Analyses of Soft Tissue from Tyrannosaurus rex Suggest the Presence of Protein

Abstract

We performed multiple analyses of Tyrannosaurus rex (specimen MOR 1125) fibrous cortical and medullary tissues remaining after demineralization. The results indicate that collagen I, the main organic component of bone, has been preserved in low concentrations in these tissues. The findings were independently confirmed by mass spectrometry. We propose a possible chemical pathway that may contribute to this preservation. The presence of endogenous protein in dinosaur bone may validate hypotheses about evolutionary relationships, rates, and patterns of molecular change and degradation, as well as the chemical stability of molecules over time.

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DOI: 10.1126/science.1138709
, 277 (2007); 316Science
et al.Mary Higby Schweitzer,
Suggest the Presence of Protein
Tyrannosaurus rexAnalyses of Soft Tissue from
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Thetimeincrementt used in the transmitted signal yields
arangeresolutionct/2, where c is the speed of light.
20. We typically define the limb-to-limb bandwidth as the
full width of the radar echo at the level of twice the root
mean square (RMS) of the off-DC, off-target noise. The
exception is the strong 2004 Arecibo data, for which we
use 10 times the RMS as the threshold to avoid
contributions from frequency sidelobes.
21. This assumes PH5 is a principal axis (PA) rotator where
the spin axis remains fixed in inertial space and aligned
with the axis of maximum moment of inertia. The spin
axis of PH5 must then be oriented such that the angles it
makes with the lines of sight satisfy the observed
bandwidths (17). The damping time scale (28)toPA
rotation for PH5 is of order 0.1 million years.
22. Materials and methods are available as supporting
material on Science Online.
23. The spin state solution is also validated by the phase
agreement of infrared lightcurves from the Spitzer Space
Telescope with synthetic lightcurves produced with our
shape (27).
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29. We thank the staffs of the Arecibo Observatory and the
Goldstone Solar System Radar for their support in
performing this research. The Arecibo Observatory is part
of the National Astronomy and Ionosphere Center, which
is operated by Cornell University under a cooperative
agreement with NSF. Some of this work was performed at
the Jet Propulsion Laboratory, California Institute of
Technology, under contract with NASA. This material is
based in part on work supported by NASA under the
Science Mission Directorate Research and Analysis
Programs. P.A.T. and J.L.M. were partially supported by
NASA grant NNG04GN31G. The work of P.P. and D.V. was
supported by the Grant Agency of the Czech Republic.
D.J.S. acknowledges support from the NASA Planetary
Geology and Geophysics Program. S.C.L. and A.F.
acknowledge support from the Leverhulme Trust and
PPARC, respectively. C.M. was partially supported by
NSF grant AST-0205975. The International Astronomical
Union has approved the name YORP for asteroid (54509)
2000 PH5.
Supporting Online Material
www.sciencemag.org/cgi/content/full/1139038/DC1
Methods
Figs. S1 to S5
Tables S1 and S2
References
19 December 2006; accepted 21 February 2007
Published online 8 March 2007;
10.1126/science.1139038
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Analyses of Soft Tissue from
Tyrannosaurus rex Suggest the
Presence of Protein
Mary Higby Schweitzer,
1,2,3
* Zhiyong Suo,
4
Recep Avci,
4
John M. Asara,
5,6
Mark A. Allen,
7
Fernando Teran Arce,
4,8
John R. Horner
3
We performed multiple analyses of Tyrannosaurus rex (specimen MOR 1125) fibrous cortical and
medullary tissues remaining after demineralization. The results indicate that collagen I, the main
organic component of bone, has been preserved in low concentrations in these tissues. The findings
were independently confirmed by mass spectrometry. We propose a possible chemical pathway that
may contribute to this preservation. The presence of endogenous protein in dinosaur bone may
validate hypotheses about evolutionary relationships, rates, and patterns of molecular change and
degradation, as well as the chemical stability of molecules over time.
I
t has long been assumed that the process
of fossilization results in the destruction of
virtually all original organic components
of an organism, and it has been hypothesized
that original molecules will be either lost or
altered to the point of nonrecognition over
relatively short time spans (well under a mil-
lion years) (1–7). However, the discovery of
intact structures retaining original transparency,
flexibility, and other characteristics in speci-
mens dating at least to the Cretaceous (8, 9)
suggested that, under certain conditions, rem-
nant organic constituents may persist across
geological time.
