Automated High-Throughput Biological Sex Identification from Archeological Human Dental Enamel Using Targeted Proteomics

Biological sex is
key information for archeological and
forensic
studies, which can be determined by proteomics. However, the lack
of a standardized 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 the 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 a high-resolution parallel
reaction monitoring (PRM) assay allowing for a throughput of 200 samples
per day (SPD) with high quantitative performance enabling confident
identification of both males and females. Additionally, we developed
a streamlined data analysis pipeline and integrated it into a Shiny
interface for ease of use. The method was first developed and optimized
using modern teeth and then validated in an independent set of deciduous
teeth of known sex. Finally, the assay was successfully applied to
archeological material, enabling the analysis of over 300 individuals.
We demonstrate unprecedented performance and scalability, speeding
up MS analysis by 10-fold compared to conventional proteomics-based
sex identification methods. This work paves the way for large-scale
archeological or forensic studies enabling the investigation of entire
populations rather than focusing on individual high-profile specimens.
Data are available via ProteomeXchange with the identifier PXD049326.