Will current technologies enable dinosaur proteomics?

Figure 1. Vessels and other fine structures revealed after partial demineralization of dinosaur bone.

Because cortical bone is so dense, it is better protected from environmental influences than trabecular bone, and is most likely to be preserved in fossilized specimens. Cortical bone has produced dinosaur cells and vessels, so it stands to reason that microenvironmental conditions may be appropriate for recovery.

Exceptional preservation has long been noted in very rare cases in the fossil record. Fossils are preserved in an exceptional manner when original mineralogy, soft-tissue detail, primary organic molecules, cellular or subcellular detail, or labile components of organisms that normally degrade too quickly to enter the fossil record persist. Here, we present a brief review with case studies of one aspect of exceptional preservation, evidence for the persistence of organic molecules in skeletal elements of extinct organisms in the fossil record.

The invention and application of PCR sparked a new era in understanding the molecular level of organization of living organisms, as well as a growing understanding of the relationship between DNA and proteins, and ultimately, the sequencing of whole organism genomes. This in turn has enabled a hypothesis to be developed regarding evolutionary relationships among living organisms, which had previously relied almost exclusively on morphological characteristics. This was soon expanded to include extinct animals through the early work of Higuchi et al.[1], Thomas et al.[2] and others. It was not long after these successes that a race began to determine the oldest DNA from the most spectacular organisms. When publications began to appear that suggested DNA could survive from the age of dinosaurs, the ultimate goal seemed to be to obtain dinosaur DNA [3,4]. Indeed, in 1994, an article was published in a high-profile peer-reviewed journal claiming to have produced just that, DNA from a fragment of bone purported to belong to a dinosaur [5]. However, upon publication of this article, there began a race of another kind, to point out the many flaws in that study [6]. Many of the earlier papers have since been re-examined and found to be flawed in analyses, interpretation or incapable of being repeated [7]. Eventually, scientific skepticism of the prospects of ancient molecular recovery became so great that there was virtually no acceptance of reports of DNA older than a few hundred thousand years.

Proteins, however, have consistently demonstrated potential for greater longevity, beginning with Abelson, who reported the identification of amino acids from fossil material as long ago as 1954 [8]. Hints of protein preservation in fossils of geological age have persisted in the literature for decades. As technologies have become more sensitive, this potential has been increasingly realized in older fossils. Bada et al. reported amino acids from 40–130 million-year-old insects in amber inclusions, possibly the oldest unaltered amino acids found thus far [9]. Previously, glycoproteins were isolated from 80 million-year-old mollusk shells, which were separated electrophoretically and in which a particular repeating amino acid sequence common to contemporary mollusk shell proteins was identified [10]. Similarly, protein residues isolated from oyster shells from the Pleistocene through Cretaceous periods showed amino acid content comparable to that of extant oysters [11].

Amino acids identified in extracts from the vertebra of a 150 million-year-old sauropod, Seismosaurus, were separated by Gurley et al. by reverse-phase high-performance liquid chromatography (HPLC) [12]. Amino acid analysis confirmed the presence of protein remnants, and one fraction had the same retention time as collagen. However, amino acid analysis suggested that none of the fractions contained a significant amount of collagen. These studies were compromised by the lack of high-resolution analytical equipment, and the fact that simple identification of amino acids associated with fossils does not address the endogeneity of their source.

In 1990, the best preserved and most complete T. rex at that time was recovered from Late Cretaceous Hell Creek deposits in eastern Montana, USA. It was noted that the internal trabecular (spongy) bone of the limb elements did not appear to be altered or permineralized. Microscopy confirmed the exceptional state of preservation of this fossil and demonstrated the presence of microstructures preserved within the vessel channels that were morphologically similar to non-mammalian vertebrate red blood cells [13]. Multiple analyses were conducted on the bone fragments, and chemical extracts of the bone were subjected to resonance Raman (RR) spectroscopy, nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR) and other analytical techniques, which when coupled with microscopic data, attested to minimal diagenetic alteration of these bone fragments and hinted at the presence of original organics, including heme [14]. Immunological techniques supported that identification.

Other dinosaur fossils showed the retention of possibly original organic material within the bone matrices. Emberly et al. reported the isolation of noncollagenous proteins in chemical extracts of compact bone from a 125–130-million-year-old Iguanodontid dinosaur [15]. Proteins ranging from 5 to 66 kDa were resolved by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and N-terminal sequencing revealed potential similarities of one component with cystatin, an inhibitor of osteoclast-mediated bone resorption. Osteocalcin immunoreactivity was also observed in Iguanodon bone extracts, and the abolition of Alcian blue reactivity following protease-free chondroitinase digestion suggested the presence of proteoglycans [16]. Preserved osteocalcin antigenicity in dinosaur bones was also independently reported by Muyzer et al.[17] and Collins et al.[18].

