Soft-Tissue, Rare Earth Element, and Molecular Analyses of Dreadnoughtus schrani, an Exceptionally Complete Titanosaur from Argentina.

Evidence that organic material preserves in deep time (>1 Ma) has been reported using a wide variety of analytical techniques. However, the comprehensive geochemical data that could aid in building robust hypotheses for how soft-tissues persist over millions of years are lacking from most paleomolecular reports. Here, we analyze the molecular preservation and taphonomic history of the Dreadnougtus schrani holotype (MPM-PV 1156) at both macroscopic and microscopic levels. We review the stratigraphy, depositional setting, and physical taphonomy of the D. schrani skeletal assemblage, and extensively characterize the preservation and taphonomic history of the humerus at a micro-scale via: (1) histological analysis (structural integrity) and X-ray diffraction (exogenous mineral content); (2) laser ablation-inductively coupled plasma mass spectrometry (analyses of rare earth element content throughout cortex); (3) demineralization and optical microscopy (soft-tissue microstructures); (4) in situ and in-solution immunological assays (presence of endogenous protein). Our data show the D. schrani holotype preserves soft-tissue microstructures and remnants of endogenous bone protein. Further, it was exposed to LREE-enriched groundwaters and weakly-oxidizing conditions after burial, but experienced negligible further chemical alteration after early-diagenetic fossilization. These findings support previous hypotheses that fossils that display low trace element uptake are favorable targets for paleomolecular analyses.

Soft Tissue and Biomolecular Preservation in Vertebrate Fossils from Glauconitic, Shallow Marine Sediments of the Hornerstown Formation, Edelman Fossil Park, New Jersey.

Endogenous biomolecules and soft tissues are known to persist in the fossil record. To date, these discoveries derive from a limited number of preservational environments, (e.g., fluvial channels and floodplains), and fossils from less common depositional environments have been largely unexplored. We conducted paleomolecular analyses of shallow marine vertebrate fossils from the Cretaceous-Paleogene Hornerstown Formation, an 80-90% glauconitic greensand from Jean and Ric Edelman Fossil Park in Mantua Township, NJ. Twelve samples were demineralized and found to yield products morphologically consistent with vertebrate osteocytes, blood vessels, and bone matrix. Specimens from these deposits that are dark in color exhibit excellent histological preservation and yielded a greater recovery of cells and soft tissues, whereas lighter-colored specimens exhibit poor histology and few to no cells/soft tissues. Additionally, a well-preserved femur of the marine crocodilian Thoracosaurus was found to have retained endogenous collagen I by immunofluorescence and enzyme-linked immunosorbent assays. Our results thus not only corroborate previous findings that soft tissue and biomolecular recovery from fossils preserved in marine environments are possible but also expand the range of depositional environments documented to preserve endogenous biomolecules, thus broadening the suite of geologic strata that may be fruitful to examine in future paleomolecular studies.

Deep Time Paleoproteomics: Looking Forward.

The goal of paleoproteomics is to characterize proteins from specimens that have been subjected to the degrading and obscuring effects of time, thus obtaining biological information about tissues or organisms both unobservable in the present and unobtainable through morphological study. Although the description of sequences from Tyrannosaurus rex and Brachylophosaurus canadensis suggested that proteins may persist over tens of millions of years, the majority of paleoproteomic analyses have focused on historical, archeological, or relatively young paleontological samples that rarely exceed 1 million years in age. However, recent advances in methodology and analyses of diverse tissues types (e.g., fossil eggshell, dental enamel) have begun closing the large window of time that remains unexplored in the fossil history of the Cenozoic. In this perspective, we discuss the history and current state of deep time paleoproteomics (DTPp), here defined as paleoproteomic study of samples ∼1 million years (1 Ma) or more in age. We then discuss the future of DTPp research, including what we see as critical ways the field can expand, advancements in technology that can be utilized, and the types of questions DTPp can address if such a future is realized.

Diagenetiforms: A new term to explain protein changes as a result of diagenesis in paleoproteomics.

