The ability to assign biological sex to human skeletal remains is a fundamental requirement in archaeology, paleoanthropology, and medico-legal sciences. While DNA sequencing can be used, it is expensive, time-consuming, and often fails due to the poor quality of the remaining DNA. An easier, more reliable, and consistently applicable method is needed. We present a method for sex determination of human remains using peptides retrieved from tooth enamel. Amelogenin is an enamel-forming protein encoded for by both chromosomes X and Y, with slight differences in their amino acid sequences. Peptides with these differences were identified by nanoflow liquid chromatography mass spectrometry and found to correctly assign sex to archaeological human remains of various chronological ages, from hundreds to thousands of years old.

Abstract

The assignment of biological sex to archaeological human skeletons is a fundamental requirement for the reconstruction of the human past. It is conventionally and routinely performed on adults using metric analysis and morphological traits arising from postpubertal sexual dimorphism. A maximum accuracy of ∼95% is possible if both the cranium and os coxae are present and intact, but this is seldom achievable for all skeletons. Furthermore, for infants and juveniles, there are no reliable morphological methods for sex determination without resorting to DNA analysis, which requires good DNA survival and is time-consuming. Consequently, sex determination of juvenile remains is rarely undertaken, and a dependable and expedient method that can correctly assign biological sex to human remains of any age is highly desirable. Here we present a method for sex determination of human remains by means of a minimally destructive surface acid etching of tooth enamel and subsequent identification of sex chromosome-linked isoforms of amelogenin, an enamel-forming protein, by nanoflow liquid chromatography mass spectrometry. Tooth enamel is the hardest tissue in the human body and survives burial exceptionally well, even when the rest of the skeleton or DNA in the organic fraction has decayed. Our method can reliably determine the biological sex of humans of any age using a body tissue that is difficult to cross-contaminate and is most likely to survive. The application of this method will make sex determination of adults and, for the first time, juveniles a reliable and routine activity in future bioarcheological and medico-legal science contexts.