The extraction and sequencing of ancient DNA (aDNA) has long been the go-to method among researchers studying human ancestors and the ancient past. For example, aDNA has played a role in the discovery of the Denisovans, an extinct cousin of Neanderthals, among countless other revelations.
But more recently, paleoproteomics, which blends the fields of chemistry and molecular biology to analyze ancient proteins, continues to gain traction as a promising method to reveal new discoveries about the history and evolution of humans and animals that aDNA cannot.
The potential of paleoproteomics
One of the most appealing aspects of paleoproteomics is that it resolves some significant limitations of aDNA. With paleoproteomics, researchers can study artifacts from deeper in time, and gain more detailed insights than from aDNA or other biomolecular techniques.
The oldest human DNA retrieved so far dates back to about 400,000 years, meaning that researchers are unable to confidently shed light on what human life on Earth was like beyond that time frame. But recently, researchers from the University of Copenhagen were able to sequence proteins containing genetic information, retrieved from the tooth enamel of 800,000-year-old human remains—a first-of-its-kind achievement—the results of which were published in Nature in April 2020. But many researchers believe paleoproteomics has the potential to go even further, and reveal information from humans from at least one million years ago.
“The preservation potential of proteins is known to be much greater than for DNA, so we can simply go further back in time and look at species that may have gone extinct beyond the capabilities of ancient DNA studies,” says Michael Buckley, Royal Society University senior research fellow at the University of Manchester. Buckley is credited with being a part of the team that developed Zooarchaeology by Mass Spectrometry (ZooMS), which has rapidly become an established method among the research community.
Buckley and colleagues first introduced ZooMS in 2008, offering a much faster and cheaper way to analyze samples than aDNA. More specifically, ZooMS uses matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-ToF-MS) to analyze collagen and determine if a bone is from an animal or a human. Buckley created ZooMS out of a PhD project, as he was looking into collagen as a research tool, and was reviewing the available sequences of a handful of mammals. “ZooMS really came into existence as a technique for species identification when I showed that we could separate the sheep from the goat using their collagen. These are the two species that are notoriously difficult to distinguish by looking at their bone morphology alone, and crucial for better understanding early animal domestication practices. Since then, we have simplified the method, and widened the species range and studies all over the planet,” he explains.
The technique proved especially useful years later when it was used to identify a hominin bone among thousands of unknown bone fragments in a Denisova cave. This ID led to the discovery that the bone was from a hybrid individual—which researchers nicknamed Denny—who was the product of a Neanderthal mother and a Denisovan father. The discovery quickly gained attention not only among fellow research groups, but also among mainstream news and media outlets.
In the last couple of decades, researchers realized that mass spectrometry, which traditionally studies modern proteins, could also work for ancient proteins. Buckley elaborates on these developments: “This shift was due to moving from a true sequencing form known as N-terminal degradation (or Edman sequencing) to a proteomic one using probability-based matching of fragmented peptides to known sequences,” he says. “This allowed for the rapid analysis of large parts of the proteins previously inaccessible. Established over four decades ago, it took nearly a decade for paleontology to take on such methodology, and almost another decade for archaeology to do so. Yet, both fields are now benefitting greatly from this technology with the number of users increasing drastically in the last decade.”
Currently, Buckley is focusing his research on biomineralized tissues, such as bones and teeth, in an attempt to better understand the limits of biomolecule preservation.
A spotlight on dental calculus and paleoproteomics
The best sources of ancient proteins are found in materials like bone, dental calculus, and shells, since these samples contain mineralized components. However, paleoproteomics is also useful in identifying food and other substances left on material artifacts like clay pots, making it a welcomed technique for a wide range of fields.
One study, published in December 2020 in the journal PNAS, reports that researchers were able to extract enough information from dental calculus to determine that exotic Asian spices like turmeric, and fruits such as bananas, were being consumed by people in the Mediterranean more than 3,000 years ago—centuries earlier than previously thought. “This is the earliest direct evidence to date of turmeric, banana, and soy outside of South and East Asia," says Philipp Stockhammer, archaeologist at Ludwig-Maximilians-Universität in Munich, in a university press release. Dental calculus is the hardened plaque found on the surface of teeth. It is rich with information for researchers, from being able to tell what an individual’s diet consisted of, how their overall health was, and in this case, the complexity of early trade routes.
"Our high-resolution study of ancient proteins and plant residues from human dental calculus is the first of its kind to study the cuisines of the ancient Near East," says Christina Warinner, a molecular archaeologist at Harvard University and the Max Planck Institute for the Science of Human History and co-senior author of the article. "Our research demonstrates the great potential of these methods to detect foods that otherwise leave few archaeological traces. Dental calculus is such a valuable source of information about the lives of ancient peoples."
Emerging trends in paleoproteomics
As with any emerging technique, there are still limitations and challenges to overcome for paleoproteomics.
Working with any ancient fossil or artifact is extremely delicate work. Conducting protein analysis for these types of applications is destructive to the precious samples, so researchers are now getting creative with how they collect the substances and proteins they need for analysis. As cited in a recent article in Science Advances, a team of researchers was able to non-invasively use ZooMS on bone point samples from Iroquoian village sites in southern Quebec, Canada, by performing ZooMS extractions on the bags storing the samples, instead of the items themselves. The bags contained enough loose collagen molecules that had separated from the bone points to be analyzed. As a result, the team was able to make taxonomic identifications of the bones, identifying bear, human, and deer. As the field of paleoproteomics continues to progress, more nondestructive sample preparation methods will likely be adopted.
There is also ample opportunity to use paleoproteomics in combination with, or complementary to, other techniques to extract the maximum amount of information from ancient samples. The authors of the Science Advances report referenced earlier suggest that techniques such as MALDI-imaging MS could be used to “examine protein preservation with objects to optimally select areas for sampling, a strategy to prevent unnecessary sample destruction.”
Paleoproteomics is a success story of how a scientific method can be used in innovative ways to serve new applications. The technique opens up an exciting new dimension for studying our ancient past, enabling researchers to look back deeper in time, and achieve more detailed analysis than ever before.