Sex Identification and Species Confirmation in Modern and Archeological Caprine Enamel

Proteomics has become a transformative tool for species and sex determination. This study introduces a novel methodology that integrates amelogenin (Amel) and enamelin (Enam) proteins extracted from the tooth enamel of caprines. Since morphologically, osteological remains of sheep and goats often cannot be easily discriminated, we developed our method on both modern domestic sheep (Ovis aries) and goats (Capra hircus) to establish unique proteomic signatures for each species for sex and species identification. Applying a targeted parallel reaction monitoring (PRM) assay, we validated the sex and species of 8 modern domestic sheep and 6 domestic goats. We then applied the same method to 10 ancient samples dating to the early eighth millennium BC Neolithic period. For sex determination, AmelY peptides were exclusively detected in modern male samples, while AmelX peptides were present in both sexes. Sex determination in 10 Neolithic samples demonstrated 40% males. For species determination, Enam species-specific peptides with single amino acid variations (SAAVs) successfully distinguished the modern caprine species. In the 10 archeological samples, only goat-specific Enam peptides were detected, validating previous zooarcheological results for this assemblage using morphology and mtDNA analysis. Robust peptide intensities and strong statistical correlations between modern and ancient data sets confirm the preservation of these unique markers in caprine enamel, expanding the application of proteomics to modern, archeological, and paleontological samples.