1 ER Paleoproteomics of Mesozoic dinosaurs and other Mesozoic fossils
2 Mary Higby Schweitzer1,2,3,4; Elena R. Schroeter1; Timothy P. Cleland5, Wenxia Zheng1
3 1Department of Biological Sciences, North Carolina State University; 2North Carolina Museum of Natural 4 Sciences, Raleigh NC; 3Museum of the Rockies, Montana State University; 4Lund University, Lund, 5 Sweden; 5Museum Conservation Institute, Smithsonian Institution, Suitland, MD
6
7
8 Address correspondence to: Mary Higby Schweitzer, Department of Biological Sciences, Campus Box 9 7617, North Carolina State University, Raleigh NC 27695
10 [email protected] 11 Phone: (919) 515-7838 12 13 Keywords: Dinosaurs, Fossil, Mesozoic, Protein, Soft tissues 14 15 Total number of words, including figure legends and citations: 16 Abbreviations: Ma, million years before present; 17 Paleoproteomics of Mesozoic dinosaurs and other Mesozoic fossils
18
19 Abstract
20 Molecular studies have contributed greatly to our understanding of evolutionary processes that
21 act upon virtually every aspect of the biology of living organisms. These studies are limited with regard
22 to extinct organisms, particularly those from the Mesozoic, because fossils pose unique challenges to
23 molecular workflows, and because prevailing wisdom suggests no endogenous molecular components
24 can persist into deep time. Here, using the lens of the iconic non-avian dinosaurs and their closest
25 relatives, we discuss molecular methods that have been applied to Mesozoic fossils, and the challenges
26 inherent in such studies. We address taphonomic processes resulting in the transition of a living
27 organism from the biosphere to the fossil record, and their possible effects on downstream analyses,
28 then consider the history of molecular studies applied to ancient remains. We evaluate these studies
29 with respect to producing phylogenetically and/or evolutionarily significant data, address the limits and
30 challenges on molecular studies in very old (>1 million years, Ma) material, and the complications of
31 such analyses induced by molecular modifications and limits on comparative databases. Finally, we
32 propose criteria for assessing the presence of endogenous biomolecules in ancient fossil remains as a
33 starting framework for such studies, and conclude by discussing the power and potential of a molecular
34 approach to Mesozoic fossils.
35 36 Introduction
37 Fossils of non-avian dinosaurs are representatives of a world long vanished—a world as foreign
38 to present day humans as an extraterrestrial planet, yet simultaneously as familiar as our own back
39 yards. The only evidence of the existence of these long-dead animals is the often fragmentary remnants
40 of the bones, teeth, and other tissues they have left behind, that have now become part of the rock
41 record. It is assumed by some that such fossils are “pseudomorphs” (e.g. [6]) in which any original
42 organics have been destroyed long ago by taphonomic processes, leaving only the crystalline framework
43 of bone mineral behind. The dearth of molecular data from Mesozoic, or even younger fossils, results in
44 part from the assumption that proteins have a strict time limit on preservation, and as such, useful
45 phylogenetic or biological information retained in these molecules is lost within a million years (e.g., [7,
46 8]).
47 Molecular studies on living organisms, however, have expanded greatly. The incorporation of
48 “omics” approaches to molecular research has revolutionized biology, and has focused on the
49 characterization and quantification of biological molecules as a way to assess molecular interactions that
50 drive the structural organization and dynamic function of living organisms. Although an “-omics”
51 approach can be applied to virtually any type of biomolecule, such studies primarily focus on DNA
52 (genomics), proteins (proteomics), or metabolites (metabolomics). Here, we limit this discussion to
53 proteins/proteomics, because proteins are predicted to have greater longevity than DNA (e.g. [9, 10] and
54 references therein); yet contain similar phylogenetic information. Therefore, proteins are the primary
55 targets for increasingly ancient specimens.
56 Both genomics and proteomics have been applied to relatively recent fossils (~100,000 years or
57 less, but see [11]), and play a prominent role in archaeological studies (e.g. [8, 10-12]). Technological
58 advances (e.g. [13]) have resulted in the ability to obtain high-resolution data, and have contributed to a
59 rapid expansion of molecular studies on recent fossils. These approaches yield molecular sequence data, 60 which is the ultimate target for evolutionary studies. However, whether these methods can be applied
61 to Mesozoic fossils is hotly debated (e.g.,[14, 15] ), because by definition, they are physically and/or
62 chemically altered from the living state.
63 Although controversial, the recovery of molecular data from very old fossils, including those
64 from the Mesozoic (~250-65 Ma), is becoming more common [16-21], and has encompassed a variety of
65 approaches that differ in their sensitivity, specificity, and methodological limitations. Here, we provide
66 an overview of the history and promise of ancient biomolecular recovery.
67
68 Fossilization modes and their impact on molecular preservation
69 Not all fossils are preserved equally. To retain proteins or the soft tissues they comprise requires
70 that they are shielded from outside influences [22], that water flow is minimal [23], and they receive some
71 degree of protection from microbial attack [24]. Thus, preservation can vary greatly, even within a single
72 bone [22, 25, 26]. Generally, the length of time a bone lies exposed at the sediment surface affects the
73 quality of its preservation; soft tissues are preserved with greater fidelity in rapidly buried bone than
74 bone with long surface exposure [27, 28]. Rapid burial can be difficult, and therefore less likely, for large
75 animals (e.g. sauropods), whereas smaller animals can be covered rapidly in their entirety. Despite the
76 relative ease of burial of small remains, however, bone type also affects preservation. Dense cortical
77 bone from larger animals generally remains more intact than fragile skull elements or the relatively
78 delicate bones of smaller creatures.
79 Despite these general rules, it is not uncommon for bones of ancient birds, small dinosaurs and
80 mammals to persist in some Mesozoic deposits (e.g.[28, 29, 30]). With these parameters in mind, then, the
81 ideal target bone for protein preservation would seem to be cortical bone from limb elements of a
82 medium-sized dinosaur, whose state of articulation indicates rapid burial, because this is the
83 intersection between speed of burial and bone density that gives the best chances for high quality 84 preservation, while providing adequate tissue for destructive analyses without obliterating informative
85 morphology. However, these criteria, or lack thereof, should not preclude the analyses of other types of
86 specimens. Recent data suggest that other modes of preservation may also yield molecular data (e. g. [21,
87 31, 32]); thus, specimens should be evaluated on a “case-by-case” basis.
