Microwave-assisted acid hydrolysis for whole-bone proteomics and paleoproteomics.

RATIONALE: Whole-bone proteomic analyses rely on lengthy sample preparation including demineralization and digestion to break bone down into peptides to recover using mass spectrometry. However, microwave-assisted acid hydrolysis, a technique used in proteomic analyses of other soft tissues and cells, will combine both demineralization and digestion and only take minutes. METHODS: To test microwave-assisted hydrolysis on whole moose bone, we microwaved five concentrations of acetic and formic acids (15%, 12.5%, 10%, 7.5% and 5%) for three times (10, 20 and 30 min) at 140°C using an ETHOS UP high performance microwave digestion system. Peptides were injected and separated using Thermo BioBasic C18 columns and detected with an LTQ Orbitrap Velos mass spectrometer. We searched the raw data on PEAKS 8.5 against the white-tailed deer database. RESULTS: Formic acid hydrolysis led to the most complete digestion, and therefore the highest number of peptide spectrum matches, more protein groups and better sequence coverage for collagenous proteins. However, for the formic acid samples there is a tradeoff with digestion completeness and a higher incidence of in vitro modifications (i.e. formylation) that are not induced using acetic acid. Acetic acid has greater cleavage specificity and higher sequence coverage for non-collagenous proteins. CONCLUSIONS: Depending on the goals of analysis, there are benefits and drawbacks to using both acetic acid and formic acid. Overall, microwave-assisted acid hydrolysis was successful in demineralizing and digesting bone fragments to considerably speed up the preparation for bottom-up proteomics analysis.

Chemical, experimental, and morphological evidence for diagenetically altered melanin in exceptionally preserved fossils.

In living organisms, color patterns, behavior, and ecology are closely linked. Thus, detection of fossil pigments may permit inferences about important aspects of ancient animal ecology and evolution. Melanin-bearing melanosomes were suggested to preserve as organic residues in exceptionally preserved fossils, retaining distinct morphology that is associated with aspects of original color patterns. Nevertheless, these oblong and spherical structures have also been identified as fossilized bacteria. To date, chemical studies have not directly considered the effects of diagenesis on melanin preservation, and how this may influence its identification. Here we use time-of-flight secondary ion mass spectrometry to identify and chemically characterize melanin in a diverse sample of previously unstudied extant and fossil taxa, including fossils with notably different diagenetic histories and geologic ages. We document signatures consistent with melanin preservation in fossils ranging from feathers, to mammals, to amphibians. Using principal component analyses, we characterize putative mixtures of eumelanin and phaeomelanin in both fossil and extant samples. Surprisingly, both extant and fossil amphibians generally exhibit melanosomes with a mixed eumelanin/phaeomelanin composition rather than pure eumelanin, as assumed previously. We argue that experimental maturation of modern melanin samples replicates diagenetic chemical alteration of melanin observed in fossils. This refutes the hypothesis that such fossil microbodies could be bacteria, and demonstrates that melanin is widely responsible for the organic soft tissue outlines in vertebrates found at exceptional fossil localities, thus allowing for the reconstruction of certain aspects of original pigment patterns.

Using Macro- and Microscale Preservation in Vertebrate Fossils as Predictors for Molecular Preservation in Fluvial Environments.

