A unified protocol for the simultaneous extraction of ancient proteins and DNA from archaeological dental calculus

Zandra Fagernäs§, Maite Iris García-Collado#, Jessica Hendy§, Courtney Hofman£, Camilla Speller ±, Christina Warinner (#,£)

§ Max Planck Institute for the Science of Human History, Jena, Germany; # University of the Basque Country, Leioa, Spain; £ University of Oklahoma, Norman, United States; ± University of York, York, United Kingdom.

Proteins in archaeological dental calculus can provide valuable information about the human past, such as insights into diet and the ancient oral microbiome. Other biomolecules and microremains can also be recovered from calculus, and offer complementary lines of evidence. However, ancient dental calculus is a finite resource, often found in small quantities, especially in earlier time periods. A protocol allowing the extraction of multiple lines of evidence from a single sample is therefore highly desirable.

Here we test a new unified protocol for protein and DNA extraction from ancient dental calculus. The protocol was systematically evaluated on calculus from archaeological sites of different ages and anticipated states of preservation. As anticipated, DNA yields were 40% lower in samples prepared using the unified protocol, with losses being slightly higher in well preserved calculus. Unexpectedly, the unified protocol yielded 74% more proteins per milligram calculus than the protein-only protocol. The number of identified proteins was consistently higher through the unified protocol, including oral bacteria, human proteins and putative dietary-derived proteins. Further testing showed that the increase in protein recovery was due to a freezing step in the unified protocol, which increased protein yield but did not change the hydrophobicity profile of downstream peptides. By contrast, when comparing the unified and protein-only protocols, peptide hydrophobicity was higher in samples prepared using the unified protocol, suggesting loss of hydrophilic proteins to the aqueous fraction – the majority of which is used for DNA extraction in the unified protocol.

Our results show that DNA and proteins can be obtained from a single sample using a unified protocol, but DNA yields are reduced, and some hydrophilic proteins may be lost. Freezing was found to be neutral for DNA recovery but beneficial for protein recovery, likely increasing protein yield by enhancing cell lysis. Freezing decalcified samples may therefore be a simple and advantageous addition to current protein extraction protocols.