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- SPIN enables high throughput species identification of archaeological bone by proteomicsPublication . Rüther, Patrick Leopold; Husic, Immanuel Mirnes; Bangsgaard, Pernille; Gregersen, Kristian Murphy; Pantmann, Pernille; Carvalho, Milena; Godinho, Ricardo Miguel; Friedl, Lukas; Cascalheira, João; Taurozzi, Alberto John; Jørkov, Marie Louise Schjellerup; Benedetti, Michael; Haws, Jonathan; Bicho, Nuno; Welker, Frido; Cappellini, Enrico; Olsen, Jesper VelgaardSpecies determination based on genetic evidence is an indispensable tool in archaeology, forensics, ecology, and food authentication. Most available analytical approaches involve compromises with regard to the number of detectable species, high cost due to low throughput, or a labor-intensive manual process. Here, we introduce “Species by Proteome INvestigation” (SPIN), a shotgun proteomics workflow for analyzing archaeological bone capable of querying over 150 mammalian species by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Rapid peptide chromatography and data-independent acquisition (DIA) with throughput of 200 samples per day reduce expensive MS time, whereas streamlined sample preparation and automated data interpretation save labor costs. We confirm the successful classification of known reference bones, including domestic species and great apes, beyond the taxonomic resolution of the conventional peptide mass fingerprinting (PMF)-based Zooarchaeology by Mass Spectrometry (ZooMS) method. In a blinded study of degraded Iron-Age material from Scandinavia, SPIN produces reproducible results between replicates, which are consistent with morphological analysis. Finally, we demonstrate the high throughput capabilities of the method in a high-degradation context by analyzing more than two hundred Middle and Upper Palaeolithic bones from Southern European sites with late Neanderthal occupation. While this initial study is focused on modern and archaeological mammalian bone, SPIN will be open and expandable to other biological tissues and taxa.
- Mandibular morphology and the Mesolithic–Neolithic transition in Westernmost IberiaPublication . Godinho, Ricardo Miguel; Umbelino, Cláudia; Valera, António Carlos; Carvalho, António; Bicho, Nuno; Cascalheira, João; Gonçalves, Célia; Smith, PatriciaNeolithic farming and animal husbandry were first developed in the Near East similar to 10,000 BCE and expanded westwards, reaching westernmost Iberia no later than 5500 BCE. It resulted in major social, cultural, economic and dietary changes. Yet, the impact of this change on human mandibular morphology in Iberia is yet to be assessed, which is regrettable because mandible form is impacted by population history and diet. In this study we used Mesolithic to Chalcolithic Iberian samples to examine the impact of this transition on mandibular morphology. We also compared these samples with a Southern Levantine Chalcolithic population to assess their relationship. Lastly, we assessed dental wear to determine if the morphological differences identified were related to the material properties of the diet. We found differences between samples in mandibular shape but not size, which we attribute to contrasting population histories between Mesolithic and later populations. Some differences in the severity of dental wear were also found between Mesolithic and later Iberian samples, and smaller between the Mesolithic Iberians and southern Levantines. Little relationship was found between wear magnitude and mandibular shape. Altogether, our results show that the Mesolithic-Neolithic Iberian transition resulted in a meaningful change in mandibular morphology, which was likely driven more by population history than by dietary change.
- Comparing extraction method efficiency for high-throughput palaeoproteomic bone species identificationPublication . Mylopotamitaki, Dorothea; Harking, Florian S.; Taurozzi, Alberto J.; Fagernäs, Zandra; Godinho, Ricardo Miguel; Smith, Geoff M.; Weiss, Marcel; Schüler, Tim; McPherron, Shannon P.; Meller, Harald; Cascalheira, João; Bicho, Nuno; Olsen, Jesper V.; Hublin, Jean-Jacques; Welker, FridoHigh-throughput proteomic analysis of archaeological skeletal remains provides information about past fauna community compositions and species dispersals in time and space. Archaeological skeletal remains are a finite resource, however, and therefore it becomes relevant to optimize methods of skeletal proteome extraction. Ancient proteins in bone specimens can be highly degraded and consequently, extraction methods for well-preserved or modern bone might be unsuitable for the processing of highly degraded skeletal proteomes. In this study, we compared six proteomic extraction methods on Late Pleistocene remains with variable levels of proteome preservation. We tested the accuracy of species identification, protein sequence coverage, deamidation, and the number of post-translational modifications per method. We find striking differences in obtained proteome complexity and sequence coverage, highlighting that simple acid-insoluble proteome extraction methods perform better in highly degraded contexts. For well-preserved specimens, the approach using EDTA demineralization and protease-mix proteolysis yielded a higher number of identified peptides. The protocols presented here allowed protein extraction from ancient bone with a minimum number of working steps and equipment and yielded protein extracts within three working days. We expect further development along this route to benefit large-scale screening applications of relevance to archaeological and human evolution research.