The skull, vertebrae, both femora and tibiae,
and other elements of an exceptionally well-
preserved Tyrannosaurus r ex [MOR 1125 (8)]
were recovered from the base of the Hell Creek
Formation in eastern Montana (USA), buried
within at least 1000 m
3
of medium-grained,
loosely consolidated sandstone interfingered with
fine-grained muds, interpreted as stream channel
sediments. Demineralization of femur and tibia
fragments revealed the preservation of fibrous,
flexible, and apparently original tissues, as well
as apparent cells and blood vessels (8), but the
endogeneity and composition of these structures
could not be ascertained without further analyses.
We present molecular and chemical (10)
analyses of tissues remaining after partial de-
mineralization (11) of the left and right femora
and associated medullary bone (12) that would,
in extant bone, represent the extracellular matrix
(osteoid) dominated by collagen I (13). Because
of its ordered structure as a triple helix (14, 15),
collagen I has unique characteristics that are
highly conserved across taxa, making validation
of its presence relatively straightforward. The
molecular composition of collagen incorporates
glycine, the smallest amino acid, at every helical
turn. Therefore, an amino acid profile of colla-
gen results in ~33% glycine content (14). This
molecular structure also results in packing of
microfibrils with a banded repeat of ~70 nm
(15, 16). Collagen also shows posttranslational
hydroxylation of about half of all proline and
some lysine residues; thus, the detection of
hydroxyproline and hydroxylysine in extracts of
organic material is viewed as strong evidence for
the presence of collagen (17, 18). Finally, colla-
gen is identified by polyclonal or monoclonal
antibody reactivity that can distinguish between
collagen types (19). We focused on identifying
collagen-like compounds because in addition to
being abundant and easily identified by multiple
1
Department of Marine, Earth and Atmospheric Sciences, North
Carolina State University, Raleigh, NC 27695, USA.
2
North
Carolina Museum of Natural Sciences, Raleigh, NC 27601,
USA.
3
Museum of the Rockies, Montana State University,
Bozeman, MT 59717, USA.
4
Image and Chemical Analysis
Laboratory Facility, Department of Physics, Montana State
University, Bozeman, MT 59717, USA.
5
Division of Signal
Transduction, Beth Israel Deaconess Medical Center, Boston,
MA 02115, USA.
6
Department of Pathology, Harvard
Medical School, Boston, MA 02115, USA.
7
Department of
Chemistry and Biochemistry, Montana State University,
Bozeman, MT 59717, USA.
8
Center for Nanomedicine,
Pulmonary and Critical Care Medicine, Department of
Medicine, University of Chicago, Chicago, IL 60637, USA.
*To whom correspondence should be addressed. E-mail:
schweitzer@ncsu.edu
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and independent methods, this protein is durable
(20, 21) and resistant to degradation.
The fibrous nature of demineralized dinosaur
tissues was demonstrated by optical (8) and elec-
tron (fig. S1) microscopy. Furthermore, regions
of dinosaur cortical and medullary (12) bone
demonstrated a repeat pattern with periodicity of
~70nmwhenexaminedbyatomicforcemicros-
copy (AFM) (Fig. 1, A to D), consistent with
collagen in extant bone (Fig. 1, E and F) and
similar to that previously observed in fragments of
demineralized Cretaceous avian bone (22). How-
ever, this periodic pattern was rarely observed in
ultrathin sections of MOR 1 125 demineralized
bone by transmission electron microscopy (TEM)
(fig. S1). This may be a methodological problem,
or the periodic features we observe (Fig. 1, A to
D) may be due to surface features generated when
demineralization removed most of the apatite
crystals emplaced during biomineralization, when
collagen acted as a template. Thus, the banded
features may represent a type of natural molecular
imprinting (23), because banded fibers have been
observed by TEM for other dinosaur tissues (24).
TEM studies confirm that, unlike extant bone,
dinosaur bone did not completely demineralize
after p rolonged incubation i n EDTA (11).
Selected-area electron diffraction (SAED) of the
tissues (fig. S1D, inset) showed that this retained
mineral is biogenic hydroxylapatite (25). It is not
possible to determine this conclusively because of
the similarity in structure between hydroxylapatite
and fluorapatite; however, the observed diffrac-
tion circle intensities are most consistent with hy-
droxylapatite. This finding suggests that the bone
mineral is virtually unchanged from the living
state and has undergone little if any alteration.
