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Additional file 5: Supplementary Figure 5. In vitro response of melanoma cells to treatment with TMZ in combination with AG-120. A] Silencing IDH1 followed by treatment with TMZ for 5 days under high glucose (25 mM) and B] low glucose (2.5 mM) in SK-MEL-28 cells; IC50 values are provided. C] Relative ROS levels after 48 hours in A375 cell line under low glucose combined with TMZ and AG-120. D] Relative ROS levels after 48 hours in SK-MEL-28 cells under low glucose combined with TMZ and AG-120. E] Cell viability of SK-MEL-28 cells, treated with the indicated doses of TMZ; IC50 values are provided. F] Cell viability of SK-MEL-28 cells treated with indicated doses of AG-120 for 6 days. IC50 values are provided. G] Drug sensitivity in SK-MEL-28 cells under low glucose, with varying concentrations of TMZ and AG-120, cultured for 5 days under low glucose. IC50 results are provided. H] Drug matrix heatmap 5 × 8 (AG-120 and TMZ) grid showing percent viability and Bliss Independence scores in SK-MEL-28 cells cultured under 2.5 mM glucose for 5 days. Positive values reflecting synergism appear green on the heatmap (Bliss volume ≥ 10). All treatments with AG-120 were carried under low glucose (2.5 mM) and low Mg2+ (0.08 mM).

Additional file 4: Supplementary Figure 4. IDH1 knockout suppresses tumor growth in vivo. A] Relative mRNA levels, normalized to mRNA levels of 18S; Western blot analysis of IDH1 expression after IDH1 knockout by CRISPR/Cas9 (IDH1.KO) compared to control (IDH1.EV) A375 cells. B] Mice were injected with IDH1.EV and IDH1.KO A375 cells (n = 5 per group) and tumor sizes were monitored for 5 weeks. Images of tumors at the end of the experiment are shown. C] Tumor volumes of IDH1.EV and IDH1.KO A375 xenografts. D] Histograms show tumor volumes with IDH1.EV and IDH1.KO A375 xenografts at the end of the experiment. E] Relative glucose levels in adjacent skin and xenograft. F] Relative free magnesium levels in skin, xenografts and serum. Each data point represents the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Additional file 3: Supplementary Figure 3. AG-120 is a potential wtIDH1 inhibitor under glucose limitation in melanoma cells. A] Representative images of colony formation assays for cells cultured under high (25 mM) and B] low glucose (2.5 mM) in the SK-MEL-28 cell line. The cells were treated with vehicle control or AG-120 (2 μM) under the indicated conditions for 9 days. Quantitation (%) is shown in the graph at the bottom. C] Cells cultured under high glucose, or D] low glucose were captured by phase-contrast imaging (4X magnification), after treatment with vehicle control or AG-120 (2 μM) for 4 days. Representative oxygen consumption rate (OCR) in A375 E] and SK-MEL-28 F] cell lines cultured in 2.5 mM glucose and treated with vehicle or AG-120 for 36 hours. G] and H] Basal mitochondrial respiration, ATP production, and maximal mitochondrial respiration of the A375 and SK-MEL-28 cells treated with vehicle or AG-120 for 36 hours. Each data point represents the mean ± SEM of three independent experiments. N.S., nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Additional file 6: Supplementary Figure 6. Treatment of mice bearing B16-F10 tumors with TMZ in combination with AG-120. A] Sanger sequencing of PCR amplicons correlated with codon 132 of the IDH1 gene in B16-F10 murine melanoma cells. B] Schematic represents the treatment model after 4 × 104 B16-F10 melanoma murine cells were injected subcutaneously into the flanks of C57BL/6 J recipient mice. After 9 days, when tumors reached 80-100 mm3, mice were divided into four groups and treated with i) Vehicle; ii) TMZ (30 mg/kg intraperitoneal once a day); iii) AG-120 (150 mg/kg orally twice a day); and iv) AG-120 + TMZ (150 mg/kg orally twice a day + 30 mg/kg intraperitoneal daily). C] Survival data of C57BL/6 J mice are represented by Kaplan-Meier curves. Significance between each group was determined using the log-rank test.

