Determining the Sex of Skeletons Hundreds of Year Old with NanoLC–MS/MS

Determining the sex of a skeleton hundreds of years old is an essential ability in the study of past human populations, as well as for establishing human identity in forensic contexts. Traditional macroscopic sex estimation methods in bioarchaeology are dependent on the presence and study of sexually dimorphic skeletal elements, including unnamed bones and the skull. Rebecca Gowland and her colleagues in the Department of Archaeology at Durham University in the United Kingdom have tested, for the first time, the applicability of a new method of sex estimation utilizing enamel peptides from a sample of permanent and deciduous teeth at different stages of mineralization, from nonadults of unknown sex, including perinates (pertaining to the time from one month prior to birth to one month after), and using a minimally destructive acid etching procedure and subsequent nano liquid chromatography– tandem mass spectrometry (nanoLC–MS/MS). Gowland spoke to us about her efforts, with additional information provided by co-authors of their resulting paper (1), Heidi Shaw of the Department of Archaeology at Durham University and Nicolas A. Stewart of the School of Pharmacy and Biomolecular Sciences at the University of Brighton in the United Kingdom.

You stated that the objective of your study was to test, for the first time, the applicability of a new method of sex estimation utilizing enamel peptides on a sample of both permanent and deciduous teeth (also known as “milk teeth” or “baby teeth”) at different stages of mineralization, from nonadults of unknown sex, including perinates. Why did you decide to test tooth enamel in your research? What is the estimated accuracy of this method of sex estimation going forward?

Dental enamel is the outermost tissue that covers the tooth crown. It is the hardest, densest, and most calcified tissue in the human body. The unique physical characteristics of enamel make it a particularly useful material for biochemical analyses. Namely, it is highly resistant to post-mortem diagenesis compared to other human tissues used in biochemical studies, such as bone and tooth dentine. Human tooth enamel also has the unique distinction of containing a small amount of proteins (<1%), which consist mainly of the heterogenous amelogenin proteins (AMELX and AMELY).

The method as currently designed enables biochemical data concerning the sex of ancient people to be reconstructed with previously unattainable accuracy, independent of preservation or sexual maturity of the skeleton, using a qualitative interpretation approach. While highly effective and reliable, the qualitative approach does not allow for the expression of statistical confidence in the results of the analysis or comparison of results within and between sample data sets. To address this, we are currently working on a project that will improve the method to provide absolute quantitative data resulting in a validated liquid chromatography–mass spectrometry (LC–MS) assay.

You pointed out that several studies in recent years have successfully demonstrated that proteins can be extracted from human tooth enamel and used as a method for identifying the sex of archaeological individuals, and went on to list a few as examples. Despite the proven robustness of this method, to date, there have only been a handful of deciduous teeth sampled using this technique, all of which were completely mineralized. Why do you suppose that deciduous teeth have not been tested as often?

The primary reason that deciduous teeth have not been tested as often is due to the method’s newness. The method was only first published in 2016. Since that time, only a small body of literature utilizing the method has been produced. Given the novelty of the method, it seemed prudent to test its validity using completely mineralized adult teeth first before introducing potential confounding variables, such as age and differing levels of mineralization.

In designing the method, the first challenge to tackle was determining if peptides could be recovered from individuals of known sex (2), followed by whether peptides could be recovered from individuals from an archaeological context (3). While there were some non-adult teeth sampled in these previous studies, it has never been systematically examined, and more to the point there was a question about how mineralized a tooth needed to be in order to recover peptides. This study presents one of the first systematic studies of tooth enamel in different stages of development.

What are the benefits of using mass spectrometry in your work?

Tooth enamel is unique; peptides are preserved in this crystalline tissue and there’s very little risk of cross-contamination. Mass spectrometry provides additional data which may be relevant to disease, diet, and so on and this is currently the direction of the field. The only other way of accurately estimating sex from the remains of past infants and children would be to use ancient DNA analysis. This method tends to be prohibitively expensive for any large-scale analysis and generally does not survive as well as the amelogenin proteins. Ancient DNA analysis also requires greater destruction of the human remains, which is problematic due to ethical reasons.

