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. 2022 Nov 4;21(11):2619-2634.
doi: 10.1021/acs.jproteome.2c00340. Epub 2022 Oct 21.

Novel Deep Eutectic Solvent-Based Protein Extraction Method for Pottery Residues and Archeological Implications

Manasij Pal Chowdhury  1   2 , Cheryl Makarewicz  3   4 , Henny Piezonka  3   4 , Michael Buckley  1   2
Affiliations

Novel Deep Eutectic Solvent-Based Protein Extraction Method for Pottery Residues and Archeological Implications

Manasij Pal Chowdhury et al. J Proteome Res. .

Abstract

Proteomic analysis of absorbed residues is increasingly used to identify the foodstuffs processed in ancient ceramic vessels, but detailed methodological investigations in this field remain rare. Here, we present three interlinked methodological developments with important consequences in paleoproteomics: the comparative absorption and identification of various food proteins, the application of a deep eutectic solvent (DES) for extracting ceramic-bound proteins, and the role of database choice in taxonomic identification. Our experiments with modern and ethnoarcheological ceramics show that DES is generally more effective at extracting ceramic-bound proteins than guanidine hydrochloride (GuHCl), and cereal proteins are absorbed and subsequently extracted and identifiedat least as readily as meat proteins. We also highlight some of the challenges in cross-species proteomics, whereby species that are less well-represented in databases can be attributed an incorrect species-level taxonomic assignment due to interspecies similarities in protein sequence. This is particularly problematic in potentially mixed samples such as cooking-generated organic residues deposited in pottery. Our work demonstrates possible proteomic separation of fishes and birds, the latter of which have so far eluded detection through lipidomic analyses of organic residue deposits in pottery, which has important implications for tracking the exploitation of avian species in various ancient communities around the globe.

Keywords: Archaeological pottery; Cross-species proteomics; DES; Deep eutectic solvent; Protein extraction; Residue analysis.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Map showing the location of Punsi and the details of the experiments. From top left (clockwise), the location of Punsi in Western Siberia, Russia; the traditional wood-fired ovens used for the cooking experiments; ceramic pots showing the result of the cooking experiments; and a black grouse that was hunted and used for the cooking experiment.
Figure 2
Figure 2
Abundance of the various proteins from meat, cereals, and milk as extracted by the two extraction methods.
Figure 3
Figure 3
Box plots showing the amount of BSA extracted (in μg mg–1) using the various extraction techniques. The various extraction codes in the x-axis are as follows: (A) 6 M GuHCl, 4 h, 65 °C; (B) urea–GuHCl DES, 4 h, 65 °C; (C) 6 M GuHCl, 66 h, 4 °C; (D) urea–GuHCl DES, 4 h, 65 °C, ultrasonication; (E) 6 M GuHCl, 4 h, 65 °C, ultrasonication; (F) urea–GuHCl DES, 8 h, 65 °C, ultrasonication; (G) urea–GuHCl DES, 6 h, 65 °C, ultrasonication; (H) urea–GuHCl DES, