Paleoproteomics: An Introduction to the Analysis of Ancient Proteins by Soft Ionisation Mass Spectrometry

  • Michael BuckleyEmail author
Part of the Population Genomics book series (POGE)


The field of proteomic research, analogous to genomic research, has only recently witnessed a rapid increase in its application to the study of ancient materials. Bone has been the most commonly used archaeological and paleontological resource for recovering biological information. This has most frequently been for ancient genomic analysis, but some of the potential advantages of proteomics lie in its ability to discriminate between sources of the molecules, rather than the particular species or individual. However, proteomes could be considered more dynamic, offering different types of information than otherwise available through DNA analyses. Proteins are also considered to survive for much longer periods of time than substantial lengths of DNA and therefore the development of proteomics allows for the possibility of being able to recover information much further back in time than previously thought possible. In this chapter, the progress of this area called ‘paleoproteomics’ is reviewed, highlighting some of its greatest achievements but also some of the current limitations in the field across proteins from a range of different materials.


Ancient proteins Collagen Extinct taxa Paleoproteomics Phylogenetics Soft ionisation mass spectrometry 



The author acknowledges the support of the Royal Society in the form of a University Research Fellowship.


  1. Abelson PH. Paleobiochemistry. Sci Am. 1956;195(1):83–92.Google Scholar
  2. Abelson PH. Geochemistry of organic substances. In: Abelson PH, editor. Researches in geochemistry, vol. 1. Chichester: Wiley; 1959. p. 79–103.Google Scholar
  3. Agnolin FL, Chimento NR. Afrotherian affinities for endemic South American “ungulates”. Mamm Biol. 2011;76(2):101–8.Google Scholar
  4. Anderson NL, Anderson NG. Proteome and proteomics: new technologies, new concepts, and new words. Electrophoresis. 1998;19(11):1853–61.PubMedGoogle Scholar
  5. Armstrong WG, Halstead LB, Reed FB, Wood L. Fossil proteins in vertebrate calcified tissues. Philos Trans R Soc Lond B Biol Sci. 1983;B301(1106):301–43.Google Scholar
  6. Asara JM, Schweitzer MH, Freimark LM, Phillips M, Cantley LC. Protein sequences from mastodon and Tyrannosaurus rex revealed by mass spectrometry. Science. 2007;316(5822):280–5.Google Scholar
  7. Bada J, Wang X, Hamilton H. Preservation of key biomolecules in the fossil record: current knowledge and future challenges. Philos Trans R Soc Lond B Biol Sci. 1999;354(1379):77–86.PubMedPubMedCentralGoogle Scholar
  8. Bailey AJ. Molecular mechanisms of ageing in connective tissues. Mech Ageing Dev. 2001;122(7):735–55.PubMedGoogle Scholar
  9. Boskey AL, Posner AS. Bone structure, composition, and mineralization. Orthop Clin North Am. 1984;15(4):597–612.PubMedGoogle Scholar
  10. Brandt LØ, Schmidt AL, Mannering U, Sarret M, Kelstrup CD, Olsen JV, et al. Species identification of archaeological skin objects from Danish bogs: comparison between mass spectrometry-based peptide sequencing and microscopy-based methods. PLoS One. 2014;9(9):e106875.PubMedPubMedCentralGoogle Scholar
  11. Breker M, Schuldiner M. The emergence of proteome-wide technologies: systematic analysis of proteins comes of age. Nat Rev Mol Cell Biol. 2014;15(7):453–64.PubMedGoogle Scholar
  12. Brown TA. How ancient DNA may help in understanding the origin and spread of agriculture. Philos Trans R Soc Lond B Biol Sci. 1999;354(1379):89–98.PubMedCentralGoogle Scholar
