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Independent analysis of controversial T. rex data confirms findings
bolstering their analysis. In September 2008, Asara released only the T. rex spectra data set into the PRIDE database. He didn’t release the control spectra from the soil sediment in the vicinity of the T. rex fossil (these events are chronicled in the JPR news story “A controversial data set stirs up even more controversy”). But Asara gave Bern and his team the
But 0.4 Da is just as good as 0.1 Da for Mascot and Sequest, whereas ByOnic gives extra weight to closely matched peaks.” Of the six collagen sequences from the Asara-Schweitzer data set in GenBank (deposited by the UniProt Consortium), Bern and his colleagues confirmed three. A case could be made that a fourth sequence is also correct, they said, because it is unlikely to be a random match for collagen. But the investigators didn’t find the last two sequences and suggested that they be removed from GenBank. However, Asara says Mascot and Sequest were able to find those two sequences. SHUTTERSTOCK
A recent JPR paper made John Asara’s day. The researcher at Harvard Medical School and Beth Israel Deaconess Medical Center and his collaborator, Mary Schweitzer of North Carolina State University, have been embroiled in a controversy over a 68-millionyear-old Tyrannosaurus rex. In 2007, they published data that indicated the dinosaur’s bones contained collagen that closely matched that of birds (Science 2007, DOI 10.1126/science. 1137614). But their study was heavily criticized on several fronts, including the accusation that peptide matches to their MS data were statistically “Bloody” controversy insignificant (see the Analytical Besides confirming the idenChemistry news story “Uproar tity of the collagen peptides, over dinosaur data”). Bern and colleagues published The JPR paper by Marshall Presence of birdlike collagen and hemoglobin peptides have been another finding: a statistically Bern and colleagues at the confirmed by a second group of researchers in the controversial T. significant match of a peptide Palo Alto Research Center, Inc., rex data set. sequence to bird hemoglobin. and the University of California Hemoglobin threatened to Davis is the first independent overshadow collagen when analysis of the Asaracontrol data set, making them the Asara released the T. rex data set. Schweitzer team’s T. rex mass spectra first to independently analyze it. That’s because Martin McIntosh and published in a peer-reviewed journal ByOnic churns through T. rex data Matthew Fitzgibbon at the Fred (2009, DOI 10.1021/pr900349r). Bern To analyze the T. rex data set, Bern Hutchinson Cancer Research Center and colleagues didn’t find anything and colleagues used the proteomics had analyzed the data set (results obviously wrong with the T. rex data. search algorithm ByOnic and its comwere not published) and found a Although the team did find numerous panion software, ComByne, both of peptide that was an exact match to common laboratory contaminants in which Bern had developed four years ostrich hemoglobin. Some critics the sample, they also identified statistiago. ByOnic works a bit differently have interpreted the match to ostrich cally significant data indicating the from the Mascot and Sequest algohemoglobin as evidence of contamipresence of peptides consistent with rithms used by Asara and his group. nation from the ostrich sample that those in old bird-like bone. All of these algorithms calculate a Asara and Schweitzer used as a con“This was a great confirmation of predicted m/z value to several decitrol; Asara and his colleagues think all our data,” says Asara. “We probmal places of accuracy. If the meathat the hemoglobin belongs to the T. ably could have done a paper like sured mass falls very close to the prerex (for more details see box in AC this as a follow-up because there dicted mass, Mascot and Sequest call news story “New analysis seen as have been so many questions about it a match. These algorithms “have a validating dinosaur proteomics the data. But I think that’s great that hard tolerance. If you are within the study”). it came from a group other than us.” tolerance, the peak is matched,” ex“There has been a lot of folklore Critics seem pleased with the paper. plains Bern. But “if you’re outside the out there about us initially finding “This paper did a thoughtful and thortolerance, there’s no credit, and the this hemoglobin peptide and the raough job of revealing new things in the peak’s not matched.” tionale for why we proposed that data set and speaking to whether the ByOnic is more forgiving because it their results could be explained by data were statistically significant or doesn’t “go for an all-or-nothing clascontamination,” says McIntosh. not,” says Tom Kaye of the Burke Musification,” says Bern. “ByOnic, Mas“There’s also Asara’s response to our seum of Natural History and Culture. cot, and Sequest all consider peaks initial claim that described arguments The critics note that Bern and colmatched if they agree within 0.4 Da. against why it may not be contamileagues took an important step in
