For archaeologists working to get a better picture of what life was like in ancient communities, determining the sex of the people who lived there is a crucial step. Traditional methods of sex estimation rely on the shape and form of the pelvis or cranium, so archaeologists are often limited when working with fragmentary or poorly-preserved remains, or individuals who are too young for their skeletal features to be reliable indicators. In the last decade, however, researchers have developed protein-based methods that require only a sample of dental enamel, advancing the ability to determine sex even when skeletal remains are insufficient. In a paper recently published in the Journal of Archaeological Science, a team led by researchers from the Smithsonian’s Museum Conservation Institute describes a new analytical protocol that increases the speed and efficiency of enamel-based sex determination.
Teeth serve as valuable archaeological recorders of biological sex thanks to sex chromosome-linked proteins called amelogenins. Amelogenins come in two distinct variants that are encoded in the X- and Y-chromosomes of humans. These variants, known as AMELX for the X-linked gene and AMELY for the Y-linked gene, both facilitate the mineralization of enamel during the development of deciduous “baby” teeth and permanent teeth. Once the tooth is formed, amelogenins are enzymatically digested into smaller peptides that remain locked up in the mineralized enamel, leaving a distinct biochemical marker of sex. “The nice thing about enamel is it’s essentially self-fossilized. […] It’s resistant to breakdown, so it really protects the proteins that are within,” saidTimothy Cleland, lead author on the study.
Previous methods to leverage the information stored in enamel relied on time-intensive protocols that required days-long extraction processes, with most analyses involving only a few teeth. Time, however, is not always on the archaeologist’s side. The method developed by Cleland and his collaborators was born from a need to process a large volume of teeth in a relatively short time frame. In the late 2010s, construction in Philadelphia unintentionally unearthed a burial ground in which the First Baptist Church of Philadelphia buried over four hundred individuals, both adults and juveniles, across the eighteenth and nineteenth centuries. With permission from the First Baptist Church of Philadelphia, the remains of eighty-six individuals underwent a variety of archaeological and anthropological analyses in efforts to better understand life in colonial and postcolonial America. But with reinterment, or reburial, scheduled for 2024, there was little time for a wider demographic study. With this timeline in mind, Cleland and his collaborators created the Sample Preparation by Easy Extraction and Digestion-free for Enamel (SPEED-E) protocol.
The SPEED-E protocol combines the strengths of the pre-existing Sample Preparation by Easy Extraction and Digestion (SPEED) protocol with advances in the analysis of proteins, called digestion-free proteomics, and applies them specifically to enamel. “It saves quite a lot of time when you don’t have to do the digestion. […] For enamel, it’s not a requirement, which makes the approach more straightforward,” Cleland said. Synthesizing these methods in the SPEED-E protocol allows researchers to extract the amelogenin peptides within minutes, as opposed to days, and substantially decreases the amount of enamel needed for conclusive analysis. This development opens the door not only for rapid analyses of hundreds of samples but also for minimally invasive sampling of rare and important archaeological materials. Using their new technique, Cleland and his colleagues analyzed eighty-nine teeth from the First Baptist Church of Philadelphia burial site, ultimately determining the biological sex of eighty-five of them: fifty-seven females and twenty-eight males. Two of the indeterminate teeth would have been identified as female if a lower cutoff for AMELX presence was used, while the other two indeterminate results may be attributable to the unintentional sampling of dentin over enamel. “There’s not much enamel on juvenile deciduous teeth, so we saw some of the other proteins from the inside of the tooth instead,” Cleland said.
Despite occasional sampling difficulties, the SPEED-E protocol’s minimal sample requirements and rapid processing make it a powerful tool for large-scale studies, particularly when working within tight timelines or with precious materials. “You can gain a lot of information from a short period of time and apply it to a variety of ages of individuals to estimate their sex, so it can help with that kind of demographic information that may not be initially obvious,” Cleland said. The SPEED-E protocol will help archaeologists bite into the mysteries of history, one tooth at a time.