Sex estimation using sexually dimorphic amelogenin protein fragments in human enamel

Amelogenin genes are located on both X and Y sex chromosomes in humans and are a major focus of DNA-based sex estimation methods. Amelogenin proteins, AMELX_HUMAN and AMELY_HUMAN, are expressed in the tooth organ and play a major role in mineralization of enamel, the most taphonomically resistant, archaeologically persistent human tissue. We describe shotgun liquid chromatography mass spectrometry analysis of 40 enamel samples representing 25 individuals, including modern third molars and archaeological teeth from open-air contexts including permanent adult (400 to 7300 BP) and deciduous teeth (100 to 1000 BP). Peptides specific to the X-chromosome isoform of amelogenin were detected in all samples. Peptides specific to the sexually dimorphic Y-chromosome isoform were also detected in 26 samples from 13 individuals, across all time periods, including previously unsexed deciduous teeth from archaeological contexts. While the signal of each gene product can vary by more than an order of magnitude, we show close agreement between osteological and amelogenin-based sex estimation and thus demonstrate that the protein-based signal can be obtained reliably from open-air archaeological contexts dating to at least 7300 years ago. While samples with AMELY_HUMAN peptides are unambiguously male, samples with no AMELY_HUMAN signal may either be low signal male false negative samples or female samples. In order to estimate sex in these samples we developed a probability curve of female sex as a function of the logarithm of AMELX_HUMAN signal (p < 0.0001) using logistic regression. This is also the first demonstration using proteomics to estimate sex in deciduous teeth and pushes back the application of the method to teeth that are at least 7300 years old