88
89 Modes of fossilization
90 The different physical and chemical pathways that contribute to fossil formation lead to a
91 variety of ‘modes,’ or types of fossilization. Many fossilization modes have been recognized, including
92 (but not limited to) natural molds and casts, compression and impression fossils, and permineralization,
93 [33-35], the latter being the most common mode observed in dinosaur bone. Permineralization occurs
94 when porosity in a bone (either gross porosity arising from degradation of the vasculature and
95 osteocytes, or microporosity from the degradation and loss of proteins and other organics in the
96 extracellular matrix) is infilled with authigenic or exogenous minerals, leaving the original mineralized
97 bony microstructure intact [34, 36]. This mode decreases the overall access of water to proteins, thus
98 reducing the probability of protein hydrolysis [22, 35, 37], and potentially increasing the likelihood that small
99 remnants of bone protein may persist. As a result, permineralized bone may be a useful target for
100 protein recovery. Unfortunately, this mode of fossilization may also introduce obstacles to the
101 extraction of original proteins/protein fragments from the bone, because it provides new mineral
102 surfaces, in addition to bone hydroxyapatite, to which proteins can bind. Because typical protein
103 extraction protocols may not result in complete demineralization, these exogenous minerals may
104 impede analytical access to tightly bound proteins [38].
105 Although permineralization is one of the most common modes of fossilization in bones or teeth,
106 this model does not fit as well for the fossilization of “soft” (i.e., originally unbiomineralized) tissues we
107 occasionally observe in Mesozoic remains. It has been hypothesized that most fossilized soft tissues are 108 preserved as impressions or
109 compressions, and that no original,
110 identifiable organic components
111 persist in these materials [39].
112 However, unlike true impressions,
113 these fossils are often histologically
114 distinct from surrounding sediments,
115 and instead retain distinct tissue
116 microstructure (e.g. Fig. 1, [18, 31, 32]).
117 In particular, fossilized feathers,
118 when not preserved in amber (e.g.
[2] Figure 1. (adapted from ) Gross (A, C) and petrographic ground section (B, [40] D) of two samples of fossil skin, preserved under differing taphonomic modes.119 ), are proposed to persist as A, B represent embryonic titanosaur skin [3, 4]. Although these were referred to as ‘impression fossils” [3], ground sections (panel B) show distinct textural120 compressions generated by heat differences between the ‘skin’ (SKN), underlying sediment grains (SED), and a forming osteoderm (ost) underlying the developing embryonic scute 121 ). C, and/or pressure, and are usually gross, and D, ground section of ‘skin’ from a mummified edmontosaur. Specimens courtesy of L.Chiappe, LACM. 122 presumed to be highly altered
123 carbonized remains of original organics, or ‘carbon films’ (e.g. [31, 41]). However, in some cases at least,
124 these fossil remains can be separated from the sediments for study [42], precluding their classification as
125 true compression or impression fossils. These specimens in particular may hold molecular information
126 from the original animal (e.g. [31, 42]). For a more comprehensive review of taphonomic modes and their
127 relationship to molecular preservation, see [2, 35] and references therein.
128
129 Environmental influences on molecular preservation
130 The various ways in which depositional environments affect the chemical composition of the
131 fossils they entomb is still being examined. Even the most well-preserved Mesozoic bone shows at least 132 some alteration. For example, fluorine can substitute for the hydroxyl group in bioapatite, forming
133 fluorapatite, or other exogenous minerals can be incorporated into the bone matrix (e.g., [22, 34, 37, 43]). It
134 has been posited that early mineralization by pyrite, phosphate minerals and/or carbonates, as well as
135 anoxia, play a role in soft tissue preservation, and thus may also impact protein preservation [44, 45].
136 Additionally, microstructural and protein preservation may be favored in a clay-rich environments,
137 because clays, like exogenous mineralization [22], may act as a sealant, preventing the movement of
138 water and/or other outside chemical influences through the bone. The high surface area and negative
139 charges on clay grains are thought to inactivate enzymes of degradation through adsorption [46]. Indeed,
140 adsorption to mineral surfaces has also been shown to contribute greatly to the stabilization of original
141 biomolecules (see [10, 47] and references therein, and discussion below). Conversely, the impermeability
142 of clays (e.g. [48]) could also negatively influence preservation at the molecular level by preventing the
143 drainage of suppurating fluids in the immediate environment, holding microbial enzymes in close
144 contact with tissues for long time periods. A survey of fossils from various time periods [49] showed that
145 at least for the recovery of still-soft tissue microstructures (e.g. blood vessels, matrix tissues) rapid burial
146 in a sandstone environment seemed preferable to other environments. This may be because the porous
147 nature of sand allows enzyme-laden, microbially rich suppurating fluids to disperse more rapidly.
148 Microbially mediated cementation [50] of sandstones can also contribute to preservation [51] by excluding
149 water or other outside influences from entombed sediments. Marine environments (e.g. [17, 21, 32, 52, 53]
150 have also produced fossils from which original molecules have been reported.
151 Other factors may also play a role in preservation of organic remains. Low thermal ages ([54] and
152 references therein) slow protein degradation, and oxidative environments [55, 56] are predicted to
153 facilitate the formation of intra- and intermolecular crosslinks, contributing to the longevity of soft
154 tissues and the molecules comprising them in vertebrate remains [55]. Close association with minerals 155 has been demonstrated to confer stability to biomolecules (e.g. [10, 57]), and incorporation of organics
156 into humics, or polycondensation reactions with sugars [58, 59] may also play a role in molecular longevity.
157
158 Exceptional preservation
159 Despite taphonomic influences and degradation processes, many exceptionally well-preserved
160 vertebrate fossils have been identified from Mesozoic environments, and the processes described above
161 cannot fully explain the level of preservation they exhibit. Fossils preserving soft tissues, including skin
162 (e.g. [3, 4, 21, 60]), feathers (e.g. [61]), internal organs (e.g. [30]), or even ‘snapshots’ of particular behaviors
163 (e.g. brooding [62], burrowing [63], sleeping [64] or fighting [65]) are not easily explained by
164 permineralization, compression, or other common modes of fossilization. Such fossils contribute to our
165 understanding of morphology, evolution, function, phylogenetic relationships, and ancient ecosystems
166 disproportionate to their rare occurrence, and are highly prized for the information they contain
167 regarding the biology of extinct organisms that are, without such preservation, unknowable. This
168 exceptional preservation requires that processes normally resulting in degradation organic material be
169 arrested [35]; thus these fossils are ideal targets for molecular analyses.
170 However, even the most exceptionally well-preserved Mesozoic fossils are altered from their
171 once-living state; thus, we can expect changes to the molecular content of fossils as well. This can
172 include the preferential preservation of some proteins [10, 66, 67], or of some regions of proteins [68].
173 Additionally, alterations to protein sequences or unusual post-translational modifications (PTMs) may be
174 induced, retained, or lost. For example, PTMs can be lost through diagenetic processes as well as
175 artificially induced by the geochemical environment [69, 70]. We discuss the effects of such alterations on
176 the search for, and identification of, preserved biomolecules below.