Force curve measurements of demineralized
dinosaur medullary and cortical bone indicate
that the elasticity of dinosaur tissues was similar
to that of demineralized extant bone. We mea-
sured both embedded sections (fig. S2A) and
unembedded whole mounts (fig. S2B) of demin-
eralized bone in both air and liquid (11). The
demineralized bone surface softened after expo-
sure to buffer, allowing the AFM tip to penetrate
deeper into the tissues with less resistance. Thus,
the modulus of elasticity (fig. S2C) was reduced
in liquid by more than three orders of magnitude
(fig. S2B). Although ~2000 nN of force was
required to penetrate ~40 nm into MOR 112 5
bone matrix in air , only ~15 nN of force was
required to depress the tip ~75 nm into the same
matrix when hydrated (fig. S2B, inset).
MOR 1 125 cortical and medullary whole-
bone extracts showed reactivity to antibodies
raised against chicken collagen I (11)whenmea-
sured by enzyme-linked immunosorbent assay
(ELISA), although the degree of binding varied
widely. Reactivity was greatly reduced in dinosaur
extracts relative to extant samples (fig. S3), but
still at least twice that observed in negative con-
Fig. 1. AFM images of partially demineralized bones of MOR 1125 (A to D)andemu(E and F). (A) Phase
image of MOR 1125 cortical bone imaged in air; (B) deflection image of MOR cortical bone imaged in
phosphate-buffered saline; (C) amplitude image of embedded and sectioned MOR medullary bone
imaged in air; (D) phase image of MOR 1125 medullary bone imaged in air (note longitudinal and cross-
sectional orientation of fiber-like structures at right angles to each other); (E) amplitude image of emu
cortical bone imaged in air; (F) amplitude image of emu medullary bone imaged in air.
Fig. 2. In situ immunochemistry on
300-nm sections of demineralized
MOR 1125 cortical bone (A to D)and
medullary bone (E to H). (A) and (E),
no primary antibodies added (negative
control); (B) and (F), antibodies to
avian collagen I; (C) and (G), anti-
bodies to actin protein (nonrelevant,
negative control); (D) and (H), anti-
bodies to avian collagen I, inhibited by
incubating with purified chicken colla-
gen before exposing to dinosaur
tissues. All data were collected using
the same parameters at 122-ms inte-
gration. (I to K) MOR 1125 cortical
tissue exposed to (I) no primary, (J) antibodies to avian collagen I, or
(K) collagenase digestion followed by antibodies to avian collagen I,
as described (11). Data in (I), (J), and (K) were collected at 149-ms
integration.
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trols of coextracted sediments and buffer without
sample, similarly treated.
We confirmed the antibody reactivity data by
in situ immunohistochemistryinaseriesof
experiments. W e exposed thin (0.3 to 0.5 mm)
sections of demineralized cortical (Fig. 2, A to D
and I to K) and medullary (Fig. 2, E to H) dinosaur
bone to antibodies raised against avian collagen I,
both before (Fig. 2, B and F) and after inhibition of
antibodies with chicken collagen (Fig. 2, D and H)
(11). Additionally , antibody reactivity (Fig. 2J) was
significantly decreased after we digested dinosaur
tissues with collagenase (Fig. 2K), although this
enzyme effect was not consistently observed. Re-
activity to antibodies, measured by fluorescence,
was significantly greater than in negative controls
(Fig. 2, A, C, E, G, and I) and was localized to
tissues. We also observed some binding of os-
teocalcin antibodies to dinosaur tissues (fig. S4).
These patterns were similar to those observed with
extant emu cortical and medullary bone (fig. S5).
Immunoreactivity in dinosaur tissues was greatly
reduced from that observed in extant bone, as illus-
trated by longer integration times and fainter signal,
but was greater than in negative controls. Immuno-
histochemistry performed on sediments was nega-
tive for binding. These results imply that the
concentration of reactive epitopes in the dinosaur
tissues is very low, consistent with the ELISA re-
sults. That antibody reactivity was more consistent-
ly observed in situ than in ELISA could be due to
greater alteration and/or loss of organic compounds
during extraction procedures, or to reduced binding
of degraded antigen to ELISA plate polymers.
