A guide to ancient protein studies

Abstract

Palaeoproteomics is an emerging neologism used to describe the application of mass spectrometry-based approaches to the study of ancient proteomes. As with palaeogenomics (the study of ancient DNA), it intersects evolutionary biology, archaeology and anthropology, with applications ranging from the phylogenetic reconstruction of extinct species to the investigation of past human diets and ancient diseases. However, there is no explicit consensus at present regarding standards for data reporting, data validation measures or the use of suitable contamination controls in ancient protein studies. Additionally, in contrast to the ancient DNA community, no consolidated guidelines have been proposed by which researchers, reviewers and editors can evaluate palaeoproteomics data, in part due to the novelty of the field. Here we present a series of precautions and standards for ancient protein research that can be implemented at each stage of analysis, from sample selection to data interpretation. These guidelines are not intended to impose a narrow or rigid list of authentication criteria, but rather to support good practices in the field and to ensure the generation of robust, reproducible results. As the field grows and methodologies change, so too will best practices. It is therefore essential that researchers continue to provide necessary details on how data were generated and authenticated so that the results can be independently and effectively evaluated. We hope that these proposed standards of practice will help to provide a firm foundation for the establishment of palaeoproteomics as a viable and powerful tool for archaeologists, anthropologists and evolutionary biologists.

Analysis of the Preserved Amino Acid Bias in Peptide Profiles of Iron Age Teeth from a Tropical Environment Enable Sexing of Individuals Using Amelogenin MRM

“…Mitigation of contamination through sample collection, handling, and analysis are an essential element of paleoproteomic workflows because the conserved protein homology across species boundaries, as well as the nature and impact of diagenesis contrasting with the biological changes that precede mortem are difficult to establish. Workflows that incorporate controls, use multiple complementary techniques and check‐stops through the project are vital in ascertaining authenticity . Our summarized approach is detailed in Figure , with details and results of all approaches taken to confirm authenticity and control for contamination available in Table S1 and Figures S1–S3, Supporting Information.…”

“…Workflows that incorporate controls, use multiple complementary techniques and check-stops through the project are vital in ascertaining authenticity. [37] Our summarized approach is detailed in Figure 1, with details and results of all approaches taken to confirm authenticity and control for contamination available in Table S1 and Figures S1-S3, Supporting Information.…”

A late Middle Pleistocene Denisovan mandible from the Tibetan Plateau

“…Example MS/MS spectra of phylogenetically informative sequences can be found in SI Figure 4. The database was supplemented with a list of proteins commonly suspected to represent contaminants proteins 58 .…”

Early Pleistocene enamel proteome sequences from Dmanisi resolve Stephanorhinus phylogeny

“…All the sample preparation procedures for palaeoproteomic analysis were conducted in laboratories dedicated to the analysis of ancient DNA and ancient proteins in clean rooms fitted with filtered ventilation and positive pressure, in line with recent recommendations for ancient protein analysis 40 . A mock “extraction blank”, containing no starting material, was prepared, processed and analysed together with each batch of ancient samples.…”