Technical Comments

Comment on "Protein Sequences from Mastodon and Tyrannosaurus rex Revealed by Mass Spectrometry"

Science  22 Aug 2008:
Vol. 321, Issue 5892, pp. 1040
DOI: 10.1126/science.1155006

Abstract

Asara et al. (Reports, 13 April 2007, p. 280) reported sequencing of Tyrannosaurus rex proteins and used them to establish the evolutionary relationships between birds and dinosaurs. We argue that the reported T. rex peptides may represent statistical artifacts and call for complete data release to enable experimental and computational verification of their findings.

Imagine a monkey typing random keys on a typewriter and let us assume that the monkey is given 100,000 attempts to generate six-letter words. One would be surprised if the monkey typed a six-letter word from Webster's dictionary on the first attempt; indeed, the probability of this is rather low. However, nobody would be surprised if some of the 100,000 words turned out to be correctly spelled English words.

Now imagine a boy who watches the monkey and discovers that 7 out of 100,000 words are actually spelled correctly. The boy is so surprised that he writes a paper called “My monkey can spell!” and publishes it in a scientific journal. Some scientists are not convinced, and they request the list of all words the monkey generated in addition to the seven correctly spelled words. The boy does not understand the reason for such requests; indeed, if all other words are just junk, what is the point of asking for them?

One often feels like a monkey (and a boy) when trying to interpret peptide mass spectra. Indeed, a randomly chosen spectrum can easily match a word in Webster (if English letters are interpreted as amino acids) or in any protein database. Scientists fail to interpret the lion's share of mass spectra generated worldwide, resulting in billions of uninterpreted or “junk” spectra. If we matched these junk spectra against Webster we would surely find that some of them spell English words. Unfortunately, we would not be able to publish a paper called “Mass spectrometers can spell!” because false protein identifications are unavoidable in the field of proteomics. Scientists learned how to cope with them by establishing the Proteomics Publication Guidelines that require authors to provide the error rates of their identifications.

Asara et al. (1) reported the sequencing of proteins from 68-million-year-old T. rex fossils and established similarities between dinosaur and chicken genomes. The authors generated seven T. rex peptides by matching mass spectra against collagen proteins. They did not reveal all generated spectra and never specified exactly how many spectra were generated. Because there are false identifications in every mass spectrometry experiment, without addressing the statistical significance problem, the results of (1) are no more convincing than the first sensational report of dinosaur DNA published in Science more than a decade ago (2).

In the spring of 2007, we notified Asara and Science of concerns about the statistical significance of some of the peptides. In a subsequent clarification letter (3), Asara et al. acknowledged some of the problems with their analysis in (1). In particular, they stated, “We have determined that one of the reported T. rex spectra for the peptide GLVGAPGLRGLPGK is statistically insignificant when searched against large protein databases....” (3, 4). By admitting this point, Asara et al. implicitly (and probably unknowingly) acknowledged a much bigger problem with their original study (1). Indeed, the statistical significance (e.g., false positive rate or FPR) is a number that needs to be computed, but Asara et al. (3) never described how they computed statistical significance, and it is not clear whether they tried. If they computed the statistical significance, they would discern that other T. rex peptides do not fare much better. For example, it turns out that there are thousands of peptides that match the fifth T. rex spectrum reported in (1, 3) even better than the alleged T. rex peptide GVVGLP*GQR [FPR or spectral probability equal to 1.3 × 10–6 (5)]. This implies that if one tries to match this spectrum against a small database of 106 amino acids, there is a good chance of matching this spectrum simply by chance. Or, equivalently, if one tries to match 1000 arbitrary spectra of similar quality against an arbitrary database of 1000 amino acids, there is a good chance to find an interpretation that is even better than the alleged T. rex peptide GVVGLP*GQR.

Asara et al. (3) must have generated at least hundreds of thousands of spectra, and their database is much larger than 1000 amino acids. This immediately characterizes the peptide GVVGLP*GQR as a statistical artifact, in addition to GLVGAPGLRGLPGK, which the authors acknowledge in (3). If Asara et al. (1) stand by the statistical significance argument given in (3), they should question all of the T. rex peptides identified in (1). Only one of these peptides was supported by chemical synthesis with a spectral correlation coefficient of 0.71, which although borderline significant, may also represent homeometric (6), but not identical, peptides. We argue that most of the peptides with GVVGLP*GQR-like spectra (e.g., 10,919 peptides with better InsPecT scores or 10,294 peptides with better X!Tandem scores than GVVGLP*GQR) would have produced spectra that are somewhat similar to the spectrum of GVVGLP*GQR, thus calling for more extensive synthesis-based verification of the results in (1). For example, one could potentially synthesize GVVGLP*GQR and discover that the resulting spectrum “looks like” one of the T. rex spectra, thus “proving” that GVVGLP*GQR is indeed a T. rex peptide. In this case, it is puzzling how Asara et al. selected the “correct” statistically insignificant peptide among hundreds of other statistically insignificant peptides. For example, peptides RVGLRAAR, RVGLPTKK, RVGP*PTKK, and thousands of others represent better InsPecT and X!Tandem spectral interpretations than GVVGLP*GQR (table S1) (supporting on-line material). If one is willing to argue that GVVGLP*GQR is a valid identification based on peptide synthesis, the peptides RVGLRAAR, RVGLPTKK, and RVGP*PTKK should also be synthesized and compared to the T. rex spectrum. Extraordinary science requires extraordinary proofs.

Since the publication of their report (1), Asara et al. have reinterpreted (3) four out of seven of the T. rex peptides originally reported. The most likely outcome of further criticism is that Asara and colleagues will continue changing their original interpretations until the critics give up. So far, five out of six of the remaining significant T. rex peptides have already emerged as identical to chicken peptides. Maybe T. rex was a chicken after all?

Recently, a group of 27 mass spectrometrists, bioinformaticians, and dinosaur experts published an insightful criticism of the T. rex protein analysis (7). Still, Asara and Schweitzer (8), refused to acknowledge the problems with their analysis. It is now the turn of the mass spectrometry community to question whether the monkey can actually spell. It is very easy to check; just ask the boy how many words (e.g., spectra) the monkey has generated and what tests of statistical significance were used to compute FPR. With this information in hand, the scientists can finally match all dinosaur proteins against Webster's dictionary to see whether mass spectrometers can spell and whether T. rex was a chicken.

Supporting Online Material

www.sciencemag.org/cgi/content/full/321/5892/1040b/DC1

SOM Text

Table S1

References

References and Notes

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