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.

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Ancient biomolecule survival remains poorly understood, even with great advancements in 'omics' technologies, both in genomics and proteomics. This study investigates the survival of ancient DNA in relation to that of proteins, taking into account proteome complexity and the relative protein abundances to improve our understanding of survival mechanisms. The results show that although protein abundance is not necessarily directly related to aDNA survival, proteome complexity appears to be.

Insects preserved in copal, the sub-fossilized resin precursor of amber, have potential value in molecular ecological studies of recently-extinct species and of extant species that have never been collected as living specimens. The objective of the work reported in this paper was therefore to determine if ancient DNA is present in insects preserved in copal. We prepared DNA libraries from two stingless bees (Apidae: Meliponini: Trigonisca ameliae) preserved in ‘Anthropocene’ Colombian copal, dated to ‘post-Bomb’ and 10,612±62 cal yr BP, respectively, and obtained sequence reads using the GS Junior 454 System. Read numbers were low, but were significantly higher for DNA extracts prepared from crushed insects compared with extracts obtained by a non-destructive method. The younger specimen yielded sequence reads up to 535 nucleotides in length, but searches of these sequences against the nucleotide database revealed very few significant matches. None of these hits was to stingless bees though one read of 97 nucleotides aligned with two non-contiguous segments of the mitochondrial cytochrome oxidase subunit I gene of the East Asia bumblebee Bombus hypocrita. The most significant hit was for 452 nucleotides of a 470-nucleotide read that aligned with part of the genome of the root-nodulating bacterium Bradyrhizobium japonicum. The other significant hits were to proteobacteria and an actinomycete. Searches directed specifically at Apidae nucleotide sequences only gave short and insignificant alignments. All of the reads from the older specimen appeared to be artefacts. We were therefore unable to obtain any convincing evidence for the preservation of ancient DNA in either of the two copal inclusions that we studied, and conclude that DNA is not preserved in this type of material. Our results raise further doubts about claims of DNA extraction from fossil insects in amber, many millions of years older than copal.

Isotope analyses on "collagen" extracted from ancient bone have been routinely used for dietary and chronological inferences worldwide for decades. These methods involve the decalcification of biomineralized tissues with acid, often followed by processes to remove exogenous contaminants, and then gelatinization of what is often described as the "collagen" fraction. However, little is known about the relative content of collagen to the many other noncollagenous proteins (NCPs) potentially present. Some of these NCPs have great longevity in ancient bone, and some, for example, fetuin-A, are useful for obtaining better taxonomic information than collagen. This study uses Orbitrap Elite LC-MS/MS to characterize the proteomes of the acid-soluble and base-soluble fractions, which are usually discarded, and the gelatinized "collagen" fraction obtained from both stable isotope and radiocarbon methods applied to several ancient bovine bones. The results showed that all fractions tested contain numerous NCPs, but the base-soluble fraction for both methods contains the greatest number of NCPs with the highest relative abundances. This study confirms that not only do the waste fractions obtained from the "collagen" extraction procedure of stable isotope or radiocarbon dating methods yield a plentiful resource of NCPs that is currently being overlooked but that they also provide proteomes as complex as those obtained from standard proteomics methods.