177
178 Histology and histochemistry as potential indicators of protein preservation in dinosaur bone 179 Protein studies on dinosaur bone are often problematic, in that they are typically both
180 destructive and expensive, are predicted to have a low rate of success, and ideal target samples for such
181 studies are often rare. Additionally, many fossil taxa are represented by the remains of only one or a few
182 individuals, which prohibits or greatly restricts destructive analyses. Thus, techniques to screen for
183 promising specimens and exclude inappropriate ones are crucial, and have included histological and
184 elemental analyses, FT-IR, or micro- or nano-computed tomography.
185 Studies on both extant and fossil bone suggest that association with bone mineral increases the
186 stability of proteins within bone [10, 47, 71], and that where the mineral phase of bone shows little
187 alteration (e.g. retaining original anisotropy, microstructure, and crystallinity) the likelihood of protein
188 preservation is greatly enhanced [72]. Thus, at least in archaeological bone, exceptional histological
189 preservation is often linked to molecular preservation (e.g., [73]), because it is an indication of minimal
190 alteration by taphonomic influences. Similarly, the histological integrity of dinosaur bone is often
191 pristine, preserving original anisotropy, orientation, and microstructure (e.g. [22, 25, 74]), to the degree that
192 inferences of growth rates and metabolic strategy can be made [75, 76]. Multiple avenues of investigation
193 have been applied to dinosaur (and non-dinosaur) fossils to ascertain histological integrity or,
194 alternatively, degree of microstructural alteration, including light microscopy on petrographic ground
195 sections [26, 75, 77], scanning electron microscopy (SEM, e.g. [2, 3, 78-80] and references therein), and, more
196 recently, transmission electron microscopy (TEM, e.g. [21, 31, 32, 42, 81, 82, 83]) and atomic force microscopy
197 (AFM, [83, 84]). In addition, analytical devices coupled to some of these methods (e.g. energy dispersive x-
198 ray spectroscopy, EDX; electron energy loss spectroscopy, EELS [79, 85]) can be used to measure alteration
199 at the elemental level, as well as degree of exogenous mineral incorporated into bone.
200 Histochemical stains have also been employed to suggest the presence of DNA [85-88] and other
201 compounds [49, 78, 85, 86, 89, 90] in well-preserved dinosaur bone, because these indicate the presence of
202 biochemicals within fossil tissues still capable of uptaking stains. These techniques are not as specific as 203 other methods, because many histochemical stains react to rather general chemical properties within
204 tissues (e.g., acidic, basic, or amine-containing groups). However, they provide strong supporting
205 evidence for the presence of compounds identified by more specific methods (see below).
206
207 Protein studies on Mesozoic fossils
208 History
209 Many examples of non-biomineralized tissues preserving in the rock record exist; but, it has only
210 been within the last few decades that these remains have been subjected to empirical chemical
211 analyses. Some analytical studies were attempted on dinosaur fossils early in the 20th century, although
212 for many years the majority of evidence for “molecular” and/or cellular preservation in dinosaurs was
213 limited to microscopic studies (e.g. [78, 91]. As early as the 1950s, however, fossils (e.g. [92, 93]), including
214 dinosaurs [78, 86, 87, 94, 95] began to be examined for organic content. In fact, some of these early studies
215 employed geochemical analyses that indicated the presence of molecular fingerprints in fossils that
216 significantly pre-dated the dinosaurs (e.g. [96]). Here we will limit our discussion to molecular evidence
217 for proteins, specifically in non-avian dinosaurs and other Mesozoic vertebrates.
218 Amino acid analyses were among the first molecular analytical methods to be applied to
219 Mesozoic fossils [97, 98], including dinosaurs (e.g. [85, 93, 95, 99]). These early studies used the presence and
220 ratios of amino acids hydrolyzed from fossils extracts (i.e., the fossil’s amino acid profile), to assess the
221 presence of endogenous proteins that could produce such profiles. However, ratios of amino acids in a
222 sample do not differentiate exogenous (e.g., human keratin, soil bacteria) from endogenous sources,
223 and do not account for loss of side chains on amino acid residues over time. These losses result in the
224 conversion of labile amino acids to the simplest—glycine—and thus can alter amino acid ratios (e.g. [44]).
225 Chirality studies were later incorporated into amino acid analyses to test the endogeneity of
226 fossil-derived amino acids, using the rationale that the ratio of D/L amino acids in proteins from a fossil 227 sample could be used to validate the geological age, and thus ancient source, of these compounds [98,
228 100]. Additionally, because racemization was correlated to rates of depurination, the degree of
229 racemization was also viewed as a proxy for DNA survival [101]. However, these studies were
230 subsequently deemed problematic [102, 103, 104]. Racemization reactions are non-linear [10] and widely
231 variant [105], can be induced or accelerated by the action of microbes [106], and have even been observed
232 in living humans with certain pathologies [107]. Furthermore, Rafalska et al. [108] and others [109, 110] found
233 that endogenous proteins may exhibit low racemization if complexed to humics, stabilized through
234 condensation reactions with other amino acids or sugars [58], or encapsulated within mineral [67, 111],
235 providing a possible mechanism for preservation of proteinaceous content over time. Thus, many
236 factors beyond absolute age of the fossils can contribute to racemization, including environmental pH
237 and burial temperatures (e.g. [54, 103] and references therein), compromising its correlation to age in even
238 relatively recent fossils [104, 105]. However, when the above caveats are considered, amino acid analyses
239 and chiral analyses (from fossils or tissues in closed systems) can be used as supporting evidence for the
240 endogeneity of molecules recovered from ancient fossils.
241
242 Current methods
243 Analytical studies conducted on Mesozoic fossil bone have employed many methods to
244 characterize remnant original molecules, including protein. However, some of these are more sensitive
245 and/or informative than others. It is imperative in designing analytical studies of fossils to understand
246 the specific analytical capabilities, and limits, of each potential method to be employed.
247
248 Vibrational and chemical imaging methods
249 Although not strictly applied to protein studies, recent analytical studies of dinosaur bone have
250 employed various forms of vibrational mode spectroscopy (e.g. Raman, FTIR [17, 18, 112-114] and reviewed in 251 [115]), as well as synchrotron-based technologies [113, 116, 117, 118] to suggest organic content in ancient bone.
252 Vibrational mode spectroscopy methods capitalize on the individual bonds within a molecule, which are
253 in turn influenced by the atoms to which they bond [119]. These techniques are capable of mapping
254 molecular signal directly to fossil surfaces. They provide very high resolution analyses and can detect
255 minute quantities of molecules, so have been used to claim the “unequivocal preservation of original
256 organismal biochemistry” [118].
