Long in the tooth

Proteins deteriorate over time, complicating their historical analysis. However, recent investigation has revealed ancient proteins in the enamel of the teeth of 18-million-year-old fossilized mammals from Kenya’s Rift Valley, granting insight into the lifestyle and evolution of these species.

In their recent publication in Nature, scientists from Harvard and the Smithsonian Museum Conservation Institute detail their discoveries.

“Teeth are akin to rocks in our mouths,” stated Daniel Green, director of field programs in the Department of Human Evolutionary Biology and the lead author of the study. “They represent the hardest formations that any organisms produce, meaning you could discover a tooth that dates back a hundred or even a hundred million years, and it will hold a geochemical history of the animal’s existence.”

This encompasses the diet and hydration of the animal, as well as its habitat.

“Previously, we believed that mature enamel, the most durable aspect of teeth, should contain very minimal proteins,” noted Green. Yet, by employing an advanced proteomics method called liquid chromatography tandem mass spectrometry, the team was able to identify “a substantial variety of proteins … in varied biological tissues.”

“The method comprises multiple stages where peptides are differentiated based on their size or chemistry, allowing for sequential analysis at higher resolutions than had been achievable with earlier techniques,” clarified Kevin T. Uno, associate professor in HEB and one of the paper’s corresponding authors.

“We and other researchers have recently discovered that there are numerous — if not hundreds — of distinct types of proteins present within tooth enamel,” Green articulated.

With the understanding that many proteins exist in modern teeth, the researchers redirected their focus to fossils, partnering with the Smithsonian and the National Museum of Kenya for access to fossilized teeth, especially from early elephants and rhinos.

As herbivores, these mammals possessed large teeth to crush the vegetation that constituted their diet. They could have enamel ranging from two to three millimeters in thickness, providing ample material for study, said Green.

What they uncovered — peptide fragments, chains of amino acids that collectively form proteins dating back 18 million years — were described by Green as “transformative.”

“No one has discovered peptide fragments this ancient before,” he remarked, labeling the findings as “rather astonishing.”

Until this point, the oldest known findings were estimated to be around 3.5 million years old, he added.

“With the collaboration of our colleague Tim Cleland, an excellent paleoproteomicist at the Smithsonian, we are extending the age range of peptide fragments five or six times beyond what was previously established.”

The newly identified peptides encompass a variety of proteins with distinct functions, collectively known as the proteome, according to Green.

“One reason we are enthusiastic about these ancient teeth is that while we do not have the complete proteome of all proteins that may have existed within the bodies of these ancient elephants or rhinoceroses, we do possess a representative sample.”

With such a repository, “There’s a possibility of obtaining more information from a collective of them than from a single protein alone.”

This study “paves the way for new advancements in paleobiology, enabling scientists to transcend beyond bones and morphology to reconstruct the molecular and physiological characteristics of extinct species and hominins,” stated Emmanuel K. Ndiema, senior research scientist at the National Museum of Kenya and a co-author of the paper. “This offers direct evidence of evolutionary ties. Together with other features of teeth, we can deduce dietary modifications, disease profiles, and even life stages at death — insights that were formerly inaccessible.”

Besides illuminating the lifeways of these organisms, it assists in situating them within historical context.

“We can utilize these peptide fragments to investigate the connections among ancient species, comparable to how contemporary DNA in humans is applied to identify familial relationships,” Uno explained.

“Even if a species is entirely vanished — with some animals examined in our research having no living descendants — it is still theoretically possible to extract proteins from their teeth and attempt to place them on a phylogenetic tree,” Green stated.

This data “might help settle long-standing disputes among paleontologists regarding the affiliations of these creatures with other mammalian lineages based on molecular evidence.”

Although this inquiry started as “a minor side project” within a much larger initiative involving numerous institutions and researchers globally, Green remarked, “We were astonished by the extent of our findings. There truly are a multitude of proteins preserved in these teeth.”

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This study was partially funded by the National Science Foundation and the Smithsonian’s Museum Conservation Institute.