Additional file 2: Supplementary Figure 2. IDH1 supports mitochondrial function under glucose limitation. A] Representative OCR tracing in A375 melanoma cells cultured under the indicated glucose concentrations for 24 hours and B] Extracellular acidification rate (ECAR) response of A375 cells under the indicated conditions. C] PCA of metabolites analyzed by LC-MS/MS performed on A375 cells under high (25 mM) and low (2.5 mM) glucose (n = 3 samples). D] A heatmap of the top 50 metabolites with the greatest changes in A375 cells (n = 3 independent samples) under 25 mM glucose. The scale is log 2 fold-change. E] Representative OCR tracing in A375 melanoma cells cultured under the indicated glucose concentrations for 24 hours.

Metabolic changes induced by oncogenic drivers of cancer contribute to tumor growth and are attractive targets for cancer treatment. Here, we found that increased growth of PTEN mutant cells was dependent on glutamine flux through the de novo pyrimidine synthesis pathway, which created sensitivity to inhibition of dihydroorotate dehydrogenase, a rate limiting enzyme for pyrimidine ring synthesis. S-phase PTEN mutant cells showed increased numbers of replication forks, and inhibitors of dihydroorotate dehydrogenase led to chromosome breaks and cell death due to inadequate ATR activation and DNA damage at replication forks. Our findings indicate that enhanced glutamine flux generates vulnerability to dihydroorotate dehydrogenase inhibition, which then causes synthetic lethality in PTEN deficient cells due to inherent defects in ATR activation. Inhibition of dihydroorotate dehydrogenase could thus be a promising therapy for patients with PTEN mutant cancers.

Decreased growth hormone (GH) and thyroid hormone (TH) signaling are associated with longevity and metabolic fitness. The mechanisms underlying these benefits are poorly understood, but may overlap with those of dietary restriction (DR), which imparts similar benefits. Recently we discovered that hydrogen sulfide (H2S) is increased upon DR and plays an essential role in mediating DR benefits across evolutionary boundaries. Here we found increased hepatic H2S production in long-lived mouse strains of reduced GH and/or TH action, and in a cell-autonomous manner upon serum withdrawal in vitro. Negative regulation of hepatic H2S production by GH and TH was additive and occurred via distinct mechanisms, namely direct transcriptional repression of the H2S-producing enzyme cystathionine γ-lyase (CGL) by TH, and substrate-level control of H2S production by GH. Mice lacking CGL failed to downregulate systemic T4 metabolism and circulating IGF-1, revealing an essential role for H2S in the regulation of key longevity-associated hormones.

The flagellated protozoan Trichomonas vaginalis is an obligate human genitourinary parasite and the most frequent cause of sexually transmitted disease worldwide. Most clinical isolates of T. vaginalis are persistently infected with one or more double-stranded RNA (dsRNA) viruses from the genus Trichomonasvirus, family Totiviridae, which appear to influence not only protozoan biology but also human disease. Here we describe the three-dimensional structure of Trichomonas vaginalis virus 1 (TVV1) virions, as determined by electron cryomicroscopy and icosahedral image reconstruction. The structure reveals a T=1 capsid comprising 120 subunits, 60 in each of two nonequivalent positions, designated A and B, as previously observed for fungal Totiviridae family members. The putative protomer is identified as an asymmetric AB dimer consistent with either decamer or tetramer assembly intermediates. The capsid surface is notable for raised plateaus around the icosahedral 5-fold axes, with canyons connecting the 2- and 3-fold axes. Capsid-spanning channels at the 5-fold axes are unusually wide and may facilitate release of the viral genome, promoting dsRNA-dependent immunoinflammatory responses, as recently shown upon the exposure of human cervicovaginal epithelial cells to either TVV-infected T. vaginalis or purified TVV1 virions. Despite extensive sequence divergence, conservative features of the capsid reveal a helix-rich fold probably derived from an ancestor shared with fungal Totiviridae family members. Also notable are mass spectrometry results assessing the virion proteins as a complement to structure determination, which suggest that translation of the TVV1 RNA-dependent RNA polymerase in fusion with its capsid protein involves-2, and not+1, ribosomal frameshifting, an uncommonly found mechanism to date. © 2013 Parent et al.