257 The vibrational spectrum of protein has two primary features, the amide I (1600–1700 cm−1) and
258 amide II (1500–1560 cm−1) bands, which arise from the C=O and C–N stretching vibrations of the peptide
259 backbone, respectively [120]. However, any amide bond will resonate in the same region, regardless of
260 the source protein (or other amide-containing compound) from which these derive [121, 122]. Because all
261 proteins and polypeptides are complex macromolecules, and contain more than one secondary
262 structural motif, they give rise to more than one amide absorption. These can overlap, resulting in the
263 formation of a composite profile of broad featureless absorbance bands that hide more detailed and
264 diagnostic information [123]. The appearance of amide bands in FTIR profiles, then, is only indicative of
265 the possible presence of peptide bonds, not specific amino acids or proteins. Additionally, because these
266 spectroscopy methods rely on vibrational patterns determined by bonding environments [121, 124] they
267 are sensitive to factors like diagenetic alteration to molecules. Thus, although these methods have been
268 used to claim (e.g. [79, 113]) or invalidate ([125]) dinosaurian proteins, it is beyond the limit of this
269 methodology to determine a source unambiguously or to provide rigorous identification of specific
270 protein molecules. However, if molecules are identified by other means (e.g., if collagen is identified
271 using mass spectrometry or antibodies), vibrational spectroscopy provides completely independent
272 supporting data for the presence of these molecules, and contributes important information regarding
273 burial environments. These latter data may, in turn, have predictive value for identifying fossils best
274 suited for molecular mining. 275 Synchrotron-based analyses of various types have been increasingly applied to fossil materials.
276 These methods can detect extremely low abundance compounds with great sensitivity and spatial
277 resolution. Thus, synchrotron studies have been used to identify remnant outlines of original soft tissues
278 in sediments, distinct from environmental signal [19], and can be applied to larger specimens [126].
279 However, synchrotron analyses are dependent upon surface chemistry, and its great sensitivity makes it
280 prone to detecting contaminants, if present, over endogenous materials [117]. Again, these methods
281 provide strong supporting evidence for proteinaceous components identified by other methods, and
282 should be used in tandem with these, when possible.
283 For paleomolecular studies on Mesozoic fossils where the goal is to detect and characterize
284 preserved protein content, the two most sensitive, reliable and informative approaches available, which
285 are being employed with increasing frequency and rigor, are immunological methods and various forms
286 of protein mass spectrometry. We discuss the benefits and limitations of each below.
287
288 Immunological methods
289 Immunological methods, primarily extraction-based methods such as enzyme linked
290 immunosorbent assays (ELISA), radioimmunoassays, or immunoblot (Western blot) have been applied to
291 Mesozoic fossil materials for many years (e.g. [98, 127, 128]). These methods rely on the well-documented
292 specificity (e.g. [129]) and sensitivity (nanogram to picogram per liter [130]) of the vertebrate immune
293 response.
294 In addition to sensitivity and specificity, immunological techniques are useful for analyses of
295 deep-time fossils because the complete protein need not be present or unaltered for identification; an
296 antigenic determinant, or epitope, need be only 5-7 amino acids in length (e.g. [130, 131]), enabling specific
297 detection of small or degraded fragments. In situ binding of antibodies to fossil tissues is thus strong
298 evidence for the detection of peptides consistent with the target protein, not simply isolated amino 299 acids. Furthermore, antibodies can be made against proteins not found in microbes or in mammals, and
300 can thus be used to eliminate these potential contaminants as a source for molecular signal [132-134].
301 The usefulness of an immunological approach to the study of fossils, when proper controls are
302 applied, cannot be overstated. Antibodies may recognize different aspects of epitopes in chemical
303 extractions of fossil tissues (ELISA and immunoblot ) [98, 127], and can, for example, differentiate between
304 the native and denatured state of a protein. ELISAs use antibodies to recognize epitopes preserved in
305 chemically extracted material, and is the most sensitive antibody assay. ELISA normally relies on
306 recognition of antibodies to epitopes in their native state, in non-denatured material [135, 136].
307 Immunoblot (Western) analyses separate antigenic components in fossil extracts by gel electrophoresis.
308 Separated fractions are transferred to a membrane, which is then exposed to the antibodies of interest
309 [136, 137]. Immunoblots give additional information over ELISA regarding molecular size of the molecules
310 detected by the antibody. Often, a reducing gel is used in the first stage, so the protein of interest is
311 denatured. In general, polyclonal antibodies that binds to both native and denatured proteins are most
312 useful for any analyses of fossils because the state of the epitope in the fossil tissue is unknown.
313 Although both ELISA and immunoblot methods are more sensitive than in situ
314 immunochemistry, they are complicated by: 1) a greater potential to introduce contaminants during the
315 process of chemical extraction (e.g. [15]); 2) a potential loss of information during extraction; and 3) the
316 possibility that co-extracted compounds (e.g., humic substances) will interfere with subsequent analyses
317 by making epitopes inaccessible to antibodies (e.g. [56, 138]). These are minimized with in situ analyses,
318 where antibodies are applied directly to tissues. Additionally, in situ assays require less material for
319 destructive analysis than do extraction-based methods, which is important when analyzing extremely
320 rare or small fossil specimens. In in situ analyses, antibodies bind directly on tissues to form antibody-
321 antigen (ab-ag) complexes, which are then detected when secondary antibodies are labeled with a
322 chromogenic (e.g., 3,3'-diaminobenzidine, DAB; alkaline phosphate, AP), fluorescent (e.g. fluorescein 323 isothiocyanate, FITC), or radioactive substrate (e.g. [127, 128]). Because most fossils are biogeochemically
324 stained a darker color than the in vivo state, chromogenic labels are not ideal for tracking antibody
325 binding. On the other hand, many fossils autofluoresce (e.g. [139]), and this can interfere with fluorescent
326 labels. Unlike chromogenic labels, however, fluorescent labels allow compensation for autofluorescence
327 by setting detectors to accommodate background, or by using labels that fluoresce at a different
328 wavelength than that at which autofluorescence is observed.
329 Finally, in situ assays using the above labels can be alternatively validated by using antibodies
330 tagged with gold beads and visualized with TEM (see Fig 2 for a comparison of in situ data). This is the
331 most sensitive and highest resolution of the in situ assays, because it allows localization at nanometer
332 resolution. Even if fossils have incorporated electron-dense components from the environment, these
333 can be distinguished from ab-ag complexes by the uniform roundness and known size of the beads, and
334 by detection/mapping of gold using a technique like electron energy loss spectroscopy (EELS [140]),
335 because gold is not usually found in dinosaur fossils.
336 The usefulness
337 of antibodies does not
338 end with the
339 identification of
Figure 2. Comparison of data from vessels recovered from B. canadensis specimens.340 A. proteinaceous Demineralizing dinosaur bone fragment showing the emergence of vessels from the mineralized matrix. B. In situ antibody binding of vessels exposed to antibodies against elastin341 compounds in fossils. If protein (Courtesy R. Mecham). Positive binding is detected using FITC label which fluoresce in green wavelengths. Binding is specific, and localized to the vessel, not other components. C. Wall of soft vessel recovered from B. canadensis, exposed to anti-elastin antibodies, as in B,342 but binding can be with antibody-antigen complexes on tissues labeled with gold beads affixed to secondary antibodies and visualized using TEM. VSL, vessel; imgld, immunogold beads on antibody. Scale343 demonstrated either in as indicated. 344 situ or in chemical
345 extracts, reactive antibodies can be used to both purify and concentrate antigenic components out of
346 bulk fossil bone extracts (e.g. [141]), making them more amenable to mass spectrometry methods. 347 Furthermore, besides the inherent cross checks for validity allowed by the combined use of extraction-
348 based and in situ assays, one can corroborate the presence of immunogenic components in fossils by
349 using extracts of reactive fossil tissues to immunize hosts (usually rabbit or mice), thereby producing an
350 anti-fossil antibody that can be applied to modern tissues of known composition to confirm antigenicity
351 (e.g. [112]).
