In-situ digestion and micro-destructive sampling for dental proteomic sex determination analysis using polyvinylalcohol-based gel

• Novel micro-invasive method for dental proteomics using PVA-based hydrogels. • Enables sex determination from enamel with minimal or no structural damage. • Compatible with degraded samples, preserving cultural and forensic value. • Combines ethical preservation with cutting-edge archaeological analysis. • Sets a new standard for micro-destructive sampling in proteomics research. Determining biological sex is pivotal for archaeological and forensic studies, providing insights into past societies, burial practices, and population demographics. Traditional methods, such as morphological analysis and DNA-based techniques, face limitations, including inconclusive results for juveniles, contamination, and genetic material degradation. Additionally, these approaches often involve significant destruction of artifacts, raising ethical concerns. Dental proteomics has emerged as a robust alternative, utilizing the stability of enamel-bound proteins, such as amelogenin isoforms (AMELx and AMELy). However, current sampling techniques are invasive, requiring substantial material removal and risking the integrity of culturally significant specimens. This study introduces a novel, minimally invasive technique for enamel protein sampling using a polyvinyl alcohol (PVA)-based highly viscous polymeric dispersion (HVPD). Acting as an elastic gel-like system, HVPD enables in-situ digestion and efficient extraction of proteins without compromising the tooth's structural integrity. The method was successfully applied to contemporary and archaeological samples, demonstrating effective recovery of amelogenin isoforms, reduced contamination risk, and compatibility with degraded specimens. This minimally invasive approach achieves reliable detection of all key amelogenin markers required for sex determination, matching the analytical outcome of conventional destructive protocols while preserving specimen integrity.’ This methodology offers transformative potential for archaeology, anthropology, and forensic science, bridging scientific innovation with ethical stewardship. By preserving the physical and cultural integrity of artifacts, it addresses a critical need for sustainable research practices. Reliable biological sex determination in challenging contexts expands the scope of proteomic applications, providing new insights into past societies while safeguarding irreplaceable heritage for future generations.