Protein sequences from bone-derived collagen as old as 68 million years have been detected by using mass spectrometry (1, 2). A BLAST search of the resulting peptide sequences found the highest similarities between the collagen peptides obtained from extracts of Tyrannosaurus rex fossil bone and those ofbirds (Gallus gallus) and between mastodon (Mammut americanum) and other mam­ mals, including elephant (Loxodonta africana). We performed phylogenetic analyses to infer the evolutionary relationships of the T. rex [Museum of the Rockies (MOR) 1125] and mastodon (MOR 605). The results extend our knowledge of trait evo­ lution within nonavian dinosaurs into the macro­ molecular level of biological organization. We used 21 extant organisms in the analyses. Collagen a 1(I) and a2(I) protein sequences from 19 extant organisms (3) were obtained from the National Center for Biotechnology Information (NCBI) and ENSEMBL databases. Collagen a1(I) and a2(I) peptide sequences from a metacarpal of Alligator mississippiensis were obtained by mi­ crocapillary liquid chromatography tandem mass spectrometry (LC/MS/MS) with an ion trap mass spectrometer using the Sequest algorithm (1, 2) and the Paragon algorithm (4). Ostrich collagen se­ quences were determined elsewhere (1, 2). Sequences from alligator (Crocodilia) and ostrich (Aves) rep­ resent 63% and 43% of the full-length collagen a1(I) protein available for other organisms, respec­ tively. The collagen a1(I) and a2(I) sequences for elephant (L. africana), tenrec (Echinops telfairi), and green anole (Anolis carolinensis) were ob­ tained by using gene translations from online databases (5). P ' Bayesian, likelihood, parsimony, and dis­ tance methods were used to generate evolu­ tionary trees (6). In the Bayesian analysis, the posterior distribution of trees reconstructed all extant groups in generally agreed-upon rela­ tionships (the posterior probability of clades ranged from 0.80 to 1.00), with the exception of green anole (A. carolinensis), which is in­ ferred here to lie at the base of amniotes in­ stead ofgrouping as the sister taxon to alligator and birds (archosaurs) (Fig. 1). LC/MS/MS from tryptic digests produces fragmentary pro­ tein sequence data; however, we found unequivocal support (posterior probability of 1.00) uniting mastodon with elephant as members of Elephantinae, which together group with tenrec (E. telfairi) as members of the mam­ malian group Afrotheria (7). Maximum likeli­ hood produces the same groupings, although with less support (approximate likelihood ratio test; aLRT = 0.855 for Elephantinae and 0.872 for Afrotheria). Maximum parsimony analysis also groups mastodon, elephant, and tenrec together (fig. S1, B to D). For the T. rex sample, we used five peptide sequences from collagen a1(I) and one from collagen a2(I) (1, 2, 8) for a total of 89 amino acids (Fig. 1). The T. rex clusters within the Archosauria (posterior probability of 0.92), more closely related to birds (chicken and ostrich, 0.9) than alligator, although a lack of informative sites in the ostrich and T. rex leaves Dinosauria unresolved. The likelihood tree is identical to the Bayesian tree, except for higher support at these locations in the tree (aLRT = 0.969 for Archosauria and 0.907 for Dinosauria). Branch lengths (expected rates of change per site) indicate a relatively stable and uniform rate of evolution, lacking evidence for a deviation from a molecular clock. Maximum par­ simony analysis also groups the T. rex with the chicken and ostrich, although bootstrap support is low (fig. S1, B to D). Neighbor joining groups the T. rex with the birds, but miscalculates the branching order and misplaces alligator, mastodon, and several extant organisms (fig. S1, B to D). The slight disagreement between the distance results compared with the Bayesian, likelihood, and parsimony results (all three of which are concordant) are predictable given that distance methods perform poorly for taxa with large amounts of missing data (9). Nevertheless, there is congruence between three out of the four methods for the mastodon and four out of four methods for the T. rex despite the problem of missing sequence data. The recovered sequences contain informative phylogenetic signal consistent with predictions based on genetic and morphological data for mastodon (10, 11) and on morphological data for T. rex (12, 13) These results support the endogenous origin of the preserved collagen molecules and confirm the prediction based on morphology that, ifbiomolecules could be retrieved from a nonavian dinosaur, they would share a higher degree of similarity with birds than with other extant vertebrates. Our findings suggest that molecular data from long-extinct organisms may have the potential for resolving relationships at critical areas of the vertebrate evolutionary tree that have, so far, been intractable. The findings presented here also bolster the use ofmorphology in phylogenetics