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Rewriting history through proteins
 
Andrew S. Wiecek
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“We weren't able to do anything with that,” says Capellini. “But no one is going to embark on the mammoth genome sequencing because it's already been published. It's not rewarding from a publication exploitation point of view. So, we had to rely on modern stuff.”

But all the latest techniques to study ancient proteins might already be, well...ancient. Since his woolly mammoth study, Capellini's team has replaced the mass spectrometer that they used with a new MS system possessing both increased sensitivity and mass accuracy.

“We changed almost everything,” says Capellini. He reservedly notes (this work is not yet published) that for sample preparation, they continue to improve ways to minimize losses and improve recovery especially in the demineralization and digestive steps. “The main point here is we're not throwing anything away.”

Not so perfect after all

Armed with these advanced protein analysis tools and methods, Corthals realized it was time to learn more about what happened during those final days for the15-year-old Inca maiden.

The remains of the maiden and her two younger companions are stored at -20°C in the Museum of High Mountain Archaeology in the city of Salta, Argentina, close to Mount Llullaillaco. Corthals convinced the archeologists to let her take a sample; in the end she was allowed to take three samples: a piece of blood-soaked cloth from the boy and cotton swabs from the lips of the maiden and the boy. Returning to her New York lab, Corthals and her team carefully handled their precious samples, using strict protocols to process the materials for analysis using a liquid chromatography tandem MS LTQ-Orbitrap system.

The results, reported in an article published earlier this year in the online journal PLoS One (4), describe the first use of “shotgun proteomics” to detect proteins involved in the anti-bacterial immune response in an ancient sample. As it turns out, the two Inca children — selected because they represented an idealized state of humanity, according to Spanish explorers — were, in fact, battling chronic lung infections.

“It calls into question, first of all, the idea of perfection, in terms of that these children need to be perfect,” says Corthals. “It also calls into question these secondary sources, which are the only sources we have of the Inca empire because they didn't have writing. And it opens a whole new field of questions of what was happening [at that moment], making things a little more exciting than we thought they were.”

In the end, Corthals believes that there might be plausible explanations for their findings. For instance, the children might have acquired infections sometime during the months of preparation for the ritual sacrifice, possibly the result of being confined to a closed house with an open fire. Such a smoky environment could have irritated the lungs, allowing for an opportunistic infection. Another possibility is that the infection could have ben obtained during the 1200-mile journey from Cusco to Mount Llullaillaco. “It's the equivalent of walking from Mexico to the Canadian border. For a 15-year-old, that's pretty tough,” says Corthals.

Moving beyond the Incas, the next step for shotgun proteomics of ancient samples will likely be the use of even older or more damaged samples. In fact, Corthals already has a target in mind here: the frozen corpse of a 1918 Spanish flu victim. “Everybody's been studying the Spanish flu virus to death, but nobody's been actually looking at the immune response,” says Corthals. But that trip back in time will have to wait its turn.

References
1.) Wilson, A. S., T. Taylor, M. C. Ceruti, J. A. Chavez, J. Reinhard, V. Grimes, W. Meier-Augenstein, L. Cartmell, B. Stern, M. P. Richards, M. Worobey, I. Barnes, and T. P. Gilbert. 2007. Stable isotope and DNA evidence for ritual sequences in inca child sacrifice. Proceedings of the National Academy of Sciences 104:16456-16461.

2.) Cappellini, E., L. J. Jensen, D. Szklarczyk, A. Ginolhac, R. A. da Fonseca, T. W. Stafford, S. R. Holen, M. J. Collins, L. Orlando, E. Willerslev, M. T. Gilbert, and J. V. Olsen. 2011. Proteomic analysis of a pleistocene mammoth femur reveals more than one hundred ancient bone proteins. Journal of Proteome Research 11:917-926.

3.) Miller, W., D. I. Drautz, A. Ratan, B. Pusey, J. Qi, A. M. Lesk, L. P. Tomsho, M. D. Packard, F. Zhao, A. Sher, A. Tikhonov, B. Raney, N. Patterson, K. Lindblad-Toh, E. S. Lander, J. R. Knight, G. P. Irzyk, K. M. Fredrikson, T. T. Harkins, S. Sheridan, T. Pringle, and S. C. Schuster. 2008. Sequencing the nuclear genome of the extinct Woolly Mammoth. Nature 456:387-390.

4.) Corthals, A., A. Koller, D. W. Martin, R. Rieger, E. I. Chen, M. Bernaski, G. Recagno, and L. M. Dávalos. 2012. Detecting the immune system response of a 500 Year-Old inca mummy. PLoS ONE 7:e41244.

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