What special sample preparation techniques are used for this method?

In brief, the tooth surface is abraded to remove any obvious surface contaminants using a dental burr. The enamel is then washed with 3% H2O2 before being rinsed with ultrapure water. A small amount of 5% (vol/vol) hydrochloric acid is then placed in the cap of an Eppendorf tube, whereby an initial etch is performed by lowering the tooth onto the hydrochloric acid; this first etch is then discarded as a precaution against potential diagenesis. The process is repeated for a second time and retained as the etch solution.

A C18 resin loaded pipette tip (MilliporeSigma) is conditioned using a combination of acetonitrile and 0.1% (vol/vol) formic acid. The proteins at this stage are bound to the pipette tip by drawing the etch solution through the pipette tip. The pipette tip is then washed with 0.1% (vol/vol) formic acid. The resin‐bound peptides are then eluted by drawing a 60% acetonitrile/0.1% formic acid elution buffer through the pipette tip and the eluted peptides subsequently lyophilized. Samples are then dissolved in 0.1% trifluoroacetic acid in water, centrifuged on a desktop centrifuge, before being transferred to glass autosampler vials.

What conclusions have you come to so far in your research?

A couple of initial conclusions that we have been able to draw from the few studies completed so far, include that amelogenin isoforms can indeed be retrieved from human tooth enamel and be used to accurately identify the chromosomal sex of an individual, including individuals from an archaeological context. Moreover, it is possible to apply this method to samples from individuals as young as 36 gestational weeks, from permanent as well as deciduous teeth, and from teeth that have minimal amounts of mineralized enamel present. There is still much to explore with this method; we’ve only begun to discover the range of its applicability.

It is your belief that the successful application of this method for estimating sex in nonadults, particularly perinates, has the potential to revolutionize the way that bioarchaeologists study infancy and childhood, including studies related to growth, epidemiology, and demography in the past. Can you please explain how?

Prior to puberty, the sexually dimorphic traits that we associate with female and male skeletons are not present. It is therefore difficult to determine with any confidence the sex of non-adult remains using only macroscopic observations of the skeleton. With the advent of this method, bioarchaeologists are now able to examine sex-specific cultural treatment and differentiate between the health of boys and girls, as well as sex specific growth trajectories and past developmental milestones, such as age of puberty and subsequent repercussions for fertility.

What are the biggest challenges that you have encountered in developing this method? What options or alternatives are available to overcome these challenges?

Since our discovery, many laboratories have adopted a version of this methodology to estimate sex from archaeological samples, consequently, factors or parameters used to estimate sex are not the same across these laboratories; no standardization exists. To overcome this, absolute amounts of peptides need to be known, limits of detection of these need to be determined to evaluate false discovery rates. Confident sex estimation is dependent on knowing these factors. A need for on-site sampling has emerged and the method for sample collection needs to be altered, this is being assessed. There will always be the issue of false positive female estimation as a deletion in the AMELY gene is probable (albeit extremely rare) and these individuals would be identified as female when they are male. aDNA, especially for juveniles, would be the only means to determine the sex with confidence, however this may not be feasible depending on the nature of preservation of the specimen and limitation stated above.

What are your next steps in this work?

The next step in this method is quantifying the results using heavy isotope-labeled synthetic peptides as internal standards from samples of individuals of known sex. We also plan to continue testing the limitations of the method, as well as optimizing the method through controlled testing.

References

• R. Gowland, N.A. Stewart, K.D. Crowder, et al., Am J Phys Anthropol. 1–11 (2021). https://doi.org/10.1002/ajpa.24231
• N.A. Stewart, G.F. Molina, J.P. Mardegan Issa, et al., RSC Adv. 6, 61673–61679 (2016). https://doi.org/10.1039/C6RA05120K
• N.A. Stewart, R.F. Gerlach, R.L. Gowland, et al., Proc. Natl. Acad. Sci. U. S. A.114, 13649–13654 (2017). https://doi.org/10.1073/pnas.1714926115

Rebecca Gowland

Rebecca Gowland is with the Department of Archeology at Durham University in the United Kingdom. Direct correspondence to: Rebecca.gowland@durham.ac.uk