352
353 Antibody cross reactivity
354 Antibody analyses have become much more specific [142] since the 1980s, when most attempts
355 at immunological characterization of fossil material were conducted. Nevertheless, it has been claimed
356 that immunological studies are not appropriate for paleomolecular research because positive antibody
357 binding in fossils could be the result of “false positives” (e.g. [15]), or more accurately, cross reactivity
358 between antibodies and proteins or components besides those they are meant to target. That
359 antibodies may cross react with proteins that are similar in three dimensional conformation to the
360 antigen used to produce them is well-known [143]; thus, rigorous controls for specificity are routinely
361 incorporated into antibody-based assays, and should always be included in paleomolecular studies.
362 Non-specific cross reactivity can arise in multiple ways; therefore, numerous specificity tests
363 should be conducted in tandem to rule out all potential sources of “false positives.”
364 1. Non-specific cross reactivity may arise from primary antibodies binding epitopes other than
365 those used to generate them, or binding non-proteinaceous tissue components (e.g. charged
366 clays). This type of non-specificity can be recognized by digestion of the fossil tissue with a
367 specific enzyme (e.g. collagenase, if collagen is the protein of interest). If digestion results in
368 reduced binding, it is likely that the antibody is binding specifically; if digestion does not reduce
369 binding, non-specific binding may be occurring because of inherent tissue properties not related
370 to preserved proteins. However, in fossils that are preserved because of diagenetic cross links [55, 371 56], binding may initially increase as these crosslinks are digested to expose hidden epitopes [144],
372 then diminish as digestion continues. In either case, if the applied antibody is binding specifically
373 to the target protein, then the enzyme, also specific for the target, will a have direct impact on
374 the observed binding intensity.
375 2. Spurious binding can occur when a secondary antibody or label bind directly to regions of the
376 tissue as well as, or instead of, to the primary antibody they are meant to exclusively recognize.
377 To control for this non-specific binding, an assay omitting incubation with the primary antibody
378 is conducted, to verify that the secondary antibody or label is only binding when the specific
379 primary is present, and bound to tissues.
380 3. Inhibition or absorbance controls detect non-specific paratopes in the polyclonal antibodies.
381 Specificity is supported if the test antibodies are first incubated with excess of the target protein
382 to block the antigen binding cite on the antibody, and then are exposed to tissues. If the
383 antibody is specific, blocking of these paratopes will result in either absent or greatly reduced
384 binding over the non-inhibited state. Conversely, if the antibody contains paratopes for proteins
385 other than the target, those will not be blocked during inhibition, and binding will remain
386 substantial in these controls.
387 These are common specificity controls, used frequently in extant studies—particularly in human and
388 biomedical research [145]. Additional controls that should be employed with fossils when possible
389 include: demonstrating a lack of binding when a pre-immune serum (derived from the host animal
390 before immunization with the immunogen of interest) or normal sera (from a non-immunized
391 conspecific animal) are applied to fossil material; and a positive control where the same antiserum, at
392 the same concentration and under the same parameters, is applied to extant tissues comparable to the
393 fossil tissue of interest. Additionally, when analyzing fossils, antibodies must be further tested against
394 two additional controls: 1) sediments simultaneously recovered in association with fossil material (when 395 available), which controls for the potential presence of cross-reactive molecules in the depositional
396 environment; and 2) all buffers and reagents used to treat/extract the fossil tissues, which controls for
397 contamination introduced by the laboratory environment.
398 Despite the many well-established ways to control for cross-reactivity, the issue remains a topic of
399 active debate in the paleomolecular community. For example, a recent study [146] claimed to refute
400 previous immunological data derived from Shuvuuia fibers [81], a study that was repeated by different
401 investigators [42, 134], using different antibodies to feather β-keratin (or corneous β-protein, CβP) [147], yet
402 which yielded similar results. However, the multiple differences in sample treatment and analytical
403 methodology between the original study and the follow up study [146] make this claim a tenuous one. In
404 particular, although Saitta et al. [146] evaluated samples derived from the same Shuvuuia specimen, these
405 samples were collected nearly two decades after the original studies, during which time the Shuvuuia
406 fibers were coated in polymers (e.g., glues, consolidants) and exposed to atmosphere. The fibers used in
407 the original and subsequent studies were separated from the rest of the fossil, treated with multiple
408 ethanol washes to remove these polymers, and the separated fibers were stored in isolation in sterile
409 containers since receipt. Thus, Saitta et al. [146] represents a study comparing results from samples
410 separated by nearly twenty years of storage in unprotected conditions and coated with preservatives
411 known to interfere with molecular analyses.
412 Furthermore, although both Saitta et al. [146] and Schweitzer et al. [81] employed ToF-SIMS, these
413 studies used different ion sources, different keV, and different sample preparations, and so are not
414 directly comparable. Additionally, the original study showed these hollow fibers to contain 3nm
415 filaments in the matrix, consistent with β-keratin proteins, and positive immunoreactivity to β-keratin
416 (CβP) polyclonal antisera, Saitta et al., did not attempt similar studies; thus they did not directly test the
417 original hypothesis of preservation. The immunological data from the original study and multiple
418 repetitions [42, 134] employing multiple controls and different antibodies, are simply not consistent with 419 random cross contamination. Caution must be employed when comparing results of studies conducted
420 using different instruments, different parameters and different methods, particularly when follow-on
421 studies are separated in time by decades.
422 Employing antibodies can make a strong case for the specific identification of a target protein in
423 Mesozoic fossils when these seven controls are rigorously incorporated. However, immunological
424 methods cannot provide sequence information, and therefore cannot wholly address
425 paleobiological/phylogenetic questions without additional supporting evidence, ideally in the form of
426 high resolution mass spectrometry.
427
428 Mass spectrometry methods
429 Mass spectrometry methods are increasingly being employed to analyze Mesozoic fossil
430 remains, and these hold great promise for providing the most detailed information on the biology,
431 physiology, and evolution of these organisms. However, as the instrumentation and data generated vary
432 greatly between MS methods, the choice of which MS method to employ when analyzing Mesozoic
433 remains must depend upon the questions being asked (for a review on various MS methods for
434 paleoproteomics see [148]).
