Species identification of fragmentary bone, such as in rendered meat and bone meal or from archaeological sites, is often difficult in the absence of clear morphological markers. Here we present a robust method of analysing genus-specific collagen peptides by mass spectrometry simply by using solid-phase extraction (a C18 ZipTip) for peptide purification, rather than liquid chromatography/mass spectrometry (LC/MS). Analysis of the collagen from 32 different mammal species identified a total of 92 peptide markers that could be used for species identification, for example, in processed food and animal feed. A set of ancient (>100 ka@10 degrees C) bone samples was also analysed to show that the proposed method has applications to archaeological bone identification.

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RATIONALEWe report the use of proteomics techniques to study how the fossil bone proteome changes in complexity over one million years.METHODSWe include the attempted use of a previously unreported methodology in proteome research, to remove the dominant bone collagens using bacterial collagenase as well as conventional shotgun proteomics methodology following digestion with the protease trypsin. In this study we expand upon a set of 19 bovine sub-fossil specimens ranging over one and a half million years that had previously been shown to possess collagen, using a total of 46 LTQ-Orbitrap liquid chromatography/tandem mass spectrometry (LC/MS/MS) analyses containing 462,186 precursor ion analyses.RESULTSAlthough many types of proteins can typically be identified in recent bone, in degraded bone we observe a rapid loss of lower abundance proteins. Abundant serum proteins such as serum albumin and alpha-2-HS-glycoprotein appear to be more easily recovered in ancient bone, both being identified in specimens dating to the Early Pleistocene, the earliest period tested in this study. Proteins belonging to the leucine-rich repeat family such as lumican, biglycan and chondroadherin also survive well, possibly because of their interactions with bone collagen.CONCLUSIONSOf these 'survivor proteins' A2HSG shows a remarkable amount of sequence variation, making it potentially one of the most useful proteins to study for species identification and phylogenetic inference in archaeological and palaeontological bone.

The mid-Pliocene was a global warm period, preceding the onset of Quaternary glaciations. Here we use cosmogenic nuclide dating to show that a fossiliferous terrestrial deposit that includes subfossil trees and the northern-most evidence of Pliocene ice wedge casts in Canada’s High Arctic (Ellesmere Island, Nunavut) was deposited during the mid-Pliocene warm period. The age estimates correspond to a general maximum in high latitude mean winter season insolation, consistent with the presence of a rich, boreal-type forest. Moreover, we report that these deposits have yielded the first evidence of a High Arctic camel, identified using collagen fingerprinting of a fragmentary fossil limb bone. Camels originated in North America and dispersed to Eurasia via the Bering Isthmus, an ephemeral land bridge linking Alaska and Russia. The results suggest that the evolutionary history of modern camels can be traced back to a lineage of giant camels that was well established in a forested Arctic.

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