Within the last decade, Titanosaurus eggs containing embryonic bone and 3D mineralized embryonic skin were described from Patagonia, Argentina [19]. The eggshells themselves demonstrated minimal alteration, with scanning electron microscopy (SEM) showing detail including surface tubercles, elongated pores and an apparent shell membrane, within which were embedded the mamillary knobs of the eggshell. When these eggshells were subjected to demineralization, an extremely strong petroliferous odor was released, indicating the association of degraded organics complexed within the mineral lattice of the shells. Rabbits immunized with titanosaurid eggshell extracts produced antibodies that reacted with chicken ovalbumin [20].

Antibody–antigen reactivity has been observed in other Mesozoic fossils. Immunohistochemical evidence of collagen preserved in embryonic skeletal tissues of an enantiornithine bird from the late Cretaceous period was supported by exceptional submicroscopic preservation. Images obtained using atomic force microscopy (AFM), illustrated significant change in submicron morphology when these demineralized tissues were subjected to collagenase digestion, verifying the observed decrease in collagen antibody binding after similar digestion [21].

In another case, an exceptionally well-preserved articulated skeleton of an enigmatic bird relative, the mononykid Shuvuuia deserti, was recovered from the Mongolian region of Ukhaa Tolgod. During preparation, small white fibers were noted along the skull and cervical regions of the skeleton, preserved in immature, poorly sorted coarse-grained sands. Transmitted light microscopy and SEM showed these structures to be hollow and to possess morphological similarities to simple hair-like feathers of extant birds [22]. Polyclonal antibodies raised against avian β-keratin were incubated with embedded sections of these hollow fibers, and were shown to bind these fibers specifically.

However, these works, though suggestive, were not compelling and certainly not universally accepted, as there was, and remains, scientific resistance to the idea that biomolecules could be retained in multi-million-year-old skeletal remains. The reigning model of fossilization is that upon the death of an organism, soft tissues, cells and finally molecules, would be degraded to completion in a relatively short period of time. Pores and microscopic spaces opened by the degradation of organics would then be secondarily filled with exogenous material, turning the bone into rock. Both theoretical kinetics and bench-top experiments suggested that all protein and DNA molecules would be degraded to completion in under 1 million years [23,24], although some kinetic studies supported the possibility that some proteins could persist, at least in theory [18].

The evidence supporting this fossilization model was soon to be challenged, at least in part. At the end of the field season of 2000, associated skeletal elements from a T. rex were discovered, buried under approximately 1000 m ³ of Hell Creek Formation sandstones, at the base of a steep cliff in northeastern Montana. Recovery of the specimen, Museum of the Rockies (MOR) 1125, took almost three field seasons and was completed in the summer of 2003. The specimen was dubbed ‘B-rex’ after its discoverer, Bob Harmon. Examination of the internal, endosteal bone fragments of the femur, broken in recovery, found a highly unusual bone tissue on the endosteal surfaces of the otherwise unremarkable compact bone. These tissues were shown to be similar in all appearances to avian reproductive bone known as medullary bone [25]. This identified the T. rex as an actively reproducing female in the process of shelling eggs. Histological studies demonstrated that the dinosaur was approximately 18 years of age (probably the oldest case of teenage motherhood on record) [26]. In order to demonstrate that this new bone tissue was consistent with medullary bone in organization, a partial etch was performed using EDTA to remove the mineral phase of this bone, revealing the organization of fibers. However, this demineralization process revealed much more: the unexpected, unpredicted presence of soft, fibrous matrix; what appeared to be transparent, hollow, flexible vessels; and at least two populations of cell-like microstructures.

Examination of the demineralized skeletal elements by multiple microscopic methods revealed pliable, hollow blood vessels containing microstructures resembling erythrocytes [27]. Transmitted light microscopy and SEM also revealed osteocytes with flexible filipodia recovered from demineralized bone fragments, and that these cell-like structures had internal contents. Several other dinosaurs, including other T. rex specimens, hadrosaurs and ceratopsians, were also shown to retain some of these components [28].

Immunohistochemical staining of demineralized and sectioned dinosaur tissues showed positive reaction with polyclonal antibodies raised against avian collagen I. Controls, including both collagen inhibition and collagenase digestion were negative. The microscopic and immunological data supported the hypothesis that collagen remained preserved in these dinosaur tissues, but in very low concentrations. However, this could only be completely confirmed through protein sequence analyses of whole bone extracts. Multiple extracts of MOR 1125 cortical bone produced seven peptides that could be definitively assigned to collagen I [29,30]. The presence of sequenceable collagen fragments implies the peptide bonds were remarkably stable [31]. Although fragmentary and extremely low in concentration, comparing the dinosaur peptides against those residing in existing databases for living organisms using BLAST showed dinosaur peptides to be most similar to extant chicken, although in some cases, the peptides sequenced had areas of overlap with many organisms. This is not unexpected for highly conserved proteins. Further interpretation has suggested that at least one collagen peptide sequence was unique to T. rex[29].

