Enamel peptides reveal the sex of the Late Antique 'Lovers of Modena'

Abstract

Recent work has disclosed the critical role played by enamel peptides in sex classification of old skeletal remains. In particular, protein AMELY (amelogenin isoform Y) is present in the enamel dental tissue of male individuals only, while AMELX (isoform X) can be found in both sexes. AMELY can be easily detected by LC-MS/MS in the ion extracted chromatograms of the SM_(ox)IRPPY peptide (monoisotopic [M + 2 H]⁺² mass = 440.2233 m/z). In this paper, we exploited the dimorphic features of the amelogenin protein to determine the sex of the so-called 'Lovers of Modena', two Late Antique individuals whose skeletons were intentionally buried hand-in-hand. Upon discovery, mass media had immediately assumed they were a male-female couple, even if bad preservation of the bones did not allow an effective sex classification. We were able to extract proteins from the dental enamel of both individuals (~1600 years old) and to confidently classify them as males. Results were compared to 14 modern and archaeological control samples, confirming the reliability of the ion chromatogram method for sex determination. Although we currently have no information on the actual relationship between the 'Lovers of Modena' (affective? Kin-based?), the discovery of two adult males intentionally buried hand-in-hand may have profound implications for our understanding of funerary practices in Late Antique Italy.

Figure 1

(a) Map of Italy with the location of the city of Modena (from NASA Visible Earth project ‒ credits to Jacques Descloitres, MODIS Rapid Response Team, NASA/GSFC); (b) Photograph of the Ciro Menotti necropolis (4th–6th century; Modena) during the archaeological excavation; the ‘Lovers of Modena’ are depicted within the inset; (c) Teeth and maxilla of individual 7_CM13; (d) Roman town of Mutina (Latin name of Modena) with the necropolis plan (modified from).

Figure 2

Ion chromatograms representing selected peptides of the ‘Lovers’ enamel proteome. Chromatograms search was performed using Xcalibur software (Thermo Scientific) with a mass tolerance of 5 ppm. Peptide sequences, protein names and retention times are reported in the graphs. The presence of peptide SM_(ox)IRPPY (AMELY; [M + 2 H]⁺² 440.2233 m/z) in both the specimens suggests that the two individuals were males.

Figure 3

(a) Ion chromatogram representing peptides SM(ox)IRPPY, M(ox)IRPPY and SMIRPPY of individual 1_CM16-5 and 2_CM16-6; the occurrence of these three specific peaks confirms the presence of AMELY; (c) fragmentation spectrum of peptide SM(ox)IRPPY (monoisotopic mass [M + 2 H]⁺² 440.2233 m/z); y-axis indicates ion intensities; (c) sequence coverage (51%) of AMELY isoform 2 for individual 1_CM16-5.

Figure 4

(a) Deamidation rate of Gln calculated as reported in the Method section; error bars are 1σ. Sample 5_FC176 seems to be an outlier of the regression; (b) example of ion peak integrated area (AA) for peptide YEVLTPLKWYQSIRPPYP, employed for the calculation of %deQ.