435 Time of flight secondary ion mass spectrometry (ToF-SIMS) was first applied to Mesozoic
436 dinosaur fossils in 1999 [80, 81]. This method, reviewed in [148], can localize amino acids, as well as di-and
437 tri-peptides and other biological compounds (melanin, e.g. [16, 21, 32, 149-151]; heme [152]) directly to tissues
438 with high resolution, but like synchrotron methods, is sensitive to surface composition and care must be
439 taken to eliminate contaminants as a source of signal. It was used to support the presence of molecules
440 consistent with protein in originally keratinous claw sheath material from Rahonavis ostromi, a very
441 basal bird [153], and as mentioned above, in hollow white filaments recovered from the cervical region of
442 an articulated specimen of the enigmatic alverezsaurid, Shuvuuia deserti, from Mongolia [27]. More 443 recently, ToF-SIMS has been applied to other Mesozoic fossils (e.g. [16, 21, 32, 53, 154]) yielding important
444 information about molecular preservation in these remains. ToF-SIMS can also detect robust PTMs that
445 are able to withstand the ionization process, such as hydroxylation of proline (HYP) or lysine (HYL)
446 residues. Hydroxylation of these residues facilitates crosslinking, which greatly enhances the stability of
447 the collagen molecule [155, 156]; thus elevated HYP and HYL, together with abundant glycine residues are
448 considered fingerprints for the presence of collagen. The ability of ToF-SIMS to detect and localize these
449 fingerprint PTMs was capitalized upon to support the presence of collagen in demineralized, fibrous
450 matrix from Tyrannosaurus rex [83]. However, because standard ToF-SIMS approaches employ a harsh
451 ionization process [157], preserved proteins are highly fragmented during analyses, obscuring the original
452 protein sequence. Thus, unless diagnostic moieties can be identified (e.g., HYP) this method is not useful
453 for determining the presence of a particular protein with confidence. ToF-SIMS is being applied to some
454 fossil materials with increasing frequency (e.g [16, 19, 32, 53, 149, 154], and is particularly appropriate for
455 detecting and mapping metal-associated functional moieties (e.g., heme [152]; melanin [16, 32, 53]), but is
456 less optimal for proteins.
457 Pyrolysis gas chromatography MS (Py-GC–MS) is another form of mass spectrometry that has
458 been applied to a wide range of fossil materials [150, 158] including dinosaurs (e.g. [151], marine reptiles [21,
459 52], ancient birds (e.g. [159], and other Mesozoic organisms. Although Py-GC–MS was first applied to fossils
460 in the 1980s and 90s [160], its previous use was limited to non-vertebrate remains. Py-GC–MS uses
461 intense heat to fragment molecules into their constituent chemical compounds [161], thereby providing a
462 chemical fingerprint for the analyzed specimen from which molecular content is inferred. However,
463 because these analyses result in thorough destruction of all molecules present, it is impossible to tell
464 with certainty from which specific compounds the detected pyrolysates derive. Furthermore, because
465 Py-GC–MS lacks the mapping capability of ToF-SIMs, it is unable to differentiate in situ protein 466 breakdown products in a specimen from those introduced by ubiquitous exogenous sources (e.g., shed
467 human keratin).
468 To recover phylogenetically informative sequence data from dinosaur fossils (or any fossils),
469 tandem mass spectrometry (MS/MS) methods are required, because the ‘soft ionization’ of tandem MS
470 allows the robust identification of peptides at the sequence level [162], as opposed to detection of single
471 component amino acids (TOF-SIMS) or fragmented molecular compounds (Py-GC/MS) [148].
472 The first protein sequence data from dinosaurs was recovered from an exceptionally well
473 preserved Tyrannosaurus rex specimen, a study which conducted MS/MS using a relatively low
474 resolution ion trap mass spectrometer [163]. Peptides consistent with collagen I, the dominant bone
475 protein, were recovered, but because of the low resolution and initial misinterpretation of some post
476 translational modifications [164], these data were met with criticism [14, 165], despite being independently
477 validated statistically [166].
478 In conjunction with multiple independent lines of molecular and histological analyses to support
479 the detection of preserved protein, additional collagen -1 and -2 peptides were reported from a
480 second, older dinosaur, Brachylophosaurus canadensis, using a higher resolution Orbitrap mass
481 spectrometer [137]. The sequences were validated using multiple search algorithms, and allowed the
482 placement of B. canadensis in proper phylogenetic context [137, 167]. Additional collagen peptides were
483 recovered from this same B. canadensis specimen eight years following the first publication. Despite
484 being performed by a different investigator, in a different lab, using different extraction methodologies
485 and different instruments than the original study, some peptides were recovered that were identical in
486 sequence to those originally reported, and the known collagen I sequence of this dinosaur was
487 expanded significantly (Fig. 3 and [5]). When mapped to a molecular model of a collagen fibril, the 488 collagen peptides detected from these dinosaurs showed a non-random distribution; peptides from
489 both dinosaurs were localized to
490 internal and shielded regions of the
491 fibril [68], shedding light on factors
492 possibly contributing to their
493 preservation.
494 To determine if the sub-
495 micron tissue differentiation
496 observed with various microscopy
Figure 3. Annotated mass spectrum of collagen I alpha 1 peptide identified497 techniques extended to the from B. canadensis using LC-MS/MS (adapted from [5]). Blue indicates the presence of hydroxylation on a proline residue, a PTM crucial to 498 the molecular level, which would be formation of a triple helix in collagen I. 499 predicted if these structures were endogenous and of vertebrate origin, structures morphologically
500 consistent with blood vessels were isolated from the B. canadensis specimen and separately analyzed
501 with LC-MS/MS [168]. The separated vessels generated a very different peptide profile when compared to
502 the MS profiles obtained from whole bone extracts from the same specimen. The former comprised
503 peptides from multiple proteins associated with vasculature in extant avian bone [168], while the latter
504 were dominated by collagen [5, 137]. Among these were multiple peptides from cytoskeletal proteins that
505 are only present in eukaryotes or metazoan taxa, (e.g. actin, tubulin) as well as histones—DNA-
506 associated proteins not produced by prokaryotes; thus, microbes were eliminated as a source for these
507 data. Despite these results, sequence data from Mesozoic fossils remains controversial [15]. In 2017,
508 Buckley et al. [15] published peptide sequences recovered from ostrich bone. Because one peptide was
509 identical to a sequence previously identified as unique to T.rex [163-165], they argued that the peptides
510 reported in 2007, 2009, and 2017 were likely contaminants. However, Buckley et al. did not address the
511 multiple immunological, morphological and chemical data, put forth to add support to the claim of 512 endogeneity of these peptides, nor does their conclusion account for the lack of similar collagen
513 sequence in any controls (e.g. sediments, buffer) that were stored and treated in tandem with the
514 dinosaur material[5, 137, 163-165], or the preferential recovery of identical sequences in 2009 and 2071 from
515 what would have had to be two separate contamination events, by two different species (ostrich and
516 alligator).. Furthermore, although Buckley et al. supported a contaminant origin for these dinosaur
517 data, they state: “Clearly, it would not be possible to disprove the hypothesis that the collagen sequences
518 produced are indeed of dinosaur origin without sequencing an intermediate species”. Because the
519 conservative nature of collagen makes identifying unique peptides from species without a known
520 proteome difficult, efforts are continually being made to expand databases of extant proteomes to
521 account for non-mammalian taxa, and to optimize LC-MS/MS and bioinformatics methods for fossils to
522 enable detection of greater and more specific regions of the bone proteome.