because our results are consistent with studies on the evolutionary relationships of fossil forms that rely on morphology (12, 13)., Published as part of Chris L. Organ, Mary H. Schweitzer, Wenxia Zheng, Lisa M. Freimark, Lewis C. Cantley & John M. Asara, 2008, Molecular phylogenetics of Mastodon and Tyrannosaurus rex, pp. 499 in Science 320 on page 499, DOI: 10.1126/science.1154284, http://zenodo.org/record/3942979, {"references":["1. J. M. Asara, M. H. Schweitzer, L. M. Freimark, M. Phillips, L. C. Cantley, Science 316, 280 (2007).","2. J. M. Asara et al., Science 317, 1324 (2007).","4. I. V. Shilov et al., Mol. Cell. Proteomics 6, 1638 (2007).","7. M. S. Springer, W. J. Murphy, E. Eizirik, S. J. O'Brien, Proc. Natl. Acad. Sci. U. S. A. 100, 1056 (2003).","8. J. M. Asara, M. H. Schweitzer, Science 319, 33 (2008).","9. J. J. Wiens, Syst. Biol. 52, 528 (2003).","12. M. J. Benton, in The Dinosauria, D. B. Weishampel, P. Dodson, H. Osmolska, Eds. (Univ. of California Press, Berkeley, 2004), pp. 7 - 19.","13. T. R. Holtz, H. Osmolska, in The Dinosauria, D. B. Weishampel, P. Dodson, H. Osmolska, Eds. (Univ. of California Press, Berkeley, 2004), pp. 21 - 24."]}

department of Marine, Earth and Atmospheric Sciences, North Carolina State University, Raleigh, NC 27695, USA. 2North Carolina Museum of Natural Sciences, Raleigh, NC 27601, USA. 3Museum of the Rockies, Montana State University, Bozeman, MT 59717, USA. 4lmage and Chemical Analysis Laboratory Facility, Department of Physics, Montana State University, Bozeman, MT 59717, USA. division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA 02115, USA. department of Pathology, Harvard Medical School, Boston, MA 02115, USA. department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717, USA. 8Center 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 The fibrous nature of demineralized dinosaur tissues was demonstrated by optical (8) and electron (fig. S1) microscopy. Furthermore, regions of dinosaur cortical and medullary (12) bone demonstrated a repeat pattern with periodicity of ~70 nm when examined by atomic force microscopy (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). However, this periodic pattern was rarely observed in ultrathin sections of MOR 1125 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 prolonged incubation in 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 diffraction circle intensities are most consistent with hydroxylapatite. 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 measured both embedded sections (fig. S2A) and unembedded whole mounts (fig. S2B) of demineralized bone in both air and liquid (11). The demineralized bone surface softened after exposure 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 1125 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 1125 cortical and medullary wholebone extracts showed reactivity to antibodies raised against chicken collagen I (11) when measured 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 controls of coextracted sediments and buffer without sample, similarly treated. We confirmed the antibody reactivity data by in situ immunohistochemistry in a series of experiments. We exposed thin (0.3 to 0.5 pm) 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. Reactivity 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 osteocalcin 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 illustrated by longer integration times and fainter signal, but was greater than in negative controls. Immunohistochemistry performed on sediments was negative for binding. These results imply that the concentration of reactive epitopes in the dinosaur tissues is very low, consistent with the ELISA results. That antibody reactivity was more consistently 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 spectrometry data. Time-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 chemical extraction. In situ TOF-SIMS analyses were performed to unambiguously detect amino acid residues consistent with the presence ofprotein in demineralized MOR 1125 tissues (Fig. 2 and fig. S6). We obtained ratios of glycine (Gly), the most abundant amino acid in collagen [-33% (74)], and alanine (Ala), which constitutes about 10% of collagenous amino acids, to support the presence of the specific collagen α1 type 1 protein in these tissues. Small peaks representing 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 desorption and ionization of some amino acid residues, 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 al type 1 sequence (27) is 2.5:1. 