Shells 

A useful rule of thumb is to keep these biominerals intact. For example, it is best to sample an articulated eggshell in its entirety in the field. 

It is also useful to minimise handling. There is no need to wash, pre-treat or glue these biominerals in the field, because modern and proteinaceous contaminants may be introduced. Use latex-free gloves to avoid touching samples with exposed skin, and sterilised containers to store plus transport samples. Bleach is corrosive to plastic, so use 70% ethanol (causing cell lysis and protein denaturation) or diluted (c. 3%) hydrogen peroxide (a strong oxidising agent) to rinse plastic containers, and leave them dry in the open air.   

If samples are targeted for multi-omics, wrap the samples using combusted tin foil (dry heated in a muffled furnace > c. 135 °C) before putting them into any plastic bags or containers. 

Please keep samples away from any potential contaminants, including field sandwiches, woollen jumpers, leather jackets, conservation glues, or anything that may contain modern proteins.

For long-term storage, it is best to store intact and articulated biominerals in a cold room (≤ 4°C). If not possible, store protein samples in a stable environment such as safelock, PCR-free, low-binding eppendorf tubes, or sealed and sterilised plastic containers. For multi-omics samples, wrapping them in combusted tin foil is preferred since plastic is a potential lipid contaminant. 

These biominerals are protein-rich and generally targeted for LC-MS/MS (liquid chromatography-tandem mass spectrometry), characterising amino acid sequences and identifying protein groups. 

For eggshells, they can be pre-treated with bleach prior analysis to remove any intercrystalline proteins. 

Since LC-MS/MS is costly and laborious, it is useful to conduct a pilot study to screen these biominerals and evaluate their protein preservation. Gas chromatography-mass spectrometry (GC-MS) or high performance liquid chromatography (HPLC) may be used to detect preserved amino acids and measure amino acid racemisation (D/L ratios). MALDI-ToF (matrix-assisted laser desorption/ionisation-time of flight) is another useful tool if peptide markers and reference databases are well-established.  

It is preferable to sub-sample and extract these biominerals in a separate and dedicated space to minimise any potential cross contamination. Extraction blanks should be included to monitor background noise. Sample size varies and published protocols are available for molluscan shells and eggshells.

While various biomolecules have been recovered from these biominerals, there are three major analytical challenges. 

1. Contamination issues

Ancient biomolecules are subject to a variety of contaminants that may be introduced during excavation, transportation, storage, extraction and analysis. Time-dependent damage patterns, especially cytosine deamination (C-to-T substitutions), have been widely used in aDNA authentication. In contrast, it is challenging to authenticate ancient proteins using post translational modifications (PTMs). There is also wide variability in deamidation rates.  

2. Extraction difficulties 

Mineralised matrices provide protection and stability into deep time. However, it is equally difficult to liberate mineral-bound organic residues from these biominerals without damage. Extraction procedures still need to be tested and optimised to maximise the recovery of proteins, DNA, metabolites and lipids. 

3. Identification problems 

High-resolution, taxa- and tissue-specific identification is dependent on the coverage of modern protein and genomic databases. These are usually predominated by model organisms such as E.coli and yeasts. Archaeological proteins and DNA sequences are often uncharacterised and remain unknown. 

Demarchi, B. et al. (2016). Protein sequences bound to mineral surfaces persist into deep time. eLife, 5. [Online]. Available at: doi:10.7554/eLife.17092.

Demarchi, B. et al. (2022). Ancient proteins resolve controversy over the identity of Genyornis eggshell. Proceedings of the National Academy of Sciences of the United States of America, p.e2109326119.

Ginolhac, A. et al. (2011). mapDamage: testing for damage patterns in ancient DNA sequences. Bioinformatics , 27 (15), pp.2153–2155. 

Hendy, J. et al. (2018). A guide to ancient protein studies. Nature ecology & evolution, 2 (5), pp.791–799.

Hendy, J. (2021). Ancient protein analysis in archaeology. Science advances, 7 (3). [Online]. Available at: doi:10.1126/sciadv.abb9314.

Ramsøe, A. et al. (2021). Assessing the degradation of ancient milk proteins through site-specific deamidation patterns. Scientific reports, 11 (1), pp.1–14. 

Sakalauskaite, J. et al. (2020). Shell palaeoproteomics: First application of peptide mass fingerprinting for the rapid identification of mollusc shells in archaeology. Journal of proteomics, 227, p.103920.

Warinner, C., Korzow Richter, K. and Collins, M. J. (2022). Paleoproteomics. Chemical reviews, 122 (16), pp.13401–13446.