The term proteoform describes all combinations of change in a protein, as elucidated through intact mass proteomics. Paleoproteomic studies have begun using digestion-free and top-down techniques to access information from ancient and historical remains. However, to discuss protein changes that uniquely occur to archaeological and paleontological proteomes as the result of diagenesis (i.e., physical and chemical change imparted by burial), a novel term is needed that both addresses issues of combinatorics and distinguishes diagenetic-specific alteration. SIGNIFICANCE: The term diagenetiform provides the opportunity to communicate clearly the sets of diagenetic changes found on preserved proteins. The diagenetiform nomenclature will allow for top-down paleoproteomic studies to accurately describe the total changes detected on ancient proteins.

Molecular tests support the viability of rare earth elements as proxies for fossil biomolecule preservation.

The rare earth element (REE) composition of a fossil bone reflects its chemical alteration during diagenesis. Consequently, fossils presenting low REE concentrations and/or REE profiles indicative of simple diffusion, signifying minimal alteration, have been proposed as ideal candidates for paleomolecular investigation. We directly tested this prediction by conducting multiple biomolecular assays on a well-preserved fibula of the dinosaur Edmontosaurus from the Cretaceous Hell Creek Formation previously found to exhibit low REE concentrations and steeply-declining REE profiles. Gel electrophoresis identified the presence of organic material in this specimen, and subsequent immunofluorescence and enzyme-linked immunosorbant assays identified preservation of epitopes of the structural protein collagen I. Our results thereby support the utility of REE profiles as proxies for soft tissue and biomolecular preservation in fossil bones. Based on considerations of trace element taphonomy, we also draw predictions as to the biomolecular recovery potential of additional REE profile types exhibited by fossil bones.

Proteomic method to extract, concentrate, digest and enrich peptides from fossils with coloured (humic) substances for mass spectrometry analyses.

Humic substances are breakdown products of decaying organic matter that co-extract with proteins from fossils. These substances are difficult to separate from proteins in solution and interfere with analyses of fossil proteomes. We introduce a method combining multiple recent advances in extraction protocols to both concentrate proteins from fossil specimens with high humic content and remove humics, producing clean samples easily analysed by mass spectrometry (MS). This method includes: (i) a non-demineralizing extraction buffer that eliminates protein loss during the demineralization step in routine methods; (ii) filter-aided sample preparation (FASP) of peptides, which concentrates and digests extracts in one filter, allowing the separation of large humics after digestion; (iii) centrifugal stage tipping, which further clarifies and concentrates samples in a uniform process performed simultaneously on multiple samples. We apply this method to a moa fossil (approx. 800-1000 years) dark with humic content, generating colourless samples and enabling the detection of more proteins with greater sequence coverage than previous MS analyses on this same specimen. This workflow allows analyses of low-abundance proteins in fossils containing humics and thus may widen the range of extinct organisms and regions of their proteomes we can explore with MS.

Paleoproteomics of Mesozoic Dinosaurs and Other Mesozoic Fossils.

Molecular studies have contributed greatly to our understanding of evolutionary processes that act upon virtually every aspect of living organisms. However, these studies are limited with regard to extinct organisms, particularly those from the Mesozoic because fossils pose unique challenges to molecular workflows, and because prevailing wisdom suggests no endogenous molecular components can persist into deep time. Here, the power and potential of a molecular approach to Mesozoic fossils is discussed. Molecular methods that have been applied to Mesozoic fossils-including iconic, non-avian dinosaurs- and the challenges inherent in such analyses, are compared and evaluated. Taphonomic processes resulting in the transition of living organisms from the biosphere into the fossil record are reviewed, and the possible effects of taphonomic alteration on downstream analyses that can be problematic for very old material (e.g., molecular modifications, limitations of on comparative databases) are addressed. Molecular studies applied to ancient remains are placed in historical context, and past and current studies are evaluated with respect to producing phylogenetically and/or evolutionarily significant data. Finally, some criteria for assessing the presence of endogenous biomolecules in very ancient fossil remains are suggested as a starting framework for such studies.

The molecular evolution of feathers with direct evidence from fossils.

Dinosaur fossils possessing integumentary appendages of various morphologies, interpreted as feathers, have greatly enhanced our understanding of the evolutionary link between birds and dinosaurs, as well as the origins of feathers and avian flight. In extant birds, the unique expression and amino acid composition of proteins in mature feathers have been shown to determine their biomechanical properties, such as hardness, resilience, and plasticity. Here, we provide molecular and ultrastructural evidence that the pennaceous feathers of the Jurassic nonavian dinosaur Anchiornis were composed of both feather ß-keratins and α-keratins. This is significant, because mature feathers in extant birds are dominated by ß-keratins, particularly in the barbs and barbules forming the vane. We confirm here that feathers were modified at both molecular and morphological levels to obtain the biomechanical properties for flight during the dinosaur-bird transition, and we show that the patterns and timing of adaptive change at the molecular level can be directly addressed in exceptionally preserved fossils in deep time.