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, subjected 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-containing species—including an alkyl amine group, C7H18N2+, located at 130 amu (fig. S6C)—in all dinosaur samples tested, but not in any surrounding sediments. We also observed a number of Fe-C-H species such as FeCH, FeCH2, and FeCH3, 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 enzymes were identified by mass spectrometry (10). We 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 phylogenetic information (28). The hypothesis that molecular fragments of original proteins are preserved in the mineralized matrix of bony elements of MOR 1125 is supported by peptide sequences recovered from dinosaur extracts, some of which align uniquely with chicken collagen al type 1 (10). The amount of protein or protein-like components in MOR 1125 is minimal. The percent yield after extraction and lyophilization was ~0.62% for cortical bone and 1.3% for medullary bone. Protein-derived material is only a small percentage 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 material (10) in a heterogeneous mixture of extracted material. Microenvironments within a single bone vary greatly, and not every fragment ofbone 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 modem environments (29). Therefore, each ofthe analyses we report has been repeated numerous times, and we have set a minimum of three repetitions 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 fragments are preserved because reactive sites on the 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), which in turn reacted with organic molecules to form polymers (6, 7, 9, 32). We propose that the unstable metal ions were derived from the post mortem degradation of iron-containing dinosaur biomolecules such as hemoglobin, myoglobin, and possibly cytochromes (9, 31). Once stabilized by these cross-linking reactions, the molecules were no longer available as substrates for further degradative reactions. The intimate relationship between apatite and the organic phase of bone also contributes to the preservation oforganic matter (16, 33-38), but we propose that the mineral phase may be stabilized by this relationship as well. The presence of biogenic 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 demineralization, 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 offossils from a variety of environments indicates that those derived from sandstones are more likely to retain soft tissues and/or cells (9). We hypothesize that the porosity of sandstones may facilitate draining of enzymes of decay and suppurating fluids as the organism degrades, whereas organisms 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 offossils according to protocols that allow future analytical studies (29). The data presented here illustrate the value ofa 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 phytogenies 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 wellpreserved 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., Published as part of Mary Higby Schweitzer, Zhiyong Suo, Recep Avci, John M. Asara, Mark A. Allen, Fernando Teran Arce & John R. Horner, 2007, Analyses of Soft Tissue from Tyrannosaurus rex Suggest the Presence of Protein, pp. 277-280 in Science 316 on pages 277-280, DOI: 10.1126/science.1138709, http://zenodo.org/record/3742836, {"references":["5. D. E. G. Briggs, R. P. Evershed, M. J. Lockheart, Paleobiology 26, 169 (2000).","10.]. M. Asara, M. H. Schweitzer, L. M. Freimark, M. Phillips, L. C. 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We sequenced collagen protein fragments derived from fossilized bones of two extinct taxa: a 160,000- to 600,000-year-old mastodon [specimen number Museum of the Rockies (MOR) 605] (9) and a 68-million-year-old dinosaur (Tyrannosaurus rex, MOR 1125) (10), results that are supported by immunological and molecular analyses published in this issue by Schweitzer et al. (11). We first looked for tryptic peptide fragments from extracts of fossilized bone that matched identically with sequences from an orthologous protein or proteins from extant taxa, thereby identifying the protein(s) of interest. This is a common procedure for conserved proteins from taxa that share genomic information. Next, we generated a protein sequence database of likely drifts in amino acids in other tryptic peptides by comparing amino acid sequences ofthe orthologs from multiple related extant taxa. This approach produced a manageable number of theoretical protein sequences. The predicted peptide fragmentation pattern from these theoretical protein sequences were then compared with the fragmentation patterns of additional peptides derived from extracts of fossilized bone that did not match peptides in public sequence databases (fig. S1)., Published as part of John M. Asara, Mary H. Schweitzer, Lisa M. Freimark, Matthew Phillips & Lewis C. Cantley, 2007, Protein sequences from mastodon and Tyrannosaurus rex revealed by mass spectrometry, pp. 280-284 in Science 316 (5822) on page 281, DOI: 10.1126/science.1137614, http://zenodo.org/record/3744436, {"references":["9. C. L. Hill, Quat. Int. 142 - 143, 87 (2006).","10. M. H. Schweitzer, J. L. Wittmeyer, J. R. Horner, J. K. Toporski, Science 307, 1952 (2005).","11. M. H. Schweitzer et al., Science 316, 277 (2007)."]}