10.1021/pr900691s
© 2009 American Chemical Society
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nation.” The Bern paper, he says, puts all the facts out into the open in a peer-reviewed format. Some critics have always wondered why Asara and colleagues haven’t published the hemoglobin data and suspect they are hiding a contamination problem. Asara says they are not ready to publish the hemoglobin data because, without further support, the MS analyses don’t count for much. Bern and colleagues “beat us to the hemoglobin finding because we put the data out there, and they decided to publish on it, which is fine,” says Asara. “There is lots of other supporting evidence that will be forthcoming by our groups and potentially other groups.” Asara points to the latest paper that he, Schweitzer, and a group of collaborators published on an 80-million-year-old hadrosaur (Science 2009, DOI 10.1126/science.1165069). “You will see lots of data in there by Mary’s group that verifies the presence of hemoglobin using antibodies.” Based on the body of work (published and unpublished) from his and Schweitzer’s groups, Asara is convinced the hemoglobin is from a dinosaur. Besides, Asara points out that in the Bern paper, the collagen hits were not restricted to a single species: they included cow, newt, human, and chicken. “You would think if this sample happened to be contaminated, giving us a wrong hemoglobin hit, that all these hits would at
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least be consistent with a single species,” he says. But McIntosh says that the current body of published data is not enough to tie the hemoglobin in the T. rex data set to dinosaur yet. “[Bern’s paper] should not permit Bern, Asara, or anybody else to assert that hemoglobin survives hundreds of millions of years,” he says. “Does this paper reassert the claims of the initial paper? The answer is yes because the initial paper only spoke to the collagen. What the paper does is put into press that there are also hemoglobin peptides in there as well. It does not make a convincing case at all about the origin or source of them.” McIntosh says if and when additional work to prove the hemoglobin’s dinosaur origins is published, the credit for the hemoglobin finding should go to Asara, Schweitzer, and their teams because they were the first to publicly assert that the hemoglobin is derived from dinosaur. Peanut: a common lab contaminant? Because Bern and colleagues painstakingly catalogued every contaminant in the T. rex mass spectraskeratins, casein, BSA, trypsin, and soil bacteriasit has become obvious to Bern that researchers need to identify the expected contaminants in their laboratories. “No one seems to know too much about what proteins are common lab contaminants,” he says. For example, he found a peanut allergen in the T. rex data set. When he
Journal of Proteome Research • Vol. 8, No. 9, 2009
told some researchers of this, they thought it sounded implausible and made the whole data set look dubious, but others confirmed that they too occasionally saw similar hits in their studies. Asara says he has seen the peanut hit and has an explanation for it. Hevia, a plant with a lot of similar sequences to the peanut plant, leaves peptide traces on latex gloves “which were possibly used in the field,” he explains. “We use nitrile gloves in the lab whenever possible to avoid this protein contaminant.” Bern is still doubtful about Asara’s attribution of the peanut allergen to latex gloves, but he says the doubt only emphasizes how researchers seriously need to document which contaminants they see when they do proteomics studies. Will this dinosaur ever rest in peace? The Bern paper, in everyone’s mind, drives a nail into the coffin of the argument that the peptide matches to spectra were statistically insignificant. But now McIntosh says the question with the T. rex data becomes: Where are these collagen-like peptides coming from? But Asara thinks the paper bolsters the case for the collagen to have a dinosaur source. He says, “The point is we have collagen in the sample. It’s birdlike. It’s not all consistent with a single species. And we have biochemical verification and negative controls. There really is nothing more to say.” —Rajendrani Mukhopadhyay
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© 2009 American Chemical Society
