Sex determination of human remains from peptides in tooth enamel Nicolas Andre Stewarta,1, Raquel Fernanda Gerlachb, Rebecca L. Gowlandc, Kurt J. Gronc, and Janet Montgomeryc aSchool of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton BN2 4AT, United Kingdom; bDepartment of Morphology, Physiology, and Basic Pathology, School of Dentistry of Ribeirão Preto, University of São Paulo, FORP/USP, 807 São Paulo, Brazil; and cDepartment of Archaeology, Durham University, Durham DH1 3LE, United Kingdom Edited by Christopher Kuzawa, Northwestern University, Evanston, IL, and accepted by Editorial Board Member C. O. Lovejoy November 13, 2017 (received for review August 23, 2017) The assignment of biological sex to archaeological human skeletons of DNA sex determination, this method has rarely been attempted is a fundamental requirement for the reconstruction of the human on any scale on human remains from archaeological sites. past. It is conventionally and routinely performed on adults using Herein we present a method for secure biological sex de- metric analysis and morphological traits arising from postpubertal termination of human remains by means of a minimally de- sexual dimorphism. A maximum accuracy of ∼95% is possible if both structive surface acid etching of tooth enamel and subsequent the cranium and os coxae are present and intact, but this is seldom identification of sex chromosome-linked isoforms of amelogenin, achievable for all skeletons. Furthermore, for infants and juveniles, an enamel-forming protein, by nanoflow liquid chromatography there are no reliable morphological methods for sex determination mass spectrometry (nanoLC-MS). Tooth enamel is the hardest without resorting to DNA analysis, which requires good DNA survival tissue in the human body and survives burial exceptionally well, and is time-consuming. Consequently, sex determination of juvenile even when the rest of the skeleton or DNA in the organic frac- remains is rarely undertaken, and a dependable and expedient tion has decayed. Therefore, this method holds promise for re- method that can correctly assign biological sex to human remains liably determining the biological sex of humans of any age using a of any age is highly desirable. Here we present a method for sex body tissue that is most likely to survive intact. It is minimally determination of human remains by means of a minimally destructive destructive, inexpensive, and reliable. surface acid etching of tooth enamel and subsequent identification of ANTHROPOLOGY sex chromosome-linked isoforms of amelogenin, an enamel-forming Results and Discussion protein, by nanoflow liquid chromatography mass spectrometry. The acid-etching procedure to extract peptides from tooth Tooth enamel is the hardest tissue in the human body and survives enamel results in a complex base peak chromatogram when burial exceptionally well, even when the rest of the skeleton or analyzed by nanoLC-MS/MS (Fig. 1). It has previously been DNA in the organic fraction has decayed. Our method can reliably shown that peptides can be identified using various acid-etching determine the biological sex of humans of any age using a body methods, and that these peptides originate from the major tooth tissue that is difficult to cross-contaminate and is most likely to enamel proteins: amelogenin, ameloblastin, and enamelin (9– survive. The application of this method will make sex determination 12). During enamel maturation, the majority of these proteins of adults and, for the first time, juveniles a reliable and routine activity in future bioarcheological and medico-legal science contexts. Significance sex determination | tooth enamel | amelogenin | human remains | mass spectrometry The ability to assign biological sex to human skeletal remains is a fundamental requirement in archaeology, paleoanthropol- ex is a fundamental primary characteristic for the analysis of ogy, and medico-legal sciences. While DNA sequencing can be Shuman skeletal remains in archaeological and medico-legal used, it is expensive, time-consuming, and often fails due to contexts. Techniques for determining other key identifying features, the poor quality of the remaining DNA. An easier, more reli- such as age at death and stature, are also sex-dependent (1). Ac- able, and consistently applicable method is needed. We pre- sent a method for sex determination of human remains using curate profiles of sex are crucial for reconstructing past societies in peptides retrieved from tooth enamel. Amelogenin is an terms of demography, identity, and epidemiology, and are also es- enamel-forming protein encoded for by both chromosomes X sential in medico-legal contexts for identifying individuals (e.g., in and Y, with slight differences in their amino acid sequences. mass disasters). In adults, sex can be determined with relative ac- – Peptides with these differences were