The big picture: Ancient proteins identified in preserved woolly mammoth bone

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  • Published: Jan 16, 2012
  • Author: Steve Down
  • Channels: Proteomics & Genomics
thumbnail image: The big picture: Ancient proteins identified in preserved woolly mammoth bone

Preserved proteins

Following the extraction and analysis of ancient DNA from a well-preserved frozen woolly mammoth several years ago, scientists have been attempting to extract and analyse proteins from similar specimens. One of the main attractions in targeting proteins rather than DNA, especially from bone, is their greater abundance and higher stability.

There have been a number of reports of ancient bone proteins, but in each case only one protein was analysed, either collagen which makes up 90% of the organic bone matrix, or osteocalcin. Identification of some of the less abundant proteins would help to expand knowledge of the biochemical processes that occurred within the bones thousands of years ago, as well as allowing the comparison of sequences between ancient and modern species.

An international team of scientists recently optimised procedures for extracting proteins from ancient bone to maximise the yields and minimise contamination. Now, the same group has applied these techniques to a bone from a woolly mammoth about 43,000 years old that had been preserved in permafrost.

Enrico Cappellini and colleagues from the Natural History Museum of Denmark, University of Copenhagen worked with coresearchers in the USA from Stafford Research, Inc., Lafayette, CO and the Denver Museum of Nature & Science, and in the UK from the University of York.

Special measures for ancient proteins

The researchers went to great lengths to avoid sample contamination. All personnel handling the samples used nitrile gloves rather than those made from latex to exclude proteins derived from the rubber tree. In addition, no modern proteomics experiments had been carried out in the work area used for sample preparation, to avoid cross-contamination. Other proteins commonly employed as calibrants or standards in proteomics were excluded completely.

Before taking the bone samples, small sections of both the exposed terminal portions of the compact bone, representing the peri- and end-osteal surfaces, were removed because they are most likely to be affected by diagenesis or infiltrated with organic matter. Then two cores of 2 mm diameter were removed with a dental drill.

Total protein extraction from the powdered cores was achieved by incubating repeatedly at elevated temperature while avoiding protein precipitation which is generally associated with protein losses. These techniques produced almost twice the protein yield than achieved for modern samples, at 4.27 mg protein/g bone, giving absolute amounts of 500-600 µg. This yield is equivalent to that required for standard shotgun sequencing methods.

In addition to the mammoth bone, the team extracted proteins from two Columbian mammoths that had been preserved in a temperate climate and were dated to 11,000 and 19,000 years old. For these samples, the yields were much lower at 150-200 µg but they were still adequate for protein sequencing.

All of the extracted proteins were enzymatically digested and the resulting peptides were extracted on stage tips, again to minimise losses before analysis by LC-tandem MS on a high resolution instrument. The identified peptides were searched against the elephant database for protein assignment.

Mammoth number of proteins identified

An unprecedented 126 proteins were identified in the ancient mammoth bone from at least two unique peptides. More than 90% had never previously been reported in mass spectrometric experiments in ancient bone. The proteome was very similar to that of modern African elephant bone, with a predominance of extracellular matrix proteins (49%) followed by plasma proteins (21%), membrane and intracellular proteins (18%), collagens (9%) and keratins (3%).

Apart from traces of human keratins, which are very difficult to exclude from proteomics studies, there were no signs of cross contamination or environmental proteins. However, the data revealed that post-mortem damage by oxidation and/or hydrolysis had affected some proteins. The team recognised that more work is required to ensure that this degradation occurred before sample preparation.

Serum albumin was the best characterised protein with 47% of the total sequence recovered, confirming its long-term stability. Its sequence contained two amino acid substitutions compared with the Indian and African elephants

The researchers carried out a molecular phylogenetic inference experiment as proof-of-concept for the use of serum albumin as a marker. The comparison encompassed data for 2 birds and 30 mammals and the serum albumin from the mammoth was clustered with that of the African and Indian elephants.

In addition, the serum albumin of other species gave completely correct assignments, indicating the potential value of this protein for recovering taxonomic data from ancient bone remains. This would be important when DNA recovery was not an option.

The mammoths preserved in the temperate climates yielded fewer protein assignments, with 35 and 19 identified for the two samples, but even these lower numbers were unparalleled before this study. So, the proteomics method has the potential to produce data from species that have not been preserved in cold environments.

These results represent a large step forward in the study of ancient tissue samples and illustrate the power of refined sample preparation methods and the latest developments in mass spectrometry. They will help in the "understanding of the knowledge of the biochemistry and physiology of bone in ancient samples and consequently to recognising possible forms of disease."

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

 A record number of ancient proteins have been isolated and identified from the femur of a 43,000 year-old woolly mammoth that had been preserved in permafrost, using specially developed techniques to minimise contamination along with high-resolution mass spectrometry 

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