Automated high-throughput biological sex identification from archaeological human dental enamel using targeted proteomics

Biological sex is key information for archaeological and forensic studies, which can be determined by proteomics. However, lack of a standardised approach for fast and accurate sex identification currently limits the reach of proteomics applications. Here, we introduce a streamlined mass spectrometry (MS)-based workflow for determination of biological sex using human dental enamel. Our approach builds on a minimally invasive sampling strategy by acid etching, a rapid online liquid chromatography (LC) gradient coupled to high-resolution parallel reaction monitoring assay allowing for a throughput of 200 samples-per-day with high quantitative performance enabling confident identification of both males and females. Additionally, we have developed a streamlined data analysis pipeline and integrated it into an R-Shiny interface for ease-of-use. The method was first developed and optimised using modern teeth and then validated in an independent set of deciduous teeth of known sex. Finally, the assay was successfully applied to archaeological material, enabling the analysis of over 300 individuals. We demonstrate unprecedented performance and scalability, speeding up MS analysis by tenfold compared to conventional proteomics-based sex identification methods. This work paves the way for large-scale archaeological or forensic studies enabling the investigation of entire populations rather than focusing on individual high-profile specimens. ### Competing Interest Statement The authors have declared no competing interest.