovery-outside permafrost areas-to specimens that are not older than approximately 0.5 million years (Myr)3. By contrast, tandem mass spectrometry has enabled the sequencing of approximately 1.5-Myr-old collagen type I4, and suggested the presence of protein residues in fossils of the Cretaceous period5-although with limited phylogenetic use6. In the absence of molecular evidence, the speciation of several extinct species of the Early and Middle Pleistocene epoch remains contentious. Here we address the phylogenetic relationships of the Eurasian Rhinocerotidae of the Pleistocene epoch7-9, using the proteome of dental enamel from a Stephanorhinus tooth that is approximately 1.77-Myr old, recovered from the archaeological site of Dmanisi (South Caucasus, Georgia)10. Molecular phylogenetic analyses place this Stephanorhinus as a sister group to the clade formed by the woolly rhinoceros (Coelodonta antiquitatis) and Merck's rhinoceros (Stephanorhinus kirchbergensis). We show that Coelodonta evolved from an early Stephanorhinus lineage, and that this latter genus includes at least two distinct evolutionary lines. The genus Stephanorhinus is therefore currently paraphyletic, and its systematic revision is needed. We demonstrate that sequencing the proteome of Early Pleistocene dental enamel overcomes the limitations of phylogenetic inference based on ancient collagen or DNA. Our approach also provides additional information about the sex and taxonomic assignment of other specimens from Dmanisi. Our findings reveal that proteomic investigation of ancient dental enamel-which is the hardest tissue in vertebrates11, and is highly abundant in the fossil record-can push the reconstruction of molecular evolution further back into the Early Pleistocene epoch, beyond the currently known limits of ancient DNA preservation.

Cretaceous dinosaur bone contains recent organic material and provides an environment conducive to microbial communities.

Fossils were thought to lack original organic molecules, but chemical analyses show that some can survive. Dinosaur bone has been proposed to preserve collagen, osteocytes, and blood vessels. However, proteins and labile lipids are diagenetically unstable, and bone is a porous open system, allowing microbial/molecular flux. These 'soft tissues' have been reinterpreted as biofilms. Organic preservation versus contamination of dinosaur bone was examined by freshly excavating, with aseptic protocols, fossils and sedimentary matrix, and chemically/biologically analyzing them. Fossil 'soft tissues' differed from collagen chemically and structurally; while degradation would be expected, the patterns observed did not support this. 16S rRNA amplicon sequencing revealed that dinosaur bone hosted an abundant microbial community different from lesser abundant communities of surrounding sediment. Subsurface dinosaur bone is a relatively fertile habitat, attracting microbes that likely utilize inorganic nutrients and complicate identification of original organic material. There exists potential post-burial taphonomic roles for subsurface microorganisms.

The chances of establishing a real-world Jurassic Park are slim. During the fossilization process, biological tissues degrade over millions of years, with some types of molecules breaking down faster than others. However, traces of biological material have been found inside some fossils. While some researchers believe these could be the remains of ancient proteins, blood vessels, and cells, traditionally thought to be among the least stable components of bone, others think that they have more recent sources. One hypothesis is that they are in fact biofilms formed by bacteria. To investigate the source of the biological material in fossil bone, Saitta et al. performed a range of analyses on the fossilized bones of a horned dinosaur called Centrosaurus. The bones were carefully excavated in a manner to reduce contamination, and the sediment the bones had been embedded in was also tested for comparison. Saitta et al. found no evidence of ancient dinosaur proteins. However, the fossils contained more organic carbon, DNA, and certain amino acids than the sediment surrounding them. Most of these appeared to have a very recent source. Sequencing the genetic material revealed that the fossil had become a habitat for an unusual community of microbes that is not found in the surrounding sediment or above ground. These buried microbes may have evolved unique ways to thrive inside fossils. Future work could investigate how these unusual organisms live and whether the communities vary in different parts of the world.

Palaeoproteomics resolves sloth relationships.

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.

'Palaeoshellomics' reveals the use of freshwater mother-of-pearl in prehistory.

The extensive use of mollusc shell as a versatile raw material is testament to its importance in prehistoric times. The consistent choice of certain species for different purposes, including the making of ornaments, is a direct representation of how humans viewed and exploited their environment. The necessary taxonomic information, however, is often impossible to obtain from objects that are small, heavily worked or degraded. Here we propose a novel biogeochemical approach to track the biological origin of prehistoric mollusc shell. We conducted an in-depth study of archaeological ornaments using microstructural, geochemical and biomolecular analyses, including 'palaeoshellomics', the first application of palaeoproteomics to mollusc shells (and indeed to any invertebrate calcified tissue). We reveal the consistent use of locally-sourced freshwater mother-of-pearl for the standardized manufacture of 'double-buttons'. This craft is found throughout Europe between 4200-3800 BCE, highlighting the ornament-makers' profound knowledge of the biogeosphere and the existence of cross-cultural traditions.

Ancient amino acids from fossil feathers in amber.

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.

Ionisation bias undermines the use of matrix-assisted laser desorption/ionisation for estimating peptide deamidation: Synthetic peptide studies demonstrate electrospray ionisation gives more reliable response ratios.

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.

Collagen proteins exchange oxygen with demineralisation and gelatinisation reagents and also with atmospheric moisture.

