Mass Spectrometry Reveals T Rex Collagen Fragments—Strengthens Link Between Dinosaurs and Modern Birds

Kristina Woodworth

    ^*Medical Writer, SciMantis Communications, Inc, Pen Argyl, Pennsylvania.    
   Address correspondence to: Kristina Woodworth, SciMantis Communications, Inc, PO Box 3, Pen Argyl, PA 18072. E-mail: kristina@scimantis.com.

Although it has been widely assumed that the fossilization process results in the destruction of all organic material in less than 1 million years,¹ researchers from North Carolina State University, Montana State University, Harvard Medical School, and the University of Chicago have successfully extracted and sequenced collagen protein from a Tyrannosaurus rex fossil recovered from eastern Montana. The investigators reported their findings in the April issue of Science.²

The authors noted that collagen was chosen as a target for analysis because the genetic structure of collagen protein is highly conserved in modern animals, and it is durable and resistant to degradation.³

Analysis of Fossilized Bone Mineral
In their article, Mary Higby Schweitzer, PhD, a scientist affiliated with North Carolina State University, the North Carolina Museum of Natural Sciences, and the Museum of the Rockies at Montana State University, and her coauthors described methods to confirm the presence of collagen in T rex fossil bone, in addition to the molecular and mass spectrometry analyses used to analyze the fossil.

They began by demonstrating the fibrous nature of the demineralized T rex tissue through optical and electron microscopy. They also confirmed that the cortical and medullary regions of T rex fossil bone had a characteristic, repeating cross-band pattern that as been found in collagen extracted from extant emus, in addition to demineralized bone of bird remains from the Cretaceous period, when viewed by atomic force microscopy.⁴ However, the researchers emphasized that transmission electron microscopy failed to reveal this periodic repeating pattern, and suggested that this could be the result of methodological problems.

Further analyses of the T rex fossil bone found that, unlike bone samples from extant species, the dinosaur fossil did not demineralize completely after prolonged incubation in EDTA. Selected-area electron diffraction studies to determine the mineral make-up of the remaining product found that the composition of the fossilized bone mineral was likely to be "virtually unchanged from the living state," according to the researchers.

Immunoreactivity Studies Link T rex to Modern Chickens
The researchers also exposed T rex cortical and medullary whole-bone extracts to chicken collagen I and demonstrated that the T rex bone reacted to the chicken collagen antibodies as measured by an enzyme-linked immunosorbent assay (ELISA) analysis. However, the authors noted that the degree of reactivity to the chicken collagen antibodies varied widely and was minor compared to samples taken from extant ostriches. Nevertheless, the dinosaur fossil extracts showed greater reactivity to chicken collagen antibodies than findings from negative controls, the researchers emphasized.

Ion Mass Spectrometry of Demineralized Fossil Bone
An important aspect of the experiments reported by the authors was the use of mass spectrometry to perform genetic analyses on the T rex fossil bone. John M. Asara, PhD, a Harvard pathology professor and director of the mass spectrometry core facility at Beth Israel Deaconess Medical Center, conducted highly sensitive time-of-flight secondary ion mass spectrometry (TOF-SIMS) analyses of the demineralized T rex fossil in an effort to detect amino acid residues in the samples.

The TOF-SIMS assay detects surface ions associated with protein fragments with a high degree of sensitivity. Furthermore, the process can detect signals in whole samples without requiring chemical extraction, according to the authors. Dr Asara noted that TOF-SIMS was specifically used in this analysis because it was one of the only options available to researchers interested in detecting the extremely low levels of protein fragments expected in fossilized bone.

To demonstrate the presence of collagen alpha-1 type 1 protein fragments in the T rex fossil bone, the researchers obtained glycine-to-alanine ratios through the TOF-SIMS analysis, as glycine is the most abundant amino acid in collagen (accounting for approximately 33% of total amino acid content) and alanine accounts for approximately 10% of the amino acid make-up of collagen, explained the authors.⁵

The results of the TOF-SIMS study revealed that the glycine-to-alanine ratio found in the demineralized T rex medullary fossil bone was 2.6:1, which was similar to the 2.5:1 ratio found in collagen alpha-1 type 1 of modern chickens. The researchers also analyzed the sandstone that had surrounded the dinosaur fossil bones as a control, and found little or no evidence of amino acids in these samples.

Based on a variety of iron-based compounds found in the TOF-SIMS analysis of the T rex samples, in addition to in sediments surrounding the bone at the excavation site, the researchers hypothesized that iron may help preserve genetic material in fossilized soft tissue by causing irreversible molecular cross-link reactions. The authors explained that reactive sites within the original protein molecules of the animal could have become irreversibly cross-linked to similar molecules or exogenous organic molecules through a free-radical reaction. Once the protein molecules were stabilized by these irreversible cross-link reactions, the authors conjectured, they were no longer available for chemical reactions that would have resulted in further degradation.