The presence of collagen-derived epitopes in
demineralized tissues is supported by mass spec-
trometry data. T ime-of-flight secondary ion mass
spectrometry (TOF-SIMS) detects surface ions
associated with molecular fragmentation with
high mass resolution, and can localize signal to
whole samples without subjecting them to chem-
ical extraction. In situ TOF-SIMS analyses were
performed to unambiguously detect amino acid
residues consistent with the presence of protein in
demineralized MOR 1125 tissues (Fig. 2 and fig.
S6). We obtained ratios of glycine (Gly), the most
abundant amino acid in collagen [~33% (14)], and
alanine (Ala), which constitutes about 10% of
collagenous amino acids, to support the presence
of the specific collagen a1type1proteininthese
tissues. Small peaks representin g proline (Pro)
at mass/charge ratio (m/z) 70 (Fig. 3C), lysine
(Lys) at m/z 84 (fig. S6A), and leucine or
isoleucine at m/z 86 (fig. S6B) were also detected.
TOF-SIMS is highly matrix dependent, and de-
sorption and ionization of some amino acid res-
idues, especially modified residues such as
hydroxylated Pro, are less efficient than for other
residues (26). These modified residues were not
detected by this method but were readily identified
by other mass spectrometry methods (10).
The Gly:Ala ratio for published chicken
collagen a1 type 1 sequence (27)is2.5:1.The
TOF-SIMS results show that the Gly:Ala ratio in
medullary bone of MOR 1125 is 2.6:1 (Fig. 3, A
and B). Sandstones entombing the dinosaur , sub-
jected to TOF-SIMS as a control, showed little or
no evidence for these amino acids (Fig. 3, D and E).
We identified a variety of nitrogen-cont ain ing
species—including an alkyl amine group, C
7
H
18
N
2
+
,
located at 130 amu (fig. S6C)—in all dinosaur
samples tested, but not in any surrounding sedi-
ments. We also observed a number of Fe-C-H
speciessuchasFeCH,FeCH
2
,andFeCH
3
,
associated with the dinosaur matrix (fig. S7) but
not seen in extant material. Similar compounds
were observed in the sediments surrounding the
dinosaur . These may be microbial products, as
sequences from iron-containing microbial en-
zymes were identified by mass spectrometry
(10). W e interpret these fragments as evidence that
iron may help preserve soft tissue through initiation
of intra- and intermolecular cross-links (9).
Dinosaur protein sequence, including collagen,
should be most similar to that of birds among
extant taxa, according to other phy logenetic
information (28). The hypothesis that molecular
fragments of original proteins are preserved in the
mineralized matrix of bony elements of MOR
1 125 is supported by peptide sequences recovered
from dinosaur extracts, some of which align
uniquely with chicken collagen a1type1(10).
The amount of protein or protein-like com-
ponents in MOR 1 125 is minimal. The percent
yield afte r extraction and lyophilization was
~0.62% for cortical bone and 1.3% for medullary
bone. Protein-derived material is only a small per-
centage of the lyophilate relative to other material
coextracted from bone, as assessed by comparison
of immunoreactivity with extant samples. This is
verified by mass spectrometry , which identifies
only femtomole amounts of sequenceable mate-
rial (10) in a heterogeneous mixture of extracted
material.
Microenvironments within a single bone vary
greatly, and not every fragment of bone examined
yielded positive results. There was a high degree
of variability between extractions, and we have
also noted progressive reduction of signal in more
recent extractions, indicating bone degradation in
modern environments (29). Therefore, each of the
analyses we report has been repeated numerous
times, and we have set a minimum of three rep-
etitions with similar results before reporting an
assay as positive. Additionally , experiments have
been conducted independently in at least three
different labs and by numerous investigators, and
the results strongly support the endogeneity of
collagen-like protein molecules.
We hypothesize that these molecular frag-
ments are preserved because reactive sites on the
Fig. 3. TOF-SIMS spectra of demineralized MOR 1125 medullary bone (A to C) and entombing
sedimentary matrix (D to F). The imonium ions for Gly (m/z = 30), Ala (m/z = 44), and Pro (m/z =
70) can be unambiguously identified for MOR 1125; no signal was observed in sediment controls
that corresponded to these amino acids. See text for discussion.
