Automated\nHigh-Throughput Biological Sex Identification\nfrom Archeological Human Dental Enamel Using Targeted Proteomics

Biological sex is\nkey information for archeological and\nforensic\nstudies, which can be determined by proteomics. However, the lack\nof a standardized approach for fast and accurate sex identification\ncurrently limits the reach of proteomics applications. Here, we introduce\na streamlined mass spectrometry (MS)-based workflow for the determination\nof biological sex using human dental enamel. Our approach builds on\na minimally invasive sampling strategy by acid etching, a rapid online\nliquid chromatography (LC) gradient coupled to a high-resolution parallel\nreaction monitoring (PRM) assay allowing for a throughput of 200 samples\nper day (SPD) with high quantitative performance enabling confident\nidentification of both males and females. Additionally, we developed\na streamlined data analysis pipeline and integrated it into a Shiny\ninterface for ease of use. The method was first developed and optimized\nusing modern teeth and then validated in an independent set of deciduous\nteeth of known sex. Finally, the assay was successfully applied to\narcheological material, enabling the analysis of over 300 individuals.\nWe demonstrate unprecedented performance and scalability, speeding\nup MS analysis by 10-fold compared to conventional proteomics-based\nsex identification methods. This work paves the way for large-scale\narcheological or forensic studies enabling the investigation of entire\npopulations rather than focusing on individual high-profile specimens.\nData are available via ProteomeXchange with the identifier PXD049326.