523
524 Challenges and limits of LC-MS/MS analyses
525 LC-MS/MS produces peptide sequence data, unlike all other previously discussed approaches,
526 and thus is the ideal method for characterizing proteins from fossil tissues. However, LC-MS/MS
527 analyses of Mesozoic fossils is subject to many challenges not present when applied to extant or even
528 Pleistocene tissues [56, 138, 148] that can potentially supress the detection of preserved proteins. These
529 include the absence of characterized proteins/peptides from sufficiently closely related extant relatives
530 of long-extinct animals in comparative protein databases. Because standard MS bioinformatics
531 approaches rely on matching experimentally detected peptides to known sequences, these gaps create
532 serious obstacles in analyzing specimens that possess no living descendants or only distantly related
533 ones, which can severely limit the capabilities of even error-tolerant database search strategies (see
534 [169]), and preclude the detection of unique (i.e., not conserved) regions of sequences that are
535 considered by some to be more rigorous evidence of endogeneity [15]. 536 Additionally, methodology is still being developed to overcome issues that decrease the
537 sensitivity of paleoproteomics analyses. These include the variable efficiency of different protein
538 extraction and sample preparation methods for MS analyses [170]; interference arising from compounds
539 within fossil extracts (e.g., humic substances) that can co-extract with proteins and which suppress
540 ionization [148, 171]; and the analytical complications that arise from unknown or unanticipated diagenetic
541 alterations to preserved sequences during bioinformatic analyses (e.g. the formation of advanced
542 glycation end products, [AGEs])[69, 172].
543 These limitations are exacerbated by the inevitable decrease in both abundance and
544 concentration of endogenous protein in ancient preserved tissues, whereas contaminants from
545 exogenous sources (e.g., human keratin, bacterial proteins from the burial environment) increase in
546 relative abundance, and can mask endogenous peptides in concentration-dependent analyses [148].
547 Furthermore, only tandem MS/MS can identify source proteins definitively within a sample, and can
548 exclude common contaminants (e.g. keratin) from other, perhaps endogenous proteins. Together, these
549 challenges make LC-MS/MS analyses a daunting prospect for Mesozoic specimens, particularly those
550 from extinct lineages, for although it is more specific than the previously discussed approaches, it is also
551 less sensitive when these methodological limitations are considered. Thus, although sequence data is
552 the ultimate goal of ancient protein research, LC-MS/MS should not be considered the only acceptable
553 validation for the identification of preserved molecules. Ultimately, the older (and thus more
554 controversial) the fossil being examined, the more critical it is to use multiple methods in analyses, and
555 to rely on total evidence to support or refute endogeneity.
556
557 Diagenetic alteration of molecules
558 Although diagenesis or taphonomic alteration of vertebrate remains is reasonably well
559 understood and amenable to experimentation (e.g., actualistic taphonomy [134, 173]), the diagenesis of 560 molecules is less well understood. Only a few diagenetic PTMs have been identified, and these derive
561 primarily from much younger fossil bone (up to 1.77 Ma; [69, 70]). To date, only deamidation [168] has been
562 identified in the limited sequence data obtained from Mesozoic remains. Thus, it is critical to expand our
563 use of molecular taphonomy to achieve a better understanding and recognition of molecules preserved
564 within fossil bones of great antiquity. For example, analytical tools applied to dinosaurs are more
565 efficient and have higher probability of success when interference from humics, Maillard products, and
566 iron or other metals are better understood [56, 108, 110, 174] and can be accounted for or mitigated.
567
568 Determining endogeneity and criteria for evaluation of very ancient samples
569 Much has been written over the last several decades regarding molecular preservation in fossils
570 in deep time, because if such information could be recovered, it would revolutionize our understanding
571 of the paleobiology, ecology, and evolution of these long extinct creatures. However, many early studies
572 were compromised by inadequate methodologies, and limited by relying on only one or two lines of
573 evidence, as well as a lack of recognition of the limitations of the methods employed. Even more recent
574 and higher resolution methodologies currently applied to the study of molecular preservation in
575 dinosaurs cannot determine absolutely the endogeneity of the molecules detected when used in
576 isolation[15]. This is problematic, because if the origin of dinosaurian proteins is in doubt, these data are
577 useless for asking larger questions of phylogeny, physiology, or molecular evolution.
578 Furthermore, because contamination of exogenous proteins is a concern for very old fossils, the
579 experimental design of any Mesozoic proteomic study must address the various forms and sources of
580 potential contamination, such as microbial or plant influences from the burial environments, shed
581 human proteins (e.g., keratin), or glues/consolidants applied during excavation, handling, and storage,
582 or transfer from other specimens in laboratory spaces during experimentation and analyses. Thus,
583 multiple, independent validations must be employed before drawing any conclusions about the 584 antiquity and/or endogeneity of recovered molecules. Criteria have been proposed for analyses of
585 archaeological bone [175], and are limited primarily to LC/MS/MS. Although these criteria should be
586 applied when analyzing ancient materials, because MS analyses are limited in sensitivity and can be
587 compromised by ion suppression, co-eluting compounds, database limitations and other factors, we
588 suggest that analyses of Mesozoic bone be subject to additional testing and criteria, in addition to those
589 proposed by Hendy et al. ([175]).We argue for a multi-pronged approach to the proteomic analysis of
590 Mesozoic fossils, realizing that claims of such preservation go beyond conventional wisdom, and
591 therefore stringent standards must apply. These suggested criteria include:
592 1. Microstructures morphologically consistent with tissues are present. The organic phase of bone
593 consists of three main components: a fibrous matrix (dominated by collagen), cells (primarily
594 osteocytes, but also blood cells, osteoblasts and osteoclasts), and blood vessels [176]. These show
595 a hierarchical organization [177, 178], and if present (e.g., Figure 4; [2, 55, 88, 168]), suggests that
596 degradation had indeed been halted before completion, making the identification of these
597 distinct tissues at the micromorphological level a first step for determining fossils appropriate
598 for further analyses. However, the relationship between microstructure and molecular
599 preservation is unclear [179]; thus even
600 for fossils exhibiting pristine
601 microstructure other studies are
602 needed to claim endogenous
603 molecular preservation. Figure 4. Light and electron micrographs of dinosaur remains. A, demineralized bone matrix of B. canadensis containing abundant604 2. Both tissues and molecular osteocytes, defined by their fusiform shape and long, interconnecting filopodia [1]. Isolated osteocyte from this same 605 signal derived from fossil bone should dinosaur, visualized using SEM. OC, osteocyte; fp, filopodia. Scale as indicated. 606 evince similar patterns to those of
607 extant bone. If endogenous, dinosaur tissues derived from demineralized bone should be 608 histologically consistent with extant tissues. For example, collagen fibers in bone are highly
609 organized into layers of parallel fibers, offset at approximately 30˚ in each layer [178], and exhibit
610 67 nm cross banding [180]. At the molecular level, the organic matrix is comprised primarily of
611 collagen I, but trace amounts of collagen III and V are also present [181], as are the non-
612 collagenous proteins osteocalcin, osteopontin, osteonectin, and bone morphogenic protein
613 (BMP), among others [182]. Thus, all of these are reasonable targets for paleomolecular studies.