  13. Buckley M. A molecular phylogeny of Plesiorycteropus reassigns the extinct mammalian order ‘Bibymalagasia’. PLoS One. 2013;8(3):e59614.PubMedPubMedCentralGoogle Scholar
  14. Buckley M. Ancient collagen reveals evolutionary history of the endemic South American ‘ungulates’. Proc Biol Sci. 2015;282(1806):20142671.PubMedPubMedCentralGoogle Scholar
  15. Buckley M, Wadsworth C. Proteome degradation in ancient bone: diagenesis and phylogenetic potential. Palaeogeogr Palaeoclimatol Palaeoecol. 2014;416:69–79.Google Scholar
  16. Buckley M, Anderung C, Penkman K, Raney BJ, Gotherstrom A, Thomas-Oates J, et al. Comparing the survival of osteocalcin and mtDNA in archaeological bone from four European sites. J Archaeol Sci. 2008a;35(6):1756–64.Google Scholar
  17. Buckley M, Walker A, Ho SY, Yang Y, Smith C, Ashton P, et al. Comment on “Protein sequences from mastodon and Tyrannosaurus rex revealed by mass spectrometry”. Science. 2008b;4(319):33c.Google Scholar
  18. Buckley M, Collins M, Thomas-Oates J, Wilson JC. Species identification by analysis of bone collagen using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Rapid Commun Mass Spectrom. 2009;23(23):3843–54.PubMedGoogle Scholar
  19. Buckley M, Larkin N, Collins M. Mammoth and Mastodon collagen sequences; survival and utility. Geochim Cosmochim Acta. 2011;75(7):2007–16.Google Scholar
  20. Buckley M, Melton ND, Montgomery J. Proteomics analysis of ancient food vessel stitching reveals >4000-year-old milk protein. Rapid Commun Mass Spectrom. 2013;27(4):531–8.PubMedGoogle Scholar
  21. Buckley M, Fraser S, Herman J, Melton N, Mulville J, Pálsdóttir A. Species identification of archaeological marine mammals using collagen fingerprinting. J Archaeol Sci. 2014;41:631–41.Google Scholar
  22. Buckley M, Gu M, Shameer S, Patel S, Chamberlain A. High-throughput collagen fingerprinting of intact microfaunal remains; a low-cost method for distinguishing between murine rodent bones. Rapid Commun Mass Spectrom. 2016;30:1–8.Google Scholar
  23. Buckley M, Harvey V, Chamberlain A. Species identification and decay assessment of Late Pleistocene fragmentary vertebrate remains from Pin Hole Cave (Creswell Crags, UK) using collagen fingerprinting. Boreas. 2017; Scholar
  24. Cappellini E, Jensen LJ, Szklarczyk D, Ginolhac A, da Fonseca RA, Stafford TW Jr, et al. Proteomic analysis of a pleistocene mammoth femur reveals more than one hundred ancient bone proteins. J Proteome Res. 2011;11(2):917–26.PubMedGoogle Scholar
  25. Colombo G, Fanti P, Yao CH, Malluche HH. Isolation and complete amino acid sequence of osteocalcin from canine bone. J Bone Miner Res. 1993;8(6):733–43.PubMedGoogle Scholar
  26. Curry GB. Amino acids and proteins from fossils. In: Eglinton G, Curry GB, editors. Molecular evolution and the fossil record. Knoxville, TN: Paleontological Society; 1988. p. 20–33.Google Scholar
  27. Delmas PD, Tracy RP, Riggs BL, Mann K. Identification of the non collagenous proteins of bovine bone by two-dimensional gel electrophoresis. Calcif Tissue Int. 1984;36:308–16.PubMedGoogle Scholar
  28. Demarchi B, Hall S, Roncal-Herrero T, Freeman CL, Woolley J, Crisp MK, et al. Protein sequences bound to mineral surfaces persist into deep time. Elife. 2016;5:e17092.PubMedPubMedCentralGoogle Scholar
  29. Di Donato A, Filippone E, Ercolano MR, Frusciante L. Genome sequencing of ancient plant remains: findings, uses and potential applications for the study and improvement of modern crops. Front Plant Sci. 2018;9:441.PubMedPubMedCentralGoogle Scholar
