Spatial analysis of the ancient proteome of archeological teeth using mass spectrometry imaging.

RATIONALE: Proteins extracted from archaeological bone and teeth are utilised for investigating the phylogeny of extinct and extant species, the biological sex and age of past individuals, as well as ancient health and physiology. However, variable preservation of proteins in archaeological materials represents a major challenge. METHODS: To better understand the spatial distribution of ancient proteins preserved within teeth, we applied matrix assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) for the first time to bioarchaeological samples to visualise the intensity of proteins in archaeological teeth thin sections. We specifically explored the spatial distribution of four proteins (collagen type I, of which the chains alpha-1 and alpha-2, alpha-2-HS-glycoprotein, haemoglobin subunit alpha and myosin light polypeptide 6). RESULTS: We successfully identified ancient proteins in archaeological teeth thin sections using mass spectrometry imaging. The data are available via ProteomeXchange with identifier PXD038114. However, we observed that peptides did not always follow our hypotheses for their spatial distribution, with distinct differences observed in the spatial distribution of several proteins, and occasionally between peptides of the same protein. CONCLUSIONS: While it remains unclear what causes these differences in protein intensity distribution within teeth, as revealed by MALDI-MSI in this study, we have demonstrated that MALDI-MSI can be successfully applied to mineralised bioarchaeological tissues to detect ancient peptides. In future applications, this technique could be particularly fruitful not just for understanding the preservation of proteins in a range of archaeological materials, but making informed decisions on sampling strategies and the targeting of key proteins of archaeological and biological interest.

Osteological, multi-isotope and proteomic analysis of poorly-preserved human remains from a Dutch East India Company burial ground in South Africa.

Skeletal remains discovered in Simon's Town, South Africa, were hypothesised as being associated with a former Dutch East India Company (VOC) hospital. We report a novel combined osteological and biochemical approach to these poorly-preserved remains. A combined strontium (87Sr/86Sr), oxygen (δ18OVPDB) and carbon (δ13CVPDB) isotope analysis informed possible childhood origins and diet, while sex-specific amelogenin enamel peptides revealed biological sex. Osteological analyses presented evidence of residual rickets, a healed trauma, dental pathological conditions, and pipe notches. The combined isotope analyses yielded results for 43 individuals which suggested a diverse range of geological origins, including at least 16% of the population being non-local. The inclusion of δ13CVPDB had intriguing implications for three individuals who likely did not have origins in the Cape Town region nor in Europe. Peptide analysis on the dental enamel of 25 tested individuals confirmed they were all biologically male. We suggest that isolated enamel may provide crucial information about individuals' pathological conditions, geographical origins, diet, and biological sex. These data further demonstrated that a combined approach using multiple osteological and biochemical methods is advantageous for human remains which are poorly preserved and can contextualise a site with little direct evidence.

Antibody-based sex determination of human skeletal remains.

Assignment of biological sex to skeletal remains is critical in the accurate reconstruction of the past. Analysis of sex-chromosome encoded AMELX and AMELY peptides from the enamel protein amelogenin underpins a minimally destructive mass spectrometry (MS) method for sex determination of human remains. However, access to such specialist approaches limits applicability. As a convenient alternative, we generated antibodies that distinguish human AMELX and AMELY. Purified antibodies demonstrated high selectivity and quantitative detection against synthetic peptides by ELISA. Using acid etches of enamel from post-medieval skeletons, antibody determinations corrected osteological uncertainties and matched parallel MS, and for Bronze Age samples where only enamel was preserved, also matched MS analyses. Toward improved throughput, automated stations were applied to analyze 19th-century teeth where sex of individuals was documented, confirming MS can be bypassed. Our immunological tools should underpin development of routine, economical, high-throughput methods for sex determination, potentially even in a field setting.