614 Bone matrix also contains glycosaminoglycans and proteoglycans, including versican,
615 chondroitin sulfate, decorin and biglycan ([182] and references therein). To make the claim for
616 endogenous proteins in dinosaurs, then, fossil bone should exhibit at least at least a subset of
617 the proteins (as supported by chemical analyses) and morphological features indicative of the
618 tissue of origin.
619 3. Target proteins derived from dinosaurs should show distinctive PTMs (e.g., hydroxylated proline
620 or lysine for collagen [156, 183],-carboxyglutamic acid for osteocalcin [184]). ToF-SIMS can
621 independently demonstrate the presence of these moieties on tissues (e.g. [83]). Furthermore, if
622 specimen components can be separated, (e.g. skin, bone matrix, vessels, cells), they should
623 show different molecular patterns when analyzed separately, consistent with the distinct
624 proteomic content present when these same tissues are compared in extant material (e.g. [21]).
625 4. Exogenous contamination should be monitored through the use of multiple, appropriate
626 controls. This includes, but is not limited to, the exclusion of bacterial biofilm as a source of
627 observed structures through chemical testing (e.g., anti-microbial antibodies show no binding
628 [132]), the simultaneous testing of entombing sediments and laboratory reagents with all assays
629 to demonstrate the lack of a proteomic signal, rigorous specificity controls for all antibodies
630 used in immunological assays, the inclusion and analyses of instrument ‘blank’ injections in LC- 631 MS/MS analyses, and peptide sequences consistent with proteins from the tissue and taxa being
632 analyzed.
633 5. Multiple independent methods should be employed. Any one method can be misinterpreted or
634 subject to artifact, thus, validation by multiple techniques that capitalize on different chemical
635 aspects of preserved molecules is critical to acceptance when analyzing fossils in deep time.
636
637 The potential of a molecular approach to Mesozoic dinosaur fossils
638 Characterizing the protein content of fossils in deep time is possible, but must be approached
639 with caution. If appropriate rigor is applied, these molecular approaches to fossils can revolutionize our
640 understanding of evolutionary biology. But why should this effort be made for dinosaurs, when the
641 estimated chances of success are so small? Our ability to predict future trends rests upon our capacity to
642 understand the patterns of the past, and the recorded history of the human lineage is simply insufficient
643 to make accurate predictions. Conversely, the dinosaur lineage spans ~150 million years [185], and during
644 that time, dinosaurs have experienced global warming and cooling, continental movements, periods of
645 volcanism, changes in vegetation, rises and lowering of atmospheric CO2 levels, and many other
646 environmental changes [186]. How they responded to these events is ultimately a molecular question.
647 Dinosaurs saw the origination of many evolutionary novelties, including feathers, gigantism, and
648 endothermy; how these changes accumulated in this lineage is a molecular question. Dinosaurs
649 biologically addressed osteoporosis during lay [89, 187], adapted to an obligate vegetarian diet [188] from a
650 carnivorous ancestry [189], and faced other issues that have also arisen in the human lineage, and they
651 responded at the molecular level. To close off these avenues of investigation because conventional
652 wisdom disallows protein preservation across deep time closes the door on a potential source of
653 information that may benefit humans. We think such an approach is worth the effort.
654 655 Acknowledgements
656 MHS would like to dedicate this review to Rune Martinson, a brave warrior in the fight against cancer 657 who left us far too soon. This research was funded by National Science Foundation (INSPIRE program 658 EAR-1344198), the David and Lucile Packard foundation, and by funding from F. and S.P. Orr, and Vance 659 and Gail Mullis (to MHS); the Arnold O. Beckman Foundation Postdoctoral Fellowship (to ERS); and the 660 Smithsonian Institution (to TPC). This work was performed in part at the AIF at North Carolina State 661 University, which is supported by the State of North Carolina, Key Laboratory of Unconventional Oil and 662 Gas Geology.
663 The authors declare no competing interests. 664 Figure 1. Gross (A, C) and ground section (B, D) micrographs of two samples of fossil skin, preserved 665 under differing taphonomic modes. A, B represent embryonic titanosaur skin [3, 4]. Although these were 666 referred to as ‘impression fossils” [3], ground sections (panel B) show distinct textural differences 667 between the ‘skin’ (SKN), underlying sediment grains (SED), and a forming osteoderm (ost) underlying 668 the developing embryonic scute (adapted from [2]). C, gross, and D, ground section of ‘skin’ from a 669 mummified edmontosaur. Specimens courtesy of L.Chiappe, LACM.
670
671 Figure 2. Comparison of data from vessels recovered from B. canadensis specimens. A. Demineralizing 672 bone fragment showing the emergence of vessels from the mineralized matrix. B. In situ antibody 673 binding of vessels exposed to antibodies against elastin protein (Courtesy R. Mecham). Positive binding 674 is detected using FITC label which fluoresce in green wavelengths. Binding is specific, and localized to 675 the vessel, not other components. C. Wall of soft vessel recovered from B. canadensis, exposed to anti- 676 elastin antibodies, as in B, but with antibody-antigen complexes on tissues labeled with gold beads 677 affixed to secondary antibodies and visualized using TEM. VSL, vessel; imgld, immunogold beads on 678 antibody. Scale as indicated.
679
680 Figure 3. Annotated mass spectrum of collagen I alpha 1 peptide identified from B. canadensis using LC- 681 MS/MS (adapted from [5]). Blue indicates the presence of hydroxylation on a proline residue, a PTM 682 crucial to the formation of a triple helix in collagen I.
683
684 Figure 4. Light and electron micrographs of dinosaur remains. A, demineralized bone matrix of B. 685 canadensis containing abundant osteocytes, defined by their fusiform shape and long, interconnecting 686 filopodia [1]. Isolated osteocyte from this same dinosaur, visualized using SEM. OC, osteocyte; fp, 687 filopodia. Scale as indicated.
688
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