  30. Doorn NL, Wilson J, Hollund H, Soressi M, Collins MJ. Site-specific deamidation of glutamine: a new marker of bone collagen deterioration. Rapid Commun Mass Spectrom. 2012;26(19):2319–27.PubMedGoogle Scholar
  31. Edman P. Mechanism of the phenyl isothiocyanate degradation of peptides. Nature. 1956;177(4510):667–8.Google Scholar
  32. Frazao C, Simes DC, Coelho R, Alves D, Williamson MK, Price PA, et al. Structural evidence of a fourth Gla residue in fish osteocalcin: biological implications. Biochemistry. 2005;44(4):1234–42.PubMedGoogle Scholar
  33. Fulton TL, Strobeck C. Multiple markers and multiple individuals refine true seal phylogeny and bring molecules and morphology back in line. Proc R Soc Lond B Biol Sci. 2010;277(1684):1065–70.Google Scholar
  34. Gendreau MA, Krishnaswamy S, Mann KG. The interaction of bone Gla protein (osteocalcin) with phospholipid vesicles. J Biol Chem. 1989;264(12):6972–8.PubMedGoogle Scholar
  35. Glowacki J, Rey C, Glimcher MJ, Cox KA, Lian J. A role for osteocalcin in osteoclast differentiation. J Cell Biochem. 1991;45(3):292–302.PubMedGoogle Scholar
  36. Hassanin A, Delsuc F, Ropiquet A, Hammer C, Jansen van Vuuren B, Matthee C, et al. Pattern and timing of diversification of Cetartiodactyla (Mammalia, Laurasiatheria), as revealed by a comprehensive analysis of mitochondrial genomes. C R Biol. 2012;335(1):32–50.PubMedGoogle Scholar
  37. Hauschka PV. Osteocalcin: the vitamin-K dependent Ca-binding protein of bone matrix. Haemostasis. 1986;16:258–72.PubMedGoogle Scholar
  38. Hauschka PV, Carr SA. Calcium-dependant a-helical structure in osteocalcin. Biochemistry. 1982;21:2538–47.PubMedGoogle Scholar
  39. Hauschka PV, Wians FH. Osteocalcin-hydroxyapatite interaction in the extracellular organic matrix of bone. Anat Rec. 1989;224:180–8.PubMedGoogle Scholar
  40. Hauschka PV, Lian JB, Cole DE, Gundberg CM. Osteocalcin and matrix Gla protein: vitamin K-dependent proteins in bone. Physiol Rev. 1989;69(3):990–1047.PubMedGoogle Scholar
  41. Hedges SB, Schweitzer MH. Detecting dinosaur DNA. Science. 1995;268(5214):1191–2.PubMedGoogle Scholar
  42. Hollemeyer K, Altmeyer W, Heinzle E, Pitra C. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry combined with multidimensional scaling, binary hierarchical cluster tree and selected diagnostic masses improves species identification of Neolithic keratin sequences from furs of the Tyrolean Iceman Oetzi. Rapid Commun Mass Spectrom. 2012;26(16):1735–45.PubMedGoogle Scholar
  43. Hulmes GM. The collagen superfamily – diverse structures and assemblies. Essays Biochem. 1992;27:49–67.PubMedGoogle Scholar
  44. Huq N, Tseng A, Chapman G. Partial amino acid sequence of osteocalcin from an extinct species of ratite bird. Biochem Int. 1989;21(3):491–6.Google Scholar
  45. Huq NL, Tseng A, Chapman GE. Partial amino acid sequence of osteocalcin from an extinct species of ratite bird. Biochem Int. 1990;21:491–6.PubMedGoogle Scholar
  46. James P. Protein identification in the post-genome era: the rapid rise of proteomics. Q Rev Biophys. 1997;30(04):279–331.PubMedGoogle Scholar
  47. Jiang X, Ye M, Jiang X, Liu G, Feng S, Cui L, et al. Method development of efficient protein extraction in bone tissue for proteome analysis. J Proteome Res. 2007;6(6):2287–94.PubMedGoogle Scholar
  48. Jones JD, Vallentyne JR. Biogeochemistry of organic matter. Geochim Cosmochim Acta. 1960;21:1–34.Google Scholar
  49. Kadler KE, Holmes DF, Trotter JA, Chapman J. Collagen fibril formation. Biochem J. 1996;316:1–11.PubMedPubMedCentralGoogle Scholar