identified by nanoflow curacy, usually estimated at 80 95% depending on such factors as liquid chromatography mass spectrometry and found to skeletal preservation and degree of sexual dimorphism within the correctly assign sex to archaeological human remains of sample (2). One of the key limiting factors of osteological analyses to various chronological ages, from hundreds to thousands of date has been an inability to reliably determine the sex of individuals years old. from skeletal features before age ∼18 y (3). Numerous methods have tried to do so, usually applying studies of the morphological and Author contributions: J.M. designed research; N.A.S., R.F.G., R.L.G., K.J.G., and J.M. per- metrical characteristics of the infant and juvenile mandible, denti- formed research; N.A.S., R.F.G., and K.J.G. contributed new reagents/analytic tools; N.A.S. tion, and ilium (e.g., refs. 4 and 5); however, no method has proven analyzed data; and N.A.S., R.F.G., R.L.G., K.J.G., and J.M. wrote the paper. sufficiently reliable when tested on documented skeletal samples (6). The authors declare no conflict of interest. Unfortunately, ancient DNA analysis is not the answer to this This article is a PNAS Direct Submission. C.K. is a guest editor invited by the Editorial Board. problem due to issues of preservation, contamination, and ex- This open access article is distributed under Creative Commons Attribution-NonCommercial- pense. For example, several DNA studies attempted to de- NoDerivatives License 4.0 (CC BY-NC-ND). termine the sex of infants from Romano-British sites with the Data deposition: The mass spectrometry data have been deposited to the ProteomeXchange aim of assessing whether preferential female infanticide was Consortium via the PRIDE partner repository with the dataset identifier PXD007856. practiced (7, 8), but in all of those studies, viable results were 1To whom correspondence should be addressed. Email: [email protected]. obtained from only a small proportion of the overall number This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. sampled. Owing to the destructive, costly, and inconclusive nature 1073/pnas.1714926115/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1714926115 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 15E6 14E6 13E6 12E6 11E6 10E6 9E6 8E6 7E6 Intensity 6E6 5E6 4E6 3E6 2E6 1E6 0 15 20 25 30 35 40 45 50 55 60 Time (min) SK339 #4768 RT: 27.36 AV: 1 NL: 3.02E6 SK339 #5789 RT: 32.67 AV: 1 NL: 1.72E6 F: FTMS + p NSI Full ms [300.00-1600.00] F: FTMS + p NSI Full ms [300.00-1600.00] 440.2232 540.2794 z=2 z=2 100 100 90 90 80 80 70 70 60 60 540.7809 z=2 50 50 440.7251 z=2 40 40 Relative Abundance Relative Abundance 30 30 541.2823 20 20 z=2 441.2266 10 z=2 10 541.7839 z=2 0 0 437 438 439 440 441 442 443 444 445 537 538 539 540 541 542 543 544 545 m/z m/z SK339 #4748 RT: 27.27 AV: 1 NL: 4.55E4 SK339 #5816 RT: 32.82 AV: 1 NL: 5.26E4 F: ITMS + c NSI d w Full ms2 [email protected] [110.00-895.00] F: ITMS + c NSI d w Full ms2 [email protected] [135.00-1095.00] y5 b6 645.4 714.4 100 100 y6 y5 y4 y3 y y y y 90 90 7 6 3 2 SM(ox) IRPPY 80 80 S I R P P Y P S Y b2 b3 b4 b5 b6 70 70 b2 b3 b6 b7 b8 2+ yMH-SOCH4 60 60 408.3 y4 50 532.3 50 b4 y3 504.3 y 40 376.2 40 3 Relative Abundance b2 Relative Abundance 366.2 30 235.0 30 y +2 7 b6 y6 879.5 20 b3 20 b +2 b b +2 8 b8 3 7 723.4 348.2 y +2 646.5 449.7 898.5 b +2 6 b 357.4 154.0 5 408.9 5 10 533.4 10 b2 y2 199.9 301.2 601.5 173.1 348.2 861.5 553.9 774.4 269.1 508.4 705.4 724.5 899.7 0 0 200 300 400 500 600 700 800 200 300 400 500 600 700 800 900 1000 m/z m/z Fig. 1. A representative base peak chromatogram (300–1,600 m/z) produced from Fewston sample SK339. (Inset) Amino acid sequences of the two dimorphic peptides of amelogenin: AMELY-(58-64) peptide and AMELX-(44-52) peptide. The reconstructed ion chromatograms (to 4 ppm) for each of these are shown in red and blue, respectively, with full-scan MS and corresponding MS/MS below. 2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1714926115 Stewart et al. Downloaded by guest on September 24, 2021 100 100 90 YX 90 YX 80 80 70 70 60 60 50 50 40 40 Relative Abundance 30 Relative Abundance 30 20 20 SK119 SK339 10 (male 38 years) 10 (male 41 years) 0 0 22 24 26 28 30 32 34 36 38 22 24 26 28 30 32 34 36 38 Time (min) Time (min) 100 100 90 YX 90 YX 80 80 70 70 60 60 50 50 40 40 Relative Abundance Relative Abundance 30 30 20 20 SK130 SK351 10 (male 66 years) 10 (male 63 years) 0 0 22 24 26 28 30 32 34 36 38 22 24 26 28 30 32 34 36 38 ANTHROPOLOGY Time (min) Time (min) 100 100 90 X 90 X 80 80 70 70 60 60 50 50 40 40 Relative Abundance Relative Abundance 30 30 20 20 SK363 SK378 10 (female 54 years) 10 (female 34 years) 0 0 22 24 26 28 30 32 34 36 38 22 24 26 28 30 32 34 36 38 Time (min) Time (min) 100 90 YX 80 70 60 50 40 Relative Abundance 30 20 SK366 10 (male 76 years) 0 22 24 26 28 30 32 34 36 38 Time (min) Fig.