The authors observed a reduction in TOF-SIMS signal strength with progressive analyses, suggesting that the fossil material was degrading after exposure to the laboratory environment. They noted that other studies have reported that freshly excavated fossil bones are ideal when the research goal is to extract genetic material, and that current methods for storing fossils may be inadequate to preserve DNA.⁶

Implications of the Research Findings
The findings of this novel, multidisciplinary approach to fossil analysis suggest a link between dinosaurs, such as T rex, and modern birds, a theory widely circulated in recent years that still meets with some degree of controversy. First of all, the fact that demineralized T rex fossil bone reacted with chicken collagen antibodies in ELISA studies points to an evolutionary link between the species. Furthermore, the mass spectrometry analysis demonstrates an alignment between amino acid residues found in the T rex fossilized bone and collagen alpha-1 type 1 in modern chickens, suggesting that these animals may share similar evolutionary ancestors.

In terms of fossil preservation for genetic analysis, the authors noted that their findings point to possible environmental factors that may play a role in the preservation of genetic material in fossilized bone, including fossils that are more than 1 million years old. The presence of iron may be important in initiating the cross-link reactions that preserved the amino acid residues. Also, the geologic composition of the excavation site may contribute to better preservation. According to the authors, the fact that intact calcium crystals were found in the T rex fossils analyzed in this study could be due to the stabilizing effect of calcite, which was found in abundance within the sandstone surrounding the fossils. This could have also contributed to the fact that the T rex fossil bone did not degrade completely in EDTA, unlike bones from extant species.

Dr Schweitzer et al emphasized that their research points to an urgent need to optimize fossil extraction and handling practices to avoid the possible degradation of available genetic material. She said that since the publication of this study, her laboratory has indeed identified improved handling and storage techniques that will be the subject of future publications.

Dr Asara noted that although TOF-SIMS has demonstrated promise in fossil analysis, the methodology is being studied with great interest in medical applications. He explained, "mass spectrometry is now being used in biomarker discovery because it is much more sensitive than MRI or X rays" in detecting early mutations that lead to cancerous tumors. Also, mass spectrometry can identify thousands of proteins in a sample, is noninvasive, and can accomplish several diagnostic goals from a molecular perspective with one analysis, he added.

"One of the goals is to bring mass spectrometry from bench to bedside," he said, emphasizing that techniques commonly used in diagnosis and tumor staging, such as ELISA, cannot detect extremely low levels of protein, nor can ELISA capture ongoing mutations or quantitate these mutations. At this point, the biggest hurdles facing mass spectrometry in regular diagnostic applications are the robustness and sensitivity of the technique, said Dr Asara.

Although the scientific community has been excited by this research, Dr Schweitzer said that most researchers "are taking a wait-and-see approach," as expected in the early communications of any new research technique. She added, "if we can demonstrate that we are consistently able to get these results with samples of varying quality and age," then there might be a shift to these research methods in the wider paleontology and archaeology communities.

In the meantime, she emphasized that exciting research continues in the imaging community with respect to fossil analysis. She noted ongoing research by Lawrence Witmer, PhD, a professor of anatomy and paleontology at Ohio University, who is using CT scanning and advanced computer visualization to image the anatomy of both extinct and extant species. She also mentioned Timothy Rowe, PhD, a geological sciences professor at the University of Texas at Austin and co-director of the university's high resolution X ray CT facility, a multiuser facility supported by the National Science Foundation. Dr Rowe uses X ray CT technology to study the evolution and development of the vertebrate skeleton, especially the skeletons of modern mammals and recently extinct relatives.

Additional Resources
Those RTs interested in additional information about mass spectrometry and other techniques to perform molecular analyses of fossils can access the following Web-based resources:

• Analyses of soft tissue from Tyrannosaurus rex suggest the presence of protein. Science. 2007; 316:277-280. Those RTs who do not subscribe to Science online can purchase the article for a fee of US $10 (http://www.sciencemag.org/cgi/content/abstract/316/5822/277).
• GeneticArchaeology.com (http://www.geneticarchaeology.com/)
• i-mass.com (mass spectrometry Web site; http://i-mass.com/)
• Lawrence M. Witmer, PhD; Laboratory and Research (http://www.oucom.ohiou.edu/dbms-witmer/lab.htm)
• The Nuclear Microprobe at the Center for Accelerator Mass Spectrometry, Lawrence Livermore National Laboratory (https://cams.llnl.gov/facilities/microprobe/index.php)
• University of Texas at Austin High Resolution X ray CT Facility (http://www.ctlab.geo.utexas.edu/index.php)

References
1. Lindahl T. Recovery of antediluvian DNA. Nature. 1993;365:700.

2. Schweitzer MH, Suo Z, Avci R, et al. Analyses of soft tissue form Tyrannosaurus rex suggest the presence of protein. Science. 2007;316:277-280.

3. Tuross N, Stathoplos L. Ancient proteins in fossil bones. Methods Enzymol. 1993;224:121-129.

4. Semal P, Orban R. Collagen extraction from recent and fossil bones: quantitative and qualitative aspects. J Archaeol Sci. 1995;22:463-467.

5. van der Rest M, Garrone R. Collagen family of proteins. FASEB J. 1991;5:2814-2823.

6. Pruvost M, Schwarz R, Correia VB, et al. Freshly excavated fossil bones are best for amplification of ancient DNA. Proc Natl Acad Sci U S A. 2007;104:739-744.