  50. Karas M, Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem. 1988;60(20):2299–301.PubMedPubMedCentralGoogle Scholar
  51. Kaye TG, Gaugler G, Sawlowicz Z. Dinosaurian soft tissues interpreted as bacterial biofilms. PLoS One. 2008;3(7):e2808.PubMedPubMedCentralGoogle Scholar
  52. Lander ES. The new genomics: global views of biology. Science. 1996;274(5287):536.PubMedGoogle Scholar
  53. Lendaro E, Ippoliti R, Bellelli A, Brunori M, Zito R, Citro G, et al. On the problem of immunological detection of antigens in skeletal remains. Am J Phys Anthropol. 1991;86(3):429–32.PubMedGoogle Scholar
  54. Liggett WH Jr, Lian JB, Greenberger JS, Glowacki J. Osteocalcin promotes differentiation of osteoclast progenitors from murine long-term bone marrow cultures. J Cell Biochem. 1994;55(2):190–9.PubMedGoogle Scholar
  55. Lockwood WW, Chari R, Chi B, Lam WL. Recent advances in array comparative genomic hybridization technologies and their applications in human genetics. Eur J Hum Genet. 2006;14(2):139–48.PubMedGoogle Scholar
  56. Logan G, Collins M, Eglinton G. Preservation of organic biomolecules. In: Allison PA, Briggs DEG, editors. Taphonomy releasing the data locked in the fossil record, vol. 9. New York: Plenum; 1991. p. 1–24.Google Scholar
  57. Lowenstein JM, Ryder OA. Immunological systematics of the extinct quagga (Equidae). Experientia. 1985;41(9):1192–3.PubMedGoogle Scholar
  58. MacPhee RD. Morphology, adaptations, and relationships of Plesiorycteropus: and a diagnosis of a new order of eutherian mammals. Bulletin of the AMNH; no. 220. 1994.Google Scholar
  59. Malone JD. Recruitment of osteoclast precursors by purified bone matrix constituents. J Cell Biol. 1982;92(1):227–30.PubMedGoogle Scholar
  60. Mardis ER. Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet. 2008;9:387–402.PubMedGoogle Scholar
  61. Matrix Science. 2016. Accessed 28 Apr 2018.
  62. Millard A. Deterioration of bone. In: Pollard AM, Brothwell D, editors. Handbook of archaeological sciences. New York: Wiley; 2001.Google Scholar
  63. Nelsestuen GL, Zytkovicz TH, Howard JB. The mode of action of vitamin K identification of caboxyglutamic acid as a component of prothrombin. J Biol Chem. 1974;249(19):6347–50.PubMedGoogle Scholar
  64. Nielsen-Marsh C. Biomolecules in fossil remains-multidisciplinary approach to endurance. Biochemist. 2002;24(3):12–4.Google Scholar
  65. Nielsen-Marsh CM, Ostrom PH, Gandhi H, Shapiro B, Cooper A, Hauschka PV, et al. Exceptional preservation of bison bones >55 ka as demonstrated by protein and DNA sequences. Geology. 2002;30(12):1099–102.Google Scholar
  66. Nogami HMD, Oohira A, Ogasawara NMD. Levels of creatine kinase activity in cartilage of tubular and nontubular bone in relation to pathogenesis of achondroplasia. Clin Orthop Relat Res. 1987;(219):308–12.Google Scholar
  67. Nomura K, Yonezawa T, Mano S, Kawakami S, Shedlock AM, Hasegawa M, et al. Domestication process of the goat revealed by an analysis of the nearly complete mitochondrial protein-encoding genes. PLoS One. 2013;8(8):e67775.PubMedPubMedCentralGoogle Scholar
  68. Ostrom PH, Schall M, Gandhi H, Shen TL, Hauschka PV, Strahler JR, et al. New strategies for characterizing ancient proteins using matrix-assisted laser desorption ionization mass spectrometry. Geochim Cosmochim Acta. 2000;64(6):1043–50.Google Scholar
  69. Ostrom PH, Gandhi H, Strahler JR, Walker AK, Andrews PC, Leykam J, et al. Unraveling the sequence and structure of the protein osteocalcin from a 42 ka fossil horse. Geochim Cosmochim Acta. 2006;70(8):2034–44.Google Scholar