A Comparison of Common Mass Spectrometry Approaches for Paleoproteomics.

The last two decades have seen a broad diversity of methods used to identify and/or characterize proteins in the archeological and paleontological record. Of these, mass spectrometry has opened an unprecedented window into the proteomes of the past, providing protein sequence data from long extinct animals as well as historical and prehistorical artifacts. Thus, application of mass spectrometry to fossil remains has become an attractive source for ancient molecular sequences with which to conduct evolutionary studies, particularly in specimens older than the proposed limit of amplifiable DNA detection. However, "mass spectrometry" covers a range of mass-based proteomic approaches, each of which utilize different technology and physical principles to generate unique types of data, with their own strengths and challenges. Here, we discuss a variety of mass spectrometry techniques that have or may be used to detect and characterize archeological and paleontological proteins, with a particular focus on MALDI-MS, LC-MS/MS, TOF-SIMS, and MSi. The main differences in their functionality, the types of data they produce, and the potential effects of diagenesis on their results are considered.

Expansion for the Brachylophosaurus canadensis Collagen I Sequence and Additional Evidence of the Preservation of Cretaceous Protein.

Sequence data from biomolecules such as DNA and proteins, which provide critical information for evolutionary studies, have been assumed to be forever outside the reach of dinosaur paleontology. Proteins, which are predicted to have greater longevity than DNA, have been recovered from two nonavian dinosaurs, but these results remain controversial. For proteomic data derived from extinct Mesozoic organisms to reach their greatest potential for investigating questions of phylogeny and paleobiology, it must be shown that peptide sequences can be reliably and reproducibly obtained from fossils and that fragmentary sequences for ancient proteins can be increasingly expanded. To test the hypothesis that peptides can be repeatedly detected and validated from fossil tissues many millions of years old, we applied updated extraction methodology, high-resolution mass spectrometry, and bioinformatics analyses on a Brachylophosaurus canadensis specimen (MOR 2598) from which collagen I peptides were recovered in 2009. We recovered eight peptide sequences of collagen I: two identical to peptides recovered in 2009 and six new peptides. Phylogenetic analyses place the recovered sequences within basal archosauria. When only the new sequences are considered, B. canadensis is grouped more closely to crocodylians, but when all sequences (current and those reported in 2009) are analyzed, B. canadensis is placed more closely to basal birds. The data robustly support the hypothesis of an endogenous origin for these peptides, confirm the idea that peptides can survive in specimens tens of millions of years old, and bolster the validity of the 2009 study. Furthermore, the new data expand the coverage of B. canadensis collagen I (a 33.6% increase in collagen I alpha 1 and 116.7% in alpha 2). Finally, this study demonstrates the importance of reexamining previously studied specimens with updated methods and instrumentation, as we obtained roughly the same amount of sequence data as the previous study with substantially less sample material. Data are available via ProteomeXchange with identifier PXD005087.

Bone protein "extractomics": comparing the efficiency of bone protein extractions of Gallus gallus in tandem mass spectrometry, with an eye towards paleoproteomics.

Proteomic studies of bone require specialized extraction protocols to demineralize and solubilize proteins from within the bone matrix. Although various protocols exist for bone protein recovery, little is known about how discrete steps in each protocol affect the subset of the bone proteome recovered by mass spectrometry (MS) analyses. Characterizing these different "extractomes" will provide critical data for development of novel and more efficient protein extraction methodologies for fossils. Here, we analyze 22 unique sub-extractions of chicken bone and directly compare individual extraction components for their total protein yield and diversity and coverage of bone proteins identified by MS. We extracted proteins using different combinations and ratios of demineralizing reagents, protein-solubilizing reagents, and post-extraction buffer removal methods, then evaluated tryptic digests from 20 µg aliquots of each fraction by tandem MS/MS on a 12T FT-ICR mass spectrometer. We compared total numbers of peptide spectral matches, peptides, and proteins identified from each fraction, the redundancy of protein identifications between discrete steps of extraction methods, and the sequence coverage obtained for select, abundant proteins. Although both alpha chains of collagen I (the most abundant protein in bone) were found in all fractions, other collagenous and non-collagenous proteins (e.g., apolipoprotein, osteonectin, hemoglobin) were differentially identified. We found that when a standardized amount of extracted proteins was analyzed, extraction steps that yielded the most protein (by weight) from bone were often not the ones that produced the greatest diversity of bone proteins, or the highest degree of protein coverage. Generally, the highest degrees of diversity and coverage were obtained from demineralization fractions, and the proteins found in the subsequent solubilization fractions were highly redundant with those in the previous fraction. Based on these data, we identify future directions and parameters to consider (e.g., proteins targeted, amount of sample required) when applying discrete parts of these protocols to fossils.

