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Proteomic Analysis IDs More Than 100 Proteins in 43,000-Year-Old Woolly Mammoth Fossil

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By Adam Bonislawski

Researchers from the University of Copenhagen, University of York, and the Denver Museum of Nature & Science have published a proteomic analysis of remains extracted from a 43,000-year-old woolly mammoth, identifying 126 unique proteins.

The study, published last month in the Journal of Proteome Research, represents one of the most extensive characterizations to date of an ancient proteome and points toward the increased use of proteomics in the analysis of such samples, said Enrico Cappellini, a postdoc at the University of Copenhagen’s Centre for GeoGenetics and lead author on the study.

Past work using proteomics to study ancient organisms has detected fairly low quantities of proteins, “around 10 or so – usually collagen and a few others,” Cappellini told ProteoMonitor. He characterized those efforts as more “mass spectrometry-based ancient protein sequencing” than actual proteomic investigations.

Advances in sample-prep methods and mass spec instrumentation enabled Cappellini’s team – which used a Thermo Scientific LTQ Orbitrap Velos for the work – to add significantly to the protein IDs made by past studies, he said. In turn, he added, their findings could raise proteins’ profile “as a valuable alternative” to biomolecules like DNA, which has been more extensively studied in analyses of ancient specimens.

The JPR study “is very exciting work because it is the first to indicate the recovery of a significant proportion of non-collagenous proteins in fossil bone,” North Carolina State University paleontology professor Mary Schweitzer told ProteoMonitor this week in an e-mail. “The discovery and sequencing of proteins other than collagen is a major advancement to the growing field of ‘paleoproteomics.’”

In 2005, Schweitzer found and confirmed the existence of protein in tissue recovered from a 68-million-year-old Tyrannosaurus rex fossil. In 2007, she collaborated with Harvard Medical School researcher John Asara to sequence these proteins by mass spectrometry (PM 4/12/2007). That work was initially met with skepticism by some scientists who doubted that proteins – particularly collagen – could survive for millions of years. However, re-evaluations of the study by outside researchers, as well as an additional study that ID'ed peptides in an 80-million-year-old hadrosaur fossil (PM 5/7/2009), bolstered Schweitzer and Asara’s claims (PM 7/30/2009).

The relevance of the JPR study, which analyzed a 43,000-year-old fossil, to samples like Schweitzer’s T. rex, which was millions of years old, remains to be seen, Cappellini said. He suggested that the question could best be approached by investigating progressively older samples to establish what proteins can be identified at what timepoints and to gain a better understanding of protein degradation over time.

The mammoth analysis benefited from sample prep procedures the researchers optimized for recovery of proteins from ancient bone, such as gelatinization and the exclusion of precipitation steps, Cappellini said. “Ancient proteins have a certain amount of damage – they are degraded, crosslinked – and so some steps that are commonly used for modern proteins can’t be used because the yield drops dramatically.”

Also key to the analysis, he said, was the use of error-tolerant searching for identifying spectra from their mass spec analysis.

“A regular search against the African elephant protein list gives you the identification of some proteins, but this finds the less interesting stuff, because you’re identifying the peptides that are identical in the mammoth and the elephant,” Cappellini said. “What you’re interested in, though, are the differences – the amino acid substitutions.”

Among the 126 proteins Cappellini and his colleagues were able to identify were lower-abundance analytes like growth factors and cytokines. Particularly interesting was the presence of serum albumin, he said, a protein that “was known to be present in bone” but which researchers hadn’t previously sequenced.

The researchers might have identified even more proteins, Cappellini suggested, if, prior to mass spec analysis, they’d depleted the samples of collagen proteins, which accounted for 58 percent of the spectra they used in making their peptide IDs.

“That’s something we need to explore, because if we can refine the purification and sample preparation even further, we can go even deeper [into the proteome],” he said.

Schweitzer likewise noted that her lab has “found that [collagen] dominates our mass spec analyses of ancient bone … and can function to mask the presence of other proteins.”

Compared to DNA-based analyses of ancient organisms, proteomics “is a field in its infancy,” Cappellini said. The hope, Schweitzer suggested, is that proteins will prove more abundant and resistant to degradation than DNA and will offer information not accessible via genetic research.

“While one can infer protein primary sequence through recovered DNA, added information about protein function is only available through studies of post-translational modifications not indicated by DNA,” she said.

Cappellini said, however, that despite the large number of proteins identified, the JPR study offers little in the way of new insights into mammoth biology.

“Unfortunately, in this case, we are not able to say anything new about the biology of the mammoth,” he said. “There was nothing related to cold adaptation, for example – or at least we were unable to find it.”

“Mostly this is a proof of concept and a good exercise that shows improvement in the methodology,” he added. “We hope that our contribution can inspire future work, that people are now aware of the possibility of getting such a rich protein list and will take this approach into serious consideration to solve new problems.”


Have topics you'd like to see covered in ProteoMonitor? Contact the editor at abonislawski [at] genomeweb [.] com.

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