Paleoproteomics

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Paleoproteomics is a relatively young and rapidly growing field of molecular science in which proteomics-based sequencing technology is used to resolve species identification and evolutionary relationships of extinct taxa. While complementary to paleogenomics in application, the study of ancient proteins has the potential to reveal older, more complete phylogenies due to the relative stability of amino acids in proteins as compared to the nucleic acids of DNA.^[1] Ancient protein studies can further reveal types and sources of recovered tissues,^[2] as well as the developmental stages of fossilized specimens.^[3] Paleoproteomics can also be extended to archaeological materials such as textiles, animal skins, food remains, and pottery.

Background[]

Philip Abelson first characterized the findings of ancient amino acid residues from fossilized materials in 1955, proposing that the peptide bonds of proteins might persist for millions of years.^[4] These initial discoveries were limited by available methodologies, and so protein sequencing remained an elusive idea for almost four decades. In 2000, mass spectrometry (MS) revealed the presence of osteocalcin in ancient bone samples^[5] and ignited a renewed interest in protein’s potential as a tool for molecular paleontology.^[6]

The development of higher resolution instruments further increased the efficiency and depth of ancient protein recovery. In 2012, the first extended fossil bone proteome from a Pleistocene mammoth femur was confidently retrieved and identified,^[7] strengthening the future of paleoproteomics research.

Paleoproteomes[]

Collagen Type I[]

The analysis of ancient bone proteomes has primarily focused on the identification of collagen type I (COL1), the dominant protein found in mineralized tissues.^[1][8][9] Collagen is highly conserved across species^[10] and comprises about 90% of organic bone compounds. Fibrillar collagens, of which COL1 is categorized, are thought to have evolved from a common metazoan ancestor,^[11] thus contributing to their abundance and importance in the fossil record.  

Collagen has also been found to survive much longer than other non-collagenous proteins in fossilized specimens,^[12] and the protein remains intact beyond the degradation of ancient DNA (aDNA).^[1][9] Its tightly coiled triple-helical structure (consisting of two genetically identical alpha-1 chains and a third genetically distinct alpha-2 chain)^[10] and hydrophobic composition^[1] also make this protein an excellent candidate for survival, even in temperate and humid climates that support the rapid break down of organic molecules.^[10]

The taxonomic resolution of collagen has been thoroughly investigated, and it is known that amino acid substitutions can be resolved to the genus level in most medium and large mammals.^[1] Species-level identification is also possible, even in small mammal remains from high thermal climates.^[13] It is for these reasons that COL1 remains a key protein in paleoproteomics and phylogenetic investigations.

Non-collagenous proteins[]

The remaining 10% of organic bone molecules are non-collagenous proteins (NCPs). The most abundant NCP, osteocalcin, is a bone and dentin protein involved in bone assembly, often used as a marker for the bone formation process. Preserved osteocalcin was first detected via mass spectrometry (MALDI-MS) in 10,000 year-old bison bone and a 53,000-year-old walrus bone,^[5] revealing phylogenetic reconstruction potential beyond the temporal limits of aDNA.

More advanced proteomic techniques have enabled the investigation of additional NCPs present in the bone extracellular matrix. Though type I collagen is the longest lived protein identified in fossilized bone specimens, the identification and sequencing of NCPs may allow for a greater taxonomic resolution than collagen-based methods.^[8][12]

Other proteins[]

Proteomic analysis has also been applied to other fossilized and ancient materials. The examination of damaged artifacts through the sequencing of their keratin peptides^[14] has allowed researchers to discriminate between horn and hoof remains of important species used at archeological sites. The keratin of textiles and animal skins worn by Ötzi, the Iceman, were also identified using peptide mass fingerprinting (PMF) from the ancient samples and from reference species.^[15] Immune response proteins have illuminated the presence of infections and diseases in multiple studies of mummified human remains.^[8] Additionally, the identification of egg proteins, caseins, whey globulins, and other proteinaceous materials used as binders in the paint of historical artworks has allowed for a better understanding of proper conservation methods.^[8]

Sequencing methods[]

Analysis of a fossil sample begins with demineralization of the bone/tooth mineral matrix. Trypsin is commonly used to digest the protein residues into peptides which can then be purified and analyzed.^[9]

Peptide mass fingerprinting[]

Peptide mass fingerprinting (PMF) is an analytical technique that can be applied to the digested protein mixture. The masses of the unknown peptides can be detected with a mass spectrometer like MALDI (matrix-assisted laser desorption/ionization) or ESI (electrospray ionization), combined with a mass analyzer,^[1] and then compared to masses of peptides that are predicted to derive from known proteins.^[9]  

Liquid Chromatography-Tandem Mass Spectrometry[]

The inclusion of liquid chromatography (LC) solvents can enhance peptide electrospray ionization.^[9] When combined with mass spectrometry, this allows for a much greater number of peptide ions to be analyzed with improved fragment spectra.^[1] Liquid chromatography-mass spectrometry LC-MS can then be performed for peptide mass fingerprinting; however, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is most often used in the case of ancient proteome analysis due to the nature of these complex samples.

De novo sequencing[]

At the present time, protein databases remain limited to particular taxa, so novel protein sequences that differ greatly from those available will not be identified via the protein search engines.^[1] Manual interpretation through de novo sequencing remains a viable solution until these databases become more robust, and this technique will allow for identification of amino acid substitutions not previously reported.^[16]

A hybrid solution of error-tolerant search algorithms that use protein sequence databases while allowing amino acid substitutions may similarly enable the identification of novel single amino acid polymorphisms (SAPs).^[9]

Challenges[]

Dinosaur collagen[]

A 2007 paleontology study reported the alleged discovery of endogenous collagen peptides in 68 mya Tyrannosaurus rex fossils.^[17] This claim purported survival beyond experimental decay rates,^[6] leading to controversy in the emerging field. The same team again reported finding similar collagen peptide sequence matches in 2009 from 80 mya hadrosaur fossils belonging to Brachylophosaurus canadensis.^[18]

Subsequent studies have reanalyzed the original T. rex sequence data to infer that the sample was predominantly laboratory contaminants, soil bacteria, and bird-like hemoglobin and collagen;^[19] the former protein is typically only seen in relatively recent samples.^[7][12] Another exceptionally preserved hadrosaur from the Hell Creek Formation (USA), yielded none of the previous findings despite extensive testing, and only the presence of protein breakdown products were detected.^[20]

Further experimentation demonstrated that contamination from other specimens present in the T. rex lab cannot be ruled out. Every peptide that was considered unique to both dinosaurs in the 2009 study could be matched to modern ostrich with much greater confidence than could be placed on their own, unique identifications.^[21]

While there have been several methods described to support the authenticity of paleoproteomics, including immunological or amino acid composition and racemization data, both of these approaches have limitations and are known to yield false-positive reactions in fossils.^[22] Great care must be taken to rule out contamination by determining whether sequences differ from those of all extant taxa present in the laboratory environments.^[21] Deamidation has also been proposed as an effective method for distinguishing between endogenous and contaminating NCPs, when extraction protocols may permit for this evaluation.^[9]

Future perspectives[]

Paleoproteomics is still a young field, with most complex proteomes only being discovered in the last decade. Current proteomic methods greatly suffer from the fact that it is not a true form of sequencing, relying on probability-matching against expected results. While several MS methods are being employed to increase the robustness of retrievable data, these techniques also increase the sensitivities to contamination.^[1]

References[]