  70. Pääbo S, Poinar H, Serre D, Jaenicke-Després V, Hebler J, Rohland N, Kuch M, Krause J, Vigilant L, Hofreiter M. Genetic analyses from ancient DNA. Annu Rev Genet. 2004;38:645–79.Google Scholar
  71. Prager EM, Wilson AC, Lowenstein JM, Sarich VM. Mammoth albumin. Science. 1980;209:287–9.PubMedGoogle Scholar
  72. Price PA, Williamson MK. Primary structure of bovine matrix Gla protein, a new vitamin K-dependent bone protein. J Biol Chem. 1985;260(28):14971–5.PubMedGoogle Scholar
  73. Procopio N, Chamberlain AT, Buckley M. Intra- and interskeletal proteome variations in fresh and buried bones. J Proteome Res. 2017;16(5):2016–29.PubMedGoogle Scholar
  74. Robbins LL, Muyzer G, Brew K. Macromolecules form living and fossil biominerals; Implications for the establishment of molecular phyolgenies. In: Engle MH, Macko SA, editors. Organic geochemistry. New York: Plenum; 1993. p. 799–816.Google Scholar
  75. Roepstorff P, Fohlman J. Letter to the editors. Biol Mass Spectrom. 1984;11(11):601.Google Scholar
  76. Rybczynski N, Gosse JC, Harington CR, Wogelius RA, Hidy AJ, Buckley M. Mid-Pliocene warm-period deposits in the High Arctic yield insight into camel evolution. Nat Commun. 2013;4:1550.PubMedPubMedCentralGoogle Scholar
  77. Sawafuji R, Cappellini E, Nagaoka T, Fotakis AK, Jersie-Christensen RR, Olsen JV, et al. Proteomic profiling of archaeological human bone. R Soc Open Sci. 2017;4(6):161004.PubMedPubMedCentralGoogle Scholar
  78. Schreiweis MA, Butler JP, Kulkarni NH, Knierman MD, Higgs RE, Halladay DL, et al. A proteomic analysis of adult rat bone reveals the presence of cartilage/chondrocyte markers. J Cell Biochem. 2007;101:466–76.PubMedGoogle Scholar
  79. Schroeter ER, DeHart CJ, Cleland TP, Zheng W, Thomas PM, Kelleher NL, Bern M, Schweitzer MH. Expansion for the Brachylophosaurus canadensis collagen I sequence and additional evidence of the preservation of cretaceous protein. J Proteome Res. 2017;16(2):920–32.PubMedPubMedCentralGoogle Scholar
  80. Schwalbe RA, Ryan J, Stern DM, Kisiel W, Dahlback B, Nelsestuen GL. Protein structural requirements and properties of membrane binding by gamma-carboxyglutamic acid-containing plasma proteins and peptides. J Biol Chem. 1989;264(34):20288–96.PubMedGoogle Scholar
  81. Schweitzer MH, Zheng W, Organ CL, Avci R, Suo Z, Freimark LM, Lebleu VS, Duncan MB, Vander Heiden MG, Neveu JM, Lane WS. Biomolecular characterization and protein sequences of the Campanian hadrosaur B. canadensis. Science. 2009;324(5927):626–31.Google Scholar
  82. Schweitzer MH, Zheng W, Cleland TP, Bern M. Molecular analyses of dinosaur osteocytes support the presence of endogenous molecules. Bone. 2013;52(1):414–23.Google Scholar
  83. Schweitzer MH, Moyer AE, Zheng W. Testing the hypothesis of biofilm as a source for soft tissue and cell-like structures preserved in dinosaur bone. PLoS One. 2016;11(2):e0150238.PubMedPubMedCentralGoogle Scholar
  84. Solazzo C, Fitzhugh WW, Rolando C, Tokarski C. Identification of protein remains in archaeological potsherds by proteomics. Anal Chem. 2008;80(12):4590–7.PubMedGoogle Scholar
  85. Solazzo C, Courel B, Connan J, Van Dongen BE, Barden H, Penkman K, Taylor S, Demarchi B, Adam P, Schaeffer P, Nissenbaum A. Identification of the earliest collagen-and plant-based coatings from Neolithic artefacts (Nahal Hemar cave, Israel). Sci Rep. 2016;6:31053.PubMedPubMedCentralGoogle Scholar