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original protein molecules became irreversibly
cross-linked, both to similar molecules and to
mineral or exogenous organic components. These
cross-linking reactions may have been initiated by
unstable metal ions that formed free radicals
(30, 31),whichinturnreactedwithorganicmol-
ecules to form polymers (6, 7, 9, 32). W e propose
that the unstable metal ions were derived from the
post mortem degradation of iron-containing di-
nosaur biomolecules such as hemoglobin, myo-
globin, and possibly cytochromes (9, 31). Once
stabilized by these cross-linking reactions, the
molecules were no longer available as substrates
for further degrad ative reactio ns.
The intimate relationship between apatite and
the organic phase of bone also contributes to the
preservation of organic matter (16, 33–38), but we
propose that the mineral phase may be stabilized
by this relationship as well. The presence of bio-
genic apatite in these 68-million-year-old bones
can only be rationalized by protection from an
intact organic phase, which in turn is only satisfied
by a synergistic relationship between collagen and
mineral phases. Whereas extant bone retains no
detectable calcium after days to weeks of de-
mineralization, dinosaur bone retains a fraction of
recognizable apatite crystals after months of
treatment (fig. S1). Another contributing factor
in the retention of original mineral may be that
apatite is stabilized in the presence of calcite (33).
Sandstones surrounding MOR 1125 contain
abundant calcite cements.
The depositional environments may affect
organic preservation in other ways. Comparison
of fossils from a variety of enviro nments indicates
that those derived from sandstones are more likely
to retain soft tissues and/or cells (9). W e hypoth-
esize that the porosity of sandstones may facilitate
draining of enzymes of decay and suppurating
fluids as the organism degrades, whereas organ-
isms buried in nonporous mudstones or clays may
be exposed to these longer and therefore may be
more completely degraded.
Our findings indicate the need for optimizing
methods of extraction and handling of fossil
material. In particular, the decrease in signal we
observed over time supports the need to establish
field collection and storage of fossils according to
protocols that allow future analytical studies (29).
The data presented here illustrate the value of a
multidisciplinary approach to the characterization
of very old fossil material and validate sequence
data reported elsewhere (10). The inclusion of
fossil-derived molecular sequences into existing
phylogenies may provide greater resolution and
may allow reconstruction of character evolution
beyond what is currently possible. Elucidating
modifications to ancient molecules may shed light
on patterns of degradation and diagenesis. The
presence of original molecular components is not
predicted for fossils older than a million years
(1–7), and the discovery of collagen in this well-
preserved dinosaur supports the use of actualistic
conditions to formulate molecular degradation
rates and models, rather than relying on theoretical
or experimental extrapolations derived from
conditions that do not occur in nature.
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1125, “B. rex.” Supported by NSF grants EAR-0541744
and EAR-0548847 and the David and Lucile Packard
Foundation (M.H.S.), NASA Experimental Program to
Stimulate Competitive Research grant NCC5-579
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Supporting Online Material
www.sciencemag.org/cgi/content/full/316/5822/277/DC1
Materials and Methods
Figs. S1 to S7
References
11 December 2006; accepted 19 March 2007
10.1126/science.1138709
Protein Sequences from Mastodon
and Tyrannosaurus Rex Revealed by
Mass Spectrometry
John M. Asara,
1,2
*MaryH.Schweitzer,
3
Lisa M. Freimark,
1
Matthew Phillips,
1
Lewis C. Cantley
1,4
Fossilized bones from extinct taxa harbor the potential for obtaining protein or DNA sequences that
could reveal evolutionary links to extant species. We used mass spectrometry to obtain protein
sequences from bones of a 160,000- to 600,000-year-old extinct mastodon (Mammut americanum)
and a 68-million-year-old dinosaur (Tyrannosaurus rex). The presence of T. rex sequences indicates
that their peptide bonds were remarkably stable. Mass spectrometry can thus be used to determine
unique sequences from ancient organisms from peptide fragmentation patterns, a valuable tool to
study the evolution and adaptation of ancient taxa from which genomic sequences are unlikely to
be obtained.
O
btaining genome sequences from a
number of taxa has dramatically en-
hanced our abilities to study the evolu-
tion and adaptation of organisms. However ,
difficulties in the acquisition of DNA or RNA
from ancient extinct taxa limit the abi lity to
examine molecular evolution. Recent advances
in mass spectrometry (MS) technologies have
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