Heritage Cases

Methodology Since the materials to be studied are up to ~100,000 years old, low preservation of organic residues is expected. Thus, the most advanced palaeoproteomics methods for the recovery and analysis of ancient protein residues will be used, following the robust experimental protocols developed and routinely used at the University of Copenhagen. To analyse the artefacts using palaeoproteomic techniques, a small quantity of material (few milligrams—mg, see details below) is taken from the sample mechanically. We will sample areas of the artefacts which are most likely to preserve organic residues, such as those not exposed to the external contaminants. When possible, 2-3 locations on each artefact will be sampled. As the exact nature and complexity of each category of starting material (ochre-rich mixtures, ostrich eggshells, sheep bones) are highly different, the first stage of sample preparation will take this difference into consideration. Proteins will be extracted from the aliquots of the samples removed from the artefacts and then digested into smaller fragments, i.e. peptides, using several chemical solutions/solvents (Please find the list of reagents in [1]). The peptides will be analysed using liquid chromatography tandem mass spectrometry (LC-MS/MS). The amount of mass spectrometric data generated in this way is so large that it needs to be processed using bioinformatic tools such as MaxQuant [8]. Finally, the identification of peptides allows the identification of the composing proteins of the sample [9], leading to a taxonomic or tissue attribution. Sample types, sampling locations and minimum sample requirement BBC c. 100 ka MSA Toolkit: Ochre-red sand mixture inside toolkits—Samples are from both the mixture itself and the orange ring residues that were scraped from the shell’s inner surface. To obtain robust results, the amount of sample needed is approximately 50 mg. BBC c. 85-72 ka MSA: Unwashed Ostrich eggshells—The inner surface of unwashed eggshells will be gently scraped. To obtain robust results, the amount of sample needed is approximately 20-50 mg. If scraping the inner surface of eggshells does not yield robust results, a subset of 5 shell fragments will be completely powdered and analysed. KDS c. 66 ka MSA: Unwashed Ostrich eggshells—The inner surface of unwashed engraved eggshells will be gently scraped. To obtain robust results, the amount of sample needed is approximately 20-50 mg. BBC c. 2 ka LSA: Sheep (Ovis aries) bones (calcaneum and left mandible)—Initial analysis of the two pieces of sheep bone will be by a non-destructive approach, where the bones are immersed in a solvent to extract proteins without altering the appearance or morphology of the artefact. This way, the destructive physical sampling step is omitted. If we do not obtain good results using this approach, then a sample will be taken from each piece using the more standard method, which involves drilling the bone to acquire approximately 50-100 mg of sample powder. Non-destructive techniques such as immersing the object in a solvent not only omits the mechanical sampling step but also helps to preserve the integrity of the object. It is good practice to start the analysis with such an approach. Since the quantity of proteins left in the ancient objects could be extremely low, this technique might not provide useful information for such old materials. If the non-destructive procedure does not yield robust results, we request permission to carry out other approaches such as extracting small samples by drilling. Soil—Soil surrounding the sampled artefacts will be analysed in parallel with the object itself, using the same laboratory procedure. The rationale is to detect possible contaminants in the soil which, in the absence of soil analysis, could hamper interpretation of data from the samples.