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Searching for hominin bones from Denisova Cave using ZooMS

Denisova Cave is one of the most important sites in the world of palaeoanthroplogy and PalaeoChron is privileged to be working there with Anatoly Derevianko and Michael Shunkov (Russian Academy of Sciences, Novosibirsk) and their team at the site, as well as Svante Pääbo and his group at the Max Planck in Leipzig.

In 2010, hominin remains excavated from the site revealed a great surprise, a new species of human. This was based on mtDNA and later nuclear DNA, extracted from a distal manual phalanx located in layer 11 of the eastern gallery (Krause et al., 2010; Reich et al., 2010). The preservation conditions at the site; stable and cool, facilitate the preservation of DNA, and so far there are two genomes from Denisovan individuals and one from a Neanderthal that have been obtained by our colleagues at the Max Planck in Leipzig. At the present time Denisova is the only place in the world where we have the physical remains of Denisovans, although we know that they must have once been quite widely distributed.

Bones from sites like Denisova are often highly fragmented. Excavations in 2014 produced several thousand such bones, which are too small to be identified to species. These were excavated from layer 11, the context in which the Denisovan type phalanx was located, and layer 12, in which Neanderthal remains were later discovered.

Sam Brown (left), an MSc student on the PalaeoChron project, is working on identifying the species to which these fragments belong using a technique called Zooarchaeology Mass Spectrometry (ZooMS). The work is being done with our collaborator and PalaeoChron member Dr Mike Buckley from the University of Manchester.

Identification through the application of ZooMS is made possible because each species has a unique combination of peptides in their collagen, acting rather like a species-specific fingerprint (Buckley et al., 2009). Tiny samples of bone (<20mg) are demineralised in HCl, heated, and then repeatedly centrifuged to allow for collagen extraction. The collagen is treated with trypsin, which undertakes digestion and enzymatically cuts the collagen into peptides. The mass-to-charge (m/z) ratios of these peptides is then measured using MALDI-MS (Matrix-Assisted Laser Ionization Mass Spectrometry) and the results are compared against an extensive reference collection of known species.

The method is high-throughput, allowing for a large number of samples to be analysed in a relatively short period of time (see left). Sam has been analysing hundreds of bones over the past few months and last week she spent 3 days again up in Manchester with Mike running her many samples. It's hard work but she's been able to build up a picture of the faunal record at the site in the key layers that contain the human fossil remains. She's also been back to Novosibirsk to get more bone fragments from the lower levels of the cave as well as more reference bones from the Altai region that can be used to compare against the unknown archaeological samples.

A key aim of the work, however, is to try and identify a fragment of a hominin from these fragmented collections of bone. If there are any there, we hope that ZooMS will allow us to find them...and then perhaps we will have another genome of a Denisovan, or a Neanderthal from this great site.

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