  86. Stewart NA, Gerlach RF, Gowland RL, Gron KJ, Montgomery J. Sex determination of human remains from peptides in tooth enamel. Proc Natl Acad Sci. 2017;114(52):13649–54.PubMedGoogle Scholar
  87. Tanaka K, Waki H, Ido Y, Akita S, Yoshida Y, Yoshida T. Protein and polymer analyses up to m/z 100,000 by laser ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom. 1988;2(8):151–3.Google Scholar
  88. Tokarski C, Martin E, Rolando C, Cren-Olivé C. Identification of proteins in renaissance paintings by proteomics. Anal Chem. 2006;78(5):1494–502.PubMedGoogle Scholar
  89. Triffitt JT, Gebauer U, Ashton BA, Owen ME, Reynolds JJ. Origin of plasma alpha2-HS-glycoprotein and its accumulation in bone. Nature. 1976;262(5565):226–7.PubMedGoogle Scholar
  90. Vuorio E, de Crombrugghe B. The family of collagen genes. Annu Rev Biochem. 1998;59:837–72.Google Scholar
  91. Wadsworth C, Buckley M. Proteome degradation in fossils: investigating the longevity of protein survival in ancient bone. Rapid Commun Mass Spectrom. 2014;28(6):605–15.PubMedPubMedCentralGoogle Scholar
  92. Wadsworth C, Procopio N, Anderung C, Carretero JM, Iriarte E, Valdiosera C, Elburg R, Penkman K, Buckley M. Comparing ancient DNA survival and proteome content in 69 archaeological cattle tooth and bone samples from multiple European sites. J Proteome. 2017;158:1–8.Google Scholar
  93. Wallace JM, Rajachar RM, Chen XD, Shi S, Allen MR, Bloomfield SA, et al. The mechanical phenotype of biglycan-deficient mice is bone-and gender-specific. Bone. 2006;39(1):106–16.PubMedGoogle Scholar
  94. Weiner S, Lowenstam HA, Hood L. Characterisation of 80-million-year-old mollusk shell proteins. Proc Natl Acad Sci U S A. 1976;73:2541–5.PubMedPubMedCentralGoogle Scholar
  95. Welker F, Collins MJ, Thomas JA, Wadsley M, Brace S, Cappellini E, et al. Ancient proteins resolve the evolutionary history of Darwin’s South American ungulates. Nature. 2015;522(7554):81–4.Google Scholar
  96. Wellner D, Panneerselvam C, Horecker B. Sequencing of peptides and proteins with blocked N-terminal amino acids: N-acetylserine or N-acetylthreonine. Proc Natl Acad Sci. 1990;87(5):1947–9.PubMedGoogle Scholar
  97. Westbury M, Baleka S, Barlow A, Hartmann S, Paijmans JL, Kramarz A, et al. A mitogenomic timetree for Darwin’s enigmatic South American mammal Macrauchenia patachonica. Nat Commun. 2017;8:15951.PubMedPubMedCentralGoogle Scholar
  98. Williams RAD, Elliot JC. Basic and applied dental biochemistry. Edinburgh: Churchill Livingstone; 1989.Google Scholar
  99. Wilson J, van Doorn NL, Collins MJ. Assessing the extent of bone degradation using glutamine deamidation in collagen. Anal Chem. 2012;84(21):9041–8.PubMedGoogle Scholar
  100. Woodward SR, Weyand NJ, Bunnell M. DNA sequence from Cretaceous period bone fragments. Science. 1994;266(5188):1229–32.Google Scholar
  101. Yamashita M, Fenn JB. Electrospray ion source. Another variation on the free-jet theme. J Phys Chem. 1984;88(20):4451–9.Google Scholar
  102. Zang X, van Heemst JDH, Dria KJ, Hatcher PG. Encapsulation of protein in humic acid from a histosol as an explanation for the occurrence of organic nitrogen in soil and sediment. Org Geochem. 2000;31(7–8):679–95.Google Scholar
  103. Zhu W, Robey PG, Boskey AL. The regulatory role of matrix proteins in mineralisation of bone. In: Feldman D, Nelson D, Rosen CJ, editors. Osteoporosis. New York: Elsevier; 2007. p. 191–240.Google Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.School of Earth and Environmental Sciences, Manchester Institute of BiotechnologyUniversity of ManchesterManchesterUK

Personalised recommendations