Exceptionally preserved fossils retain soft tissues and often the biomolecules that were present in an animal during its life. The majority of terrestrial vertebrate fossils are not traditionally considered exceptionally preserved, with fossils falling on a spectrum ranging from very well-preserved to poorly preserved when considering completeness, morphology and the presence of microstructures. Within this variability of anatomical preservation, high-quality macro-scale preservation (e.g., articulated skeletons) may not be reflected in molecular-scale preservation (i.e., biomolecules). Excavation of the Hayden Quarry (HQ; Chinle Formation, Ghost Ranch, NM, USA) has resulted in the recovery of thousands of fossilized vertebrate specimens. This has contributed greatly to our knowledge of early dinosaur evolution and paleoenvironmental conditions during the Late Triassic Period (~212 Ma). The number of specimens, completeness of skeletons and fidelity of osteohistological microstructures preserved in the bone all demonstrate the remarkable quality of the fossils preserved at this locality. Because the Hayden Quarry is an excellent example of good preservation in a fluvial environment, we have tested different fossil types (i.e., bone, tooth, coprolite) to examine the molecular preservation and overall taphonomy of the HQ to determine how different scales of preservation vary within a single locality. We used multiple high-resolution mass spectrometry techniques (TOF-SIMS, GC-MS, FT-ICR MS) to compare the fossils to unaltered bone from extant vertebrates, experimentally matured bone, and younger dinosaurian skeletal material from other fluvial environments. FT-ICR MS provides detailed molecular information about complex mixtures, and TOF-SIMS has high elemental spatial sensitivity. Using these techniques, we did not find convincing evidence of a molecular signal that can be confidently interpreted as endogenous, indicating that very good macro- and microscale preservation are not necessarily good predictors of molecular preservation.

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 preservation in mammoth bone and variation based on burial environment.

Biomolecules preserved in fossils are expanding our understanding of the biology and evolution of ancient animals. Molecular taphonomy seeks to understand how these biomolecules are preserved and how they can be interpreted. So far, few studies on molecular preservation have considered burial context to understand its impact on preservation or the potentially complementary information from multiple biomolecular classes. Here, we use mass spectrometry and other analytical techniques to detect the remains of proteins and lipids within intact fossil mammoth bones of different ages and varied depositional setting. By combining these approaches, we demonstrate that endogenous amino acids, amides and lipids can preserve well in fossil bone. Additionally, these techniques enable us to examine variation in preservation based on location within the bone, finding dense cortical bone better preserves biomolecules, both by slowing the rate of degradation and limiting the extent of exogenous contamination. Our dataset demonstrates that biomolecule loss begins early, is impacted by burial environment and temperature, and that both exogenous and endogenous molecular signals can be both present and informative in a single fossil.

Assessing ontogenetic maturity in extinct saurian reptiles.

Morphology forms the most fundamental level of data in vertebrate palaeontology because it is through interpretations of morphology that taxa are identified, creating the basis for broad evolutionary and palaeobiological hypotheses. Assessing maturity is one of the most basic aspects of morphological interpretation and provides the means to study the evolution of ontogenetic changes, population structure and palaeoecology, life-history strategies, and heterochrony along evolutionary lineages that would otherwise be lost to time. Saurian reptiles (the least-inclusive clade containing Lepidosauria and Archosauria) have remained an incredibly diverse, numerous, and disparate clade through their ~260-million-year history. Because of the great disparity in this group, assessing maturity of saurian reptiles is difficult, fraught with methodological and terminological ambiguity. We compiled a novel database of literature, assembling >900 individual instances of saurian maturity assessment, to examine critically how saurian maturity has been diagnosed. We review the often inexact and inconsistent terminology used in saurian maturity assessment (e.g. 'juvenile', 'mature') and provide routes for better clarity and cross-study coherence. We describe the various methods that have been used to assess maturity in every major saurian group, integrating data from both extant and extinct taxa to give a full account of the current state of the field and providing method-specific pitfalls, best practices, and fruitful directions for future research. We recommend that a new standard subsection, 'Ontogenetic Assessment', be added to the Systematic Palaeontology portions of descriptive studies to provide explicit ontogenetic diagnoses with clear criteria. Because the utility of different ontogenetic criteria is highly subclade dependent among saurians, even for widely used methods (e.g. neurocentral suture fusion), we recommend that phylogenetic context, preferably in the form of a phylogenetic bracket, be used to justify the use of a maturity assessment method. Different methods should be used in conjunction as independent lines of evidence when assessing maturity, instead of an ontogenetic diagnosis resting entirely on a single criterion, which is common in the literature. Critically, there is a need for data from extant taxa with well-represented growth series to be integrated with the fossil record to ground maturity assessments of extinct taxa in well-constrained, empirically tested methods.