The preservation of proteinaceous materials over millions of years has caused paleobiologists to reconsider current models of fossilization. Conventional wisdom held that, under normal circumstances, decomposition occurs so rapidly and completely that, after a relatively short period of time, no molecular fragments (let alone cells or tissues) would remain. However, the observation of these components in multiple specimens of geological age, supported by amino acid sequence data from collagen preserved in the skeletal elements of T. rex, provide evidence for molecular preservation over millions of years.

Challenges of modern bone proteomics

The analyses of proteins derived from dinosaur bone will logically draw on what we have learned from bone from extant species. Unfortunately, proteomic analysis of extant bone has largely been limited to the study of proteins derived from cultured osteoblasts and osteoclasts. Proteomics conducted on whole bone extracts is much more complex, partly because of the intrinsic nature of the organic phase of bone (e.g., 35% calcium, 19% phosphate in mammals) and in part to the fact that compact bone usually requires prolonged demineralization over several days to enable full access to cellular components. The kinetics of bone demineralization have been proposed by Horneman et al.[32].

Recently, Jiang et al. used 2D chromatography and tandem mass spectrometry to identify more than 1000 peptides released from mammalian compact bone following acid demineralization, the most common method for removing the mineral phase of bone [33]. This process is aided by the fact that acid induces Asp-specific cleavages in proteins, favoring the fragmentation of intact proteins [34]. Another study demonstrated that acid demineralization using hydrochloric, acetic or formic acids resulted in significant protein losses from mammalian and avian bone [35]. Protein losses were minimized using rapid cycles of high and low hydrostatic pressure, facilitating the extraction of intact proteins from bone without prior demineralization [36].

Only femtomolar quantities of protein are likely to be available from grams of dinosaur bone. Therefore, the proteins must be concentrated to reach levels within the detection limits of downstream analyses. However, exogenous contaminants as well as endogenous minerals associated with the organic fraction are concentrated to the same degree in the process, making interpretation difficult. In addition, methods employed to achieve increased concentration and/or purification of the proteinaceous material, while found only in permineralized specimens thus far, may result in unaffordable loss of protein. Current protein precipitation methods are ineffectual when the protein concentration is very low, because contaminating ions such as phosphate are concentrated rather than removed, and interfere with some sensitive analytical techniques or mask extremely low concentrations of biomolecular remnants.

A common dilemma in proteomics is that the reagents most compatible with mass spectrometry are frequently not suitably stringent to ensure solubility of all protein constituents. Hence, the analysis may be skewed toward the preferential recognition of hydrophilic proteins. More stringent reagents, such as chaotropes and detergents, typically yield more proteins, including hydrophobic ones, but frequently render the sample incompatible with downstream processes.

Since exogenous proteins, common in all laboratory preparations as background, are possible contaminants in these preparations, the burden of positively identifying putative dinosaur proteins falls on the researcher. If the eventual goal is to apply proteomic technologies to identify and characterize bone-derived proteins from dinosaurs and other fossils, a database must be derived from extant animals that bracket these organisms. Of all living taxa today, ostrich is probably most closely related to theropod dinosaurs.

What can be learned from dinosaurs?

There are several reasons to pursue molecular recovery from dinosaurs and other fossils, but just a few of these are considered here. Molecules recovered from fossils will offer insight into evolution at the molecular level. Phylogenetic estimation of divergence times and the direction and rate of evolutionary change has been limited to the extant members of crown clades. Crocodiles and birds are the two crown group clades representing Archosauria, but both are highly derived and may not be the best models for predicting certain aspects of dinosaur biology and evolution.

Because we have well-supported molecular and morphological phylogenies of living taxa, we can estimate molecular clocks with some confidence. However, sites become saturated over time due to limited character state options (four for DNA, 20 for proteins). Recovery of informative fragments from fossil material of geological age, if validated, can be used to root molecular clocks and to polarize character state evolution in estimating direction of molecular change.

Third, some molecules may hold clues to the physiology of organisms, either in overall content (i.e., GC ratios are higher in endothermic animals overall) or by recognition of specific genetic changes in molecules that may be tied to metabolic upregulation, such as allosteric binding pocket changes in hemoglobin molecules [37].

While only found in permineralized specimens thus far, morphological anomalies identified as osteomas and other tumors, including metastatic cancer, have been reported in dinosaur bones [38]. Using x-ray and computerized tomography, Rothschild et al. have screened over 10,000 dinosaur vertebrae specimens in the largest epidemiological study of its kind [39]. With the exception of a suspected brain tumor found in a 72 million year old Gorgosaurus, the tumors appeared exclusively in Late Cretaceous hadrosaurs.

Acknowledgement

The authors thank Ada Kwan of Pressure BioSciences for her assistance during the preparation of this manuscript.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.