RATIONALE: The oxygen (O) isotope composition of collagen proteins is a potential indicator of adult residential location, useful for provenancing in ecology, archaeology and forensics. In acidic solution, proteins can exchange O from carboxylic acid moieties with reagent O. This study investigated whether this exchange occurs during demineralisation and gelatinisation preparation of bone/ivory collagen. METHODS: EDTA and HCl demineralisation or gelatinisation reagents were made up in waters with different δ18 O values, and were used to extract collagen from four skeletal tissue samples. Aliquots of extracted collagen were exposed to two different atmospheric waters, at 120°C and ambient temperature, and subsequently dried in a vacuum oven at 40°C or by freeze drying. Sample δ18 O values were measured by HT-EA pyrolysis/IRMS using a zero-blank autosampler. RESULTS: Collagen samples exchanged O with both reagent waters and atmospheric water, which altered sample δ18 O values. Exchange with reagent waters occurred in all extraction methods, but was greater at lower pH. Damage to the collagen samples during extraction increased O exchange. The nature of exchange of O with atmospheric water depended on the temperature of exposure: kinetic fractionation of O was identified at 120°C but not at ambient temperature. Exchange was difficult to quantify due to the high variability of δ18 O values between experimental replicates. CONCLUSIONS: Studies of δ18 O values in collagen proteins should avoid extraction methods using acidic solutions.

Palaeogenomes of Eurasian straight-tusked elephants challenge the current view of elephant evolution.

The straight-tusked elephants Palaeoloxodon spp. were widespread across Eurasia during the Pleistocene. Phylogenetic reconstructions using morphological traits have grouped them with Asian elephants (Elephas maximus), and many paleontologists place Palaeoloxodon within Elephas. Here, we report the recovery of full mitochondrial genomes from four and partial nuclear genomes from two P. antiquus fossils. These fossils were collected at two sites in Germany, Neumark-Nord and Weimar-Ehringsdorf, and likely date to interglacial periods ~120 and ~244 thousand years ago, respectively. Unexpectedly, nuclear and mitochondrial DNA analyses suggest that P. antiquus was a close relative of extant African forest elephants (Loxodonta cyclotis). Species previously referred to Palaeoloxodon are thus most parsimoniously explained as having diverged from the lineage of Loxodonta, indicating that Loxodonta has not been constrained to Africa. Our results demonstrate that the current picture of elephant evolution is in need of substantial revision.

Dietary modulation of cortical excitation and inhibition.

The balance of excitatory and inhibitory neurotransmitters in the brain affects both neural responses and behaviour in humans and animals. Here we investigated whether dietary intervention aimed at increasing levels of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) can influence neural responses to basic sensory stimuli. Using a steady-state electroencephalography (EEG) paradigm, we found that the neural response to visual patterns was reduced in individuals who consumed a yeast extract product rich in substances associated with the production of GABA (glutamate and B vitamins), but not in a control group who consumed a placebo substance ( n = 14 per group). This demonstrates that the balance of excitation and inhibition in the brain can be influenced by dietary interventions, suggesting possible clinical benefits in conditions (e.g. epilepsy) where inhibition is abnormal.

Comparing ancient DNA survival and proteome content in 69 archaeological cattle tooth and bone samples from multiple European sites.

Ancient DNA (aDNA) is the most informative biomolecule extracted from skeletal remains at archaeological sites, but its survival is unpredictable and its extraction and analysis is time consuming, expensive and often fails. Several proposed methods for better understanding aDNA survival are based upon the characterisation of some aspect of protein survival, but these are typically non-specific; proteomic analyses may offer an attractive method for understanding preservation processes. In this study, in-depth proteomic (LC-Orbitrap-MS/MS) analyses were carried out on 69 archaeological bovine bone and dentine samples from multiple European archaeological sites and compared with mitochondrial aDNA and amino acid racemisation (AAR) data. Comparisons of these data, including estimations of the relative abundances for seven selected non-collagenous proteins, indicate that the survival of aDNA in bone or dentine may correlate with the survival of some proteins, and that proteome complexity is a more useful predictor of aDNA survival than protein abundance or AAR. The lack of a strong correlation between the recovery of aDNA and the proteome abundance may indicate that the survival of aDNA is more closely linked to its ability to associate with bone hydroxyapatite crystals rather than to associate with proteins. SIGNIFICANCE: Ancient biomolecule survival remains poorly understood, even with great advancements in 'omics' technologies, both in genomics and proteomics. This study investigates the survival of ancient DNA in relation to that of proteins, taking into account proteome complexity and the relative protein abundances to improve our understanding of survival mechanisms. The results show that although protein abundance is not necessarily directly related to aDNA survival, proteome complexity appears to be.

Lessons from Star Carr on the vulnerability of organic archaeological remains to environmental change.

Examples of wetland deposits can be found across the globe and are known for preserving organic archaeological and environmental remains that are vitally important to our understanding of past human-environment interactions. The Mesolithic site of Star Carr (Yorkshire, United Kingdom) represents one of the most influential archives of human response to the changing climate at the end of the last glacial in Northern Europe. A hallmark of the site since its discovery in 1948 has been the exceptional preservation of its organic remains. Disturbingly, recent excavations have suggested that the geochemistry of the site is no longer conducive to such remarkable survival of organic archaeological and environmental materials. Microcosm (laboratory-based) burial experiments have been undertaken, alongside analysis of artifacts excavated from the site, to assess the effect of these geochemical changes on the remaining archaeological material. By applying a suite of macroscopic and molecular analyses, we demonstrate that the geochemical changes at Star Carr are contributing to the inexorable and rapid loss of valuable archaeological and paleoenvironmental information. Our findings have global implications for other wetland sites, particularly archaeological sites preserved in situ.