One ordinary day back in the Cretaceous period, the genes of a brachylophosaurus produced some collagen proteins inside the dinosaur’s bones. When the plant-munching giant died, its corpse was entombed in the sediments of a river that ran through today’s Montana. Now, in work that challenges paleontology dogma, researchers have identified traces of these proteins clinging to one of the bones—80 million years after they were expressed from the dinosaur DNA.
The eight fragments of collagen protein from a Brachylophosaurus canadensis are the oldest to be extracted from a fossil, said Harvard and North Carolina State University researchers who led the study, published in the May 1 issue of Science. The protein fragments show that the 30-foot-long brachylophosaurus (meaning short-crested lizard) was actually closer to a chicken than a lizard on the evolutionary tree.
The findings build upon a previous study by the same team that in 2007 found similar results in a 68-million-year-old Tyrannosaurus rex. “This helps verify that our first discovery was not a one-hit wonder,” said John Asara, who coauthored the studies on the T. rex and the “brachy,” as he dubs B. canadensis. Asara is an instructor in pathology at HMS and Beth Israel Deaconess Medical Center and director of BID’s Mass Spectrometry Core.
Basics RevisitedThe study questions the wisdom of paleontology-101. Current models say that soft tissue, DNA, proteins and any other biological material is totally replaced by minerals in old fossils, especially those dating back more than a million years.
Experts are going to receive this study with much skepticism, said Matthew Collins, an archaeologist at the University of York in the UK, who was not involved in the research.
After the T. rex study was published, Collins was one of the many authors who raised concerns that the T. rex samples could be contaminated with proteins from other sources.
“This is a more convincing study than the previous one, but still, it’s so incredible that proteins can survive in this kind of environment,” he said. “It’s hard to understand how it’s possible.”
The authors of the B. canadensis study cannot explain how those fragments of collagen made it through the ages, but they say it is unlikely they come from ostrich, microbes or other nondinosaur sources because all of the controls for contamination were clean.
The explanation could lie where the three-foot-long brachy femur was found in 2007. The fossil was dug out from the side of a washed-out ridge at the Judith River Formation in Montana, one of the largest fossil havens in the United States. Unlike other remains, the bone was buried 20 feet under and encased in sandstone, which might have softened the wear of 80 million years of aging.
Revolution in the Making“Deep burial in sandstone seems to favor exceptional conservation,” said Mary Schweitzer, a paleontologist at North Carolina State University who coauthored both studies. In the field, she and other authors supervised stringent handling procedures that included wrapping the fossil along with surrounding sediments in a plaster cast to be sawed off only at her lab. “It’s just as if you had broken your arm,” she said.
Once samples were extracted and dipped into acid to erode away the minerals, Schweitzer found a pattern she had already observed in T. rex bone. The microscope techniques revealed possible vestiges of bone cells, blood cells and vessels entwined within a fibrous structure that looked like collagen. Further analysis with antibodies confirmed there was collagen and other proteins mingling with those structures.
Schweitzer sent samples to Asara, who usually works at detecting very low levels of signaling proteins in cancer cells through analysis by mass spectrometry. “We have tuned up the lab for sensitivity,” he said.
He scanned the light colored powder coming from Schweitzer’s lab with the same machines he had used for sequencing T. rex collagen. This time, he also used a brand-new toy known as Orbitrap, which allowed him to acquire sequences through much more accurate mass spectrometry than in the previous study.
The search yielded eight fragments of collagen, a ubiquitous protein that is present in many animals and makes up to 90 percent of bone. In total, the protein sequences were 149 amino acids long, almost twice the amount extracted from the T. rex, but still under 10 per cent the size of a full-length collagen sequence. “We are just getting past the tip of the iceberg,” said Asara.
Even with such a minute quantity of protein, the team could place the brachy in the tree of evolution. As if they were DNA sequences, the protein fragments were compared to collagen from 21 living species and the fossils of the T. rex and a 300,000-year-old mastodon. The B. canadensis falls within the group of archosaurs, closer to modern chickens and ostriches than to alligators and lizards, said Chris Organ, a postdoctoral fellow at Harvard University, who reconstructed the evolutionary tree in this study. The B. canadensis and T. rex data provide the first molecular proof of the well-established theory that birds evolved from dinosaurs, said Asara.
“This is going to open a huge field of investigation,” said Raghu Kalluri, an HMS professor of medicine who is chief of the Division of Matrix Biology at BID. Kalluri is head of one of the two labs at Harvard that reproduced parts of Schweitzer’s experiments along with Lewis Cantley, the William Bosworth Castle professor of medicine, who is chief of the Division of Signal Transduction at BID. Asara’s tests were reproduced by William Lane’s lab at Harvard and the sequence data was independently verified by John Cottrell at Matrix Science, a software company in the UK.
“This should put in the minds of all the skeptics that a protein really survives millions of years,” said Asara. He hopes that as sequencing techniques become more precise, more details on dinosaurs and other extinct species will be extracted from larger pieces of collagen and other proteins. It will be the dawn of what Asara and colleagues call “fossilomics.”
Other researchers said more studies are needed to determine whether these proteins come from dinosaurs or contaminated samples. “The more data that become available, the better we will be able to asses the probability that they are real,” said Beth Shapiro, an ancient DNA expert at Pennsylvania State University. “I look forward to what happens next.”
Students may contact John Asara at jasara@bidmc.harvard.edu for more information.
Conflict Disclosure: The authors declare no conflicts of interest.
Funding Sources: The National Science Foundation, the David and Lucile Packard Foundation, the Merck Postdoctoral Science Research Fellowship, the National Institutes of Health, and the Taplin Funds for Discovery at HMS; the content of the work is the responsibility solely of the authors.
