Analysis of the preserved amino acid bias in peptide profiles of Iron Age teeth from a tropical environment enabling sexing of individuals using amelogen in MRM

We describe the first dental proteomic profiles of Iron Age individuals (c2000-1000 years B.P), collected from the site of Long Long Rak rock shelter (LLR) in northwest Thailand. A bias toward the preservation of small, acidic and hydrophobic amino acids is observed. It is evident that the 212 proteins identified (2 peptide, FDR <1%) comprise a palimpsest of alterations that occurred both ante-mortem and post-mortem. Conservation of particular amino acids has contributed to the identification of amelogenin peptides. A novel MRM method for sexing individuals using the amelogenin protein is described, with four teeth indicating male origin. Stable isotope analysis using carbon and oxygen isotopes highlights the strongly C3 based (~80%) diet of the Long Long Rak cemetery people, which probably comprised rice combined with protein from freshwater fish among other food items. The combination of pathway and isotopic analysis adds weight to the relatively simple C3 based diets having contributed to the enrichment of pathways associated with metabolic conditions and shows capacity for harboring these conditions prior to death. The combination of proteomics and stable isotope analysis provides a complementary strategy for assessing the demography, diet, lifestyle and possible diseases experienced by ancient populations.