Dental proteomic analyses and Raman spectroscopy for the estimation of the biological sex and age of human remains from the Greek cemetery of San Giorgio Extra, Reggio Calabria (Italy)

Sex and age estimation is one of the most fundamental steps in mortuary studies and bioarchaeology. It is essential for a deeper understanding of ancient societies, and has wide applications in gender archaeology. The aim of this paper is to create a new and reliable protocol for unambiguous sex estimation of the deceased, comparing proteomic analyses, archaeological evidence, and anthropological data from a Greek cemetery located in Reggio Calabria (or the ancient Rhegion), as well as a first approach to estimate the age of the deceased by Raman spectroscopy in archaeology. Excavations carried out in the San Giorgio Extra district, headed by the Superintendence for the Archaeological Heritage of Calabria during the years 2004 and 2007, led to the discovery of the most significant Hellenistic cemetery in the city. Specifically, archaeological campaigns brought to light thirty Greek inhumation burials and their related funerary objects. Through proteomic analyses, we monitored a total of eight characteristic peptides for the amelogenin isoform variants AMELX and AMELY from the dental enamel of twelve selected adult individuals. The presence or absence of the AMELY variant (exclusively present in male subjects, being encoded by the Y gene) allowed us to estimate the sex of the analyzed individuals with high accuracy. Raman spectroscopy was also applied to study the enamel and dentin crystallinity to determine other environmental and biological parameters. At the same time, archaeological studies based on artifacts discovered inside the graves and double-blind bio-anthropological sex estimation of the twelve subjects were performed in order to compare these evaluations with data from the proteomic analyses. Comparison between these different approaches produced totally congruent results for the majority of individuals. In addition, the proteomic analysis allowed us to estimate the sex of four poorly preserved subjects for whom sex estimation was somewhat doubtful, as well as that of one undetermined individual. Finally, proteomic results were produced here with a faster protocol than those found in the literature and are potentially scalable to much lower sample amounts.