How ZooMS can be used to discover Palaeolithic human bones

The publication of our recent paper in Scientific Reports has capped a very exciting process of research. To cut a long story short, we found a tiny Neanderthal bone, 2.5 cm long, from amongst 2000+ of bone fragments excavated from Denisova Cave in the Siberian Altai. The technique used to do this is called Zooarchaeology by Mass Spectrometry, or ZooMS (you can read about the method further on another page on this website) and we are fortunate that one of our team on PalaeoChron is Mike Buckley, one of the pioneers of the method.

Katerina Douka and I came up with the idea of looking for hominin remains using this method while we were at Denisova Cave in 2014, but we never thought that it would one day work as well as it did. The idea of applying it at Denisova was simple; the site has amazing preservation, as well as the (previously) identified remains of Denisovans and Neanderthals. Perhaps, if we found more, these would produce more of the high quality genomes that the Leipzig group of Svante Paabo had already obtained. We talked with our Russian colleagues Michael Shunkov and Anatoly Derevianko while we were the site for a collaborators meeting, and they were interested in letting us try ZooMS. Then we discussed it with Svante, who was also at the meeting, and he was very keen too.

Michael Shunkov got us a large bag of bones later in the year. Preparing the samples for ZooMS down in the lab is time consuming work...because we were aiming to be very careful in case we did find something, we needed to clean our gear between sampling of each bone. Initial sampling was done by Dan Comeskey, who fitted it in between his other work.

We decided that perhaps one of our MSc students might be interested in taking on the project, so I pitched the idea at the end of 2014. Students have more time and can dedicate themselves more fully to their own projects. One student of the 18 we had that year came forward and wanted to take it on; Sam Brown, one of our international students, from Australia. Sam was hugely enthusiastic and once given the go ahead dived straight in to the lab...for days and days and days she would disappear stopping only to pop up occasionally and report that she'd prepped 454 samples, then another 256, then more and more etc etc. Slowly she had enough for a large ZooMS batch and went up on a train to Manchester to do the wet chemistry and mass spec work with Mike Buckley.

The first 1000 samples sadly produced no hominins, just lots of nicely identified animals. Mammoths, rhinos, hyaenas, wolves, reindeer, etc, the typical Ice Age fauna, all identified using collagen fingerprinting. 95% of the bone from Denisova is not able to identified, so it was lovely to see the technology shining a new path in archaeozoology. Sam was able to plot the results and show the different numbers of bones and the variety of species. Very cool.

I said to Sam that I thought she had enough data for her MSc and that she could stop now and write up her results. Too bad that she didn't get to find a hominin, but that's life right? Sam had other ideas and was keen to do more. Thank goodness she did! By this stage we'd run out of bones from the first batch, so we arranged for Sam to go to Novosibirsk in May to search for some more bags of bones with our Russian colleagues.

More samples to prepare, more time in the lab and then back to Manchester to run another 1000 or so samples, bringing the overall total over 2000. There were so many this time that Sam hadn't the time to analyse the data before coming home. Later next week we still didn't have the results of the batch so I sent an email to Mike asking what was going on. He told me that, amazingly, one of the samples in the batch had produced a hominin profile!

The bone that was idenfiied was small, just 2.5 cm long, but appeared fairly dense and large enough, if well-preserved, for DNA work. Later, more detailed ZooMS and proteomics showed that it was indeed a human bone!

We arranged for photography to be done in the Institute of Archaeology by Ian Cartwright, producing the lovely image at the top of this page, and also for CT scanning, which was done by Fiona Brock in Cranfield University, near to Oxford.

Dressing up in full DNA projective suits for these events was necessary to reduce any contamination!!

We also did some 3D imaging with Jamie Cameron (to the right in the picture above) and Sam ran an AMS date and got some stable isotope results for the sample too.

Later, she took the bone to Leipzig, to Matthias Meyer, Viviane Slon and Svante Paabo, who sampled the bone and later worked on its DNA. We waited excitedly for the results before the ESHE conference in London in 2015.

We hoped it would be a Denisovan, but a few weeks later we found out that our little bone was actually a Neanderthal! We also found out that it is closely related to the Ohkladnikov Neanderthal, differing in only 4 base positions.

On March 29 2016 our paper "Identification of a new hominin bone from Denisova Cave, Siberia using collagen fingerprinting and mitochondrial DNA analysis" was published in Scientific Reports.

Now we are hoping to extend the work. For this we need funding and money for Sam. Watch this space....ZooMS is going to be massive for archaeology, and particularly for the Palaeolithic, offering as it does the opportunity to find tiny human bones. Now all we have to do is to find some more...

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