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Going to Pot: Team Using Proteomics to Examine What Ancient Populations Ate

Can proteomics provide clues to the fate of lost civilizations?
In a novel use of technology and methods that are more used to study tissue and fluid samples, an interdisciplinary team of researchers will use mass spectrometry to look at centuries-old pots, pans, and dishes to search out protein residues to look for clues about how ancient populations survived — or didn’t.
During the summer, the researchers from the universities of North Texas and South Florida received a one-year, $95,000 award from the National Science Foundation to fund the work.

“We’re not even there yet in terms of even knowing if we can … reliably extract protein residues.”

While prior research has delved into the analysis of cooking residues on archaeological pottery, “these had used some pretty coarse-scale techniques,” Steve Wolverton, the principal investigator on the award and an assistant professor of geography at the University of North Texas, told ProteoMonitor this week.
Most of the earlier work, he said, focused on lipids and fatty acids, which tend to preserve “pretty well” but offer only very general information about what is preserved in the pottery, Wolverton said.
And those studies that have focused on proteins have usually relied on immunoassays to identify them, which have the opposite problem. While immunoassays offer “precise, reliable, quantitative data for this kind of analysis … [they are] not suitable for studying general subsistence trends or broad analysis of pottery function,” the researchers write in their grant proposal.
“A large array of immunoassays would have to be designed to cover the range of taxa represented in prehistoric (or historic) diets, which would be analytically cumbersome,” they added.
By using proteomics technology, they hope to get a more precise picture of ancient cooking and eating patterns, which in turn could shed light on how these populations evolved.
“This can weigh in on questions like, ‘Under what conditions does pottery evolve through time?’” Wolverton said. Evidence of pottery artifacts is “often associated with people settling down and becoming less mobile, no longer hunting and gathering. Often it’s when they settle down and start to practice farming. So these are questions that provide a contextual basis … for broader scale cultural evolutionary changes,” he said.
Their work can also be the first step toward trying to decipher what happened to populations that seemingly and suddenly vanished. Wolverton and his colleagues will be looking at pottery from the ancient Pueblo populations of the United States as well as the inhabitants of the Mesa Verde, he said.
“In the Southwest where we’re working right now, what is the context surrounding not just pottery use but diet as that community starts to fade away rather rapidly and that people leave that region around AD 1325?” Wolverton said. “It’s not that this is the line of evidence, but it’s a new line of inquiry into some of the more established questions.”
While proteomics technologies have been recently used for other archaeological studies, including one that looked at the remains of a Tyrannosaurus rex [See PM 04/12/07] and another at the remains of a 2,700- year old Scythian king [See PM 01/03/08], they have been overlooked in the study of ancient pottery, Wolverton said. Indeed, a search on PubMed using keywords such as “proteomics,” “protein,” “mass spectrometer,” and “pottery” turned up just five results.
“We’re not even there yet in terms of even knowing if we can … reliably extract protein residues,” Wolverton said. “Then there’s the whole question of how well these things preserve. ... Preservation conditions of any organic tissue is going to be diverse across the archaeological world. It’s going to depend on sediment characteristics, it’s going depend on time, how long it’s been sitting there, how fast it was buried, [and] what the cooking conditions were.”
Extracting proteins from pottery has proven to be a formidable challenge due to covalent bonding between the protein and the pottery surface, which prevents extraction by conventional methods.
Indeed, according to Stanley Stevens, who is in charge of the proteomics research on the project, the challenge is the “actual matrix [of the pottery]. The major difficulty is going to be in the extraction and actually generating peptides for mass spec analysis. From that point on, it’s just the basic methodology that you use in proteomics work,” he said.
Stevens had been Wolverton’s colleague at the University of North Texas, but recently moved to the University of South Florida where he is an assistant professor of biology.
A technique using hydrofluoric acid to digest the ceramic matrix had been developed as an extraction technique and remains in common use as a method of protein residue extraction. However, the method destroys much of the protein residue, reducing what can be an already limited amount available for study.
In the work being funded by the NSF award, Wolverton’s team is addressing two basic questions: Can proteomics be used for such an archaeological analysis? And if so, what will it yield?
And in order to answer them, they will need to develop a method of extracting proteins that doesn’t destroy them; and develop a method of protein characterization that is broad and sensitive enough to identify a range of taxa that may be present in pottery that is hundreds of years old.
“What we hope to find is … taxonomically identifiable, or even down to the tissue level, identifiable protein residues so that we can characterize more clearly what people ate in the past and what they cooked in the pots,” Wolverton said.
Whether they are able to extract proteins from the vessels and how well they can characterize them, in part, will turn on luck, he said. “I don’t think it’s always going to be the case where we can just grab pottery and reliably extract proteins.”
Two years ago, the researchers did a preliminary study loading bovine serum albumin onto ground prehistoric archaeological pottery which was then cooked for seven days at 85 degrees Celsius. Using hydrofluoric acid to extract the protein residue from the clay matrix, followed by routine trypsin digestion, they were able to identify two BSA peptide sequences.
Then using an alternative extraction technique based on prolonged straight trypsin digestion of BSA from cooked pottery without hydrofluoric acid digestion, they “were much more successful … identifying 10 BSA peptides,” they said in their grant proposal.
Moving ahead, the focus will be on refining their extraction methods and mass-spec analysis, said Wolverton and Stevens.
Various sample-prep methods will be evaluated, including enzymatic digestion with trypsin, the use of urea, formic acid, and microwave irradiation, Stevens said.
In the prior work, mass spec analysis was done on the LTQ-FTICR instrument from Thermo Fisher Scientific, but will now be carried out on the LTQ-FT Orbitrap, which though it has lower resolving power has higher sensitivity and is more suited to the project, he added.
Most of the analysis in the next year will be done on unglazed pottery rather than archaeological vessels in order to evaluate their methods, but Wolverton said that he hopes that work will also be done on archaeological pottery that will be donated by the Crow Canyon Archaeological Center.

“I think we all feel that we haven’t been successful unless we get to the point where we’ve had some identifiable protein residue at some taxonomic scale,” he said. “We want to be careful that until we’ve done as much experimental work to iron out a methodology as we can.”

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