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Deep-time phylogenetic inference by paleoproteomic analysis of dental enamel

Nature Protocols (2024)Cite this article

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Abstract

In temperate and subtropical regions, ancient proteins are reported to survive up to about 2 million years, far beyond the known limits of ancient DNA preservation in the same areas. Accordingly, their amino acid sequences currently represent the only source of genetic information available to pursue phylogenetic inference involving species that went extinct too long ago to be amenable for ancient DNA analysis. Here we present a complete workflow, including sample preparation, mass spectrometric data acquisition and computational analysis, to recover and interpret million-year-old dental enamel protein sequences. During sample preparation, the proteolytic digestion step, usually an integral part of conventional bottom-up proteomics, is omitted to increase the recovery of the randomly degraded peptides spontaneously generated by extensive diagenetic hydrolysis of ancient proteins over geological time. Similarly, we describe other solutions we have adopted to (1) authenticate the endogenous origin of the protein traces we identify, (2) detect and validate amino acid variation in the ancient protein sequences and (3) attempt phylogenetic inference. Sample preparation and data acquisition can be completed in 3–4 working days, while subsequent data analysis usually takes 2–5 days. The workflow described requires basic expertise in ancient biomolecules analysis, mass spectrometry-based proteomics and molecular phylogeny. Finally, we describe the limits of this approach and its potential for the reconstruction of evolutionary relationships in paleontology and paleoanthropology.

Key points

  • Paleoproteomics has shown that it is possible to obtain useful phylogenetic information from dental enamel proteins up to 2 million years old. They are heavily fragmented and chemically modified, making their recovery and analysis challenging.

  • The protocol describes how to (1) extract million-year-old dental enamel protein remains while minimizing contamination, (2) sequence them using high-resolution tandem mass spectrometry and (3) attempt otherwise so far impossible molecular-based phylogenetic inference.

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Fig. 1: Artistic representation of a cross-section of a human tooth.
Fig. 2: Schematic representation of the data analysis process.
Fig. 3: Authentication of the ancient, endogenous origin of the retrieved peptides.
Fig. 4: Validation of ancient protein sequence reconstruction.
Fig. 5: Polymer contamination.

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Data availability

All raw data points underlying Fig. 3, originally published in ref. 12, are accessible through the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD011008.

Code availability

Scripts required for the protein database reconstruction and phylogenetic analysis can be found at the following github repository: https://github.com/johnpatramanis/Nature_Prot_Enamel

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Acknowledgements

E.C., F.W. and J.R.-M. were supported by the VILLUM FONDEN (grant no. 17649). E.C., P.L.R. and J.V.O. were supported by the European Commission through the MSC European Training Network ‘TEMPERA’ (grant agreement no. 722606). E.C., I.P., C.K., R.S.P., P.P.M., F.S.H. and J.V.O. are supported by the European Commission through the MSC European Training Network ‘PUSHH’ (grant agreement no. 861389). E.C., A.J.T., M.M. and J.V.O. were supported by the European Research Council under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 101021361). F.W. has received funding from the European Research Council under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 948365). A.J.T. was supported by the Danish National Research Foundation award PROTEIOS (DNRF128). Work at The Novo Nordisk Foundation Center for Protein Research was funded in part by a generous donation from the Novo Nordisk Foundation (NNF14CC0001). We thank F. Racimo for critical reading of the manuscript and for the valuable comments and feedback.

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Authors and Affiliations

  1. Globe Institute, University of Copenhagen, Copenhagen, Denmark

    Alberto J. Taurozzi, Ioannis Patramanis, Ryan Sinclair Paterson, Palesa P. Madupe, Frido Welker, Meaghan Mackie, Jazmín Ramos-Madrigal & Enrico Cappellini

  2. Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark

    Patrick L. Rüther, Claire Koenig, Florian Simon Harking, Meaghan Mackie & Jesper V. Olsen

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  1. Alberto J. Taurozzi
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Contributions

E.C. devised the sample preparation methodology. J.R.-M. and I.P. respectively devised and further developed the phyloproteomic pipeline. P.L.R. devised the quantitative analysis of low-abundance modifications. R.S.P. and P.P.M. specifically focused on the experimental design of ancient protein authentication and sequence validation. P.L.R., M.M., C.K., F.S.H. and J.V.O. specifically focused on the description of the MS analysis steps. Figures 2 and 5 were designed by C.K. Figure 3 was created by R.S.P., who also reanalyzed the data underlying it. Figure 4 was designed by R.S.P. and C.K. A.J.T. and E.C. wrote the manuscript with contributions from P.L.R., I.P., C.K., R.S.P., P.M., F.S.K., F.W., M.M. and J.R.-M. All authors read and commented on the manuscript.

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Correspondence to Jazmín Ramos-Madrigal or Enrico Cappellini.

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Nature Protocols thanks Katerina Douka, Glendon Parker, Brett Phinney and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Cappellini, E. et al. Nature 574, 103–107 (2019): https://doi.org/10.1038/s41586-019-1555-y

Welker, F. et al. Nature 576, 262–265 (2019): https://doi.org/10.1038/s41586-019-1728-8

Welker, F. et al. Nature 580, 235–238 (2020): https://doi.org/10.1038/s41586-020-2153-8

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Taurozzi, A.J., Rüther, P.L., Patramanis, I. et al. Deep-time phylogenetic inference by paleoproteomic analysis of dental enamel. Nat Protoc (2024). https://doi.org/10.1038/s41596-024-00975-3

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