Molecular evidence of keratin and melanosomes in feathers of the Early Cretaceous bird Eoconfuciusornis.

Microbodies associated with feathers of both nonavian dinosaurs and early birds were first identified as bacteria but have been reinterpreted as melanosomes. Whereas melanosomes in modern feathers are always surrounded by and embedded in keratin, melanosomes embedded in keratin in fossils has not been demonstrated. Here we provide multiple independent molecular analyses of both microbodies and the associated matrix recovered from feathers of a new specimen of the basal bird Eoconfuciusornis from the Early Cretaceous Jehol Biota of China. Our work represents the oldest ultrastructural and immunological recognition of avian beta-keratin from an Early Cretaceous (∼130-Ma) bird. We apply immunogold to identify protein epitopes at high resolution, by localizing antibody-antigen complexes to specific fossil ultrastructures. Retention of original keratinous proteins in the matrix surrounding electron-opaque microbodies supports their assignment as melanosomes and adds to the criteria employable to distinguish melanosomes from microbial bodies. Our work sheds new light on molecular preservation within normally labile tissues preserved in fossils.

Peptide sequences from the first Castoroides ohioensis skull and the utility of old museum collections for palaeoproteomics.

Vertebrate fossils have been collected for hundreds of years and are stored in museum collections around the world. These remains provide a readily available resource to search for preserved proteins; however, the vast majority of palaeoproteomic studies have focused on relatively recently collected bones with a well-known handling history. Here, we characterize proteins from the nasal turbinates of the first Castoroides ohioensis skull ever discovered. Collected in 1845, this is the oldest museum-curated specimen characterized using palaeoproteomic tools. Our mass spectrometry analysis detected many collagen I peptides, a peptide from haemoglobin beta, and in vivo and diagenetic post-translational modifications. Additionally, the identified collagen I sequences provide enough resolution to place C. ohioensis within Rodentia. This study illustrates the utility of archived museum specimens for both the recovery of preserved proteins and phylogenetic analyses.

Glutamine deamidation: an indicator of antiquity, or preservational quality?

RATIONALE: Much credence has been given in the paleoproteomic community to glutamine deamidation as a proxy for the age of proteins derived from fossil and subfossil material, and this modification has been invoked as a means for determining the endogeneity of molecules recovered from very old fossil specimens. METHODS: We re-evaluated the relationship between glutamine deamidation and geologic time by examining previously published data from five recent mass spectrometry studies of archeaological fossils. Deamidation values recovered for fossils were graphed against their reported chronologic age using WebPlotDigitizer. RESULTS: The experimental data that has been produced from fossil material to date show that the extent of glutamine deamidation does not correspond to the absolute age of the specimens being examined, but rather show extreme variation between specimens of similar age and taxonomic affinity. CONCLUSIONS: Because deamidation rates and levels can be greatly affected by numerous chemical and environmental factors, we propose that glutamine deamidation is better suited as an indicator of preservational quality and/or environmental conditions than a mark of the endogeneity or authenticity of ancient proteins.

Mass Spectrometry and Antibody-Based Characterization of Blood Vessels from Brachylophosaurus canadensis.

Structures similar to blood vessels in location, morphology, flexibility, and transparency have been recovered after demineralization of multiple dinosaur cortical bone fragments from multiple specimens, some of which are as old as 80 Ma. These structures were hypothesized to be either endogenous to the bone (i.e., of vascular origin) or the result of biofilm colonizing the empty osteonal network after degradation of original organic components. Here, we test the hypothesis that these structures are endogenous and thus retain proteins in common with extant archosaur blood vessels that can be detected with high-resolution mass spectrometry and confirmed by immunofluorescence. Two lines of evidence support this hypothesis. First, peptide sequencing of Brachylophosaurus canadensis blood vessel extracts is consistent with peptides comprising extant archosaurian blood vessels and is not consistent with a bacterial, cellular slime mold, or fungal origin. Second, proteins identified by mass spectrometry can be localized to the tissues using antibodies specific to these proteins, validating their identity. Data are available via ProteomeXchange with identifier PXD001738.

Biologically and diagenetically derived peptide modifications in moa collagens.

The modifications that occur on proteins in natural environments over time are not well studied, yet characterizing them is vital to correctly interpret sequence data recovered from fossils. The recently extinct moa (Dinornithidae) is an excellent candidate for investigating the preservation of proteins, their post-translational modifications (PTMs) and diagenetic alterations during degradation. Moa protein extracts were analysed using mass spectrometry, and peptides from collagen I, collagen II and collagen V were identified. We also identified biologically derived PTMs (i.e. methylation, di-methylation, alkylation, hydroxylation, fucosylation) on amino acids at locations consistent with extant proteins. In addition to these in vivo modifications, we detected novel modifications that are probably diagenetically derived. These include loss of hydroxylation/glutamic semialdehyde, carboxymethyllysine and peptide backbone cleavage, as well as previously noted deamidation. Moa collagen sequences and modifications provide a baseline by which to evaluate proteomic studies of other fossils, and a framework for defining the molecular relationship of moa to other closely related taxa.

A gigantic, exceptionally complete titanosaurian sauropod dinosaur from southern Patagonia, Argentina.

Titanosaurian sauropod dinosaurs were the most diverse and abundant large-bodied herbivores in the southern continents during the final 30 million years of the Mesozoic Era. Several titanosaur species are regarded as the most massive land-living animals yet discovered; nevertheless, nearly all of these giant titanosaurs are known only from very incomplete fossils, hindering a detailed understanding of their anatomy. Here we describe a new and gigantic titanosaur, Dreadnoughtus schrani, from Upper Cretaceous sediments in southern Patagonia, Argentina. Represented by approximately 70% of the postcranial skeleton, plus craniodental remains, Dreadnoughtus is the most complete giant titanosaur yet discovered, and provides new insight into the morphology and evolutionary history of these colossal animals. Furthermore, despite its estimated mass of about 59.3 metric tons, the bone histology of the Dreadnoughtus type specimen reveals that this individual was still growing at the time of death.

Lamniform shark teeth from the late cretaceous of southernmost South America (Santa Cruz province, Argentina).

Here we report multiple lamniform shark teeth recovered from fluvial sediments in the (Campanian-Maastrichtian) Cerro Fortaleza Formation, Santa Cruz Province, Argentina. This small tooth assemblage is compared to various lamniform sharks possessing similar dental morphologies, including Archaeolamna, Cretalamna, Dwardius, Dallasiella, and Cretodus. Although the teeth share numerous morphological features with the genus Archaeolamna, including a developed neck that maintains a relatively consistent width along the base of the crown, the small sample size and incomplete nature of these specimens precludes definitive taxonomic assignment. Regardless, the discovery of selachian teeth unique from those previously described for the region broadens the known diversity of Late Cretaceous South American sharks. Additionally, the discovery of the teeth in fluvial sandstone may indicate a euryhaline paleobiology in the lamniform taxon or taxa represented by this tooth assemblage.

Protein molecular data from ancient (>1 million years old) fossil material: pitfalls, possibilities and grand challenges.

Advances in resolution and sensitivity of analytical techniques have provided novel applications, including the analyses of fossil material. However, the recovery of original proteinaceous components from very old fossil samples (defined as >1 million years (1 Ma) from previously named limits in the literature) is far from trivial. Here, we discuss the challenges to recovery of proteinaceous components from fossils, and the need for new sample preparation techniques, analytical methods, and bioinformatics to optimize and fully utilize the great potential of information locked in the fossil record. We present evidence for survival of original components across geological time, and discuss the potential benefits of recovery, analyses, and interpretation of fossil materials older than 1 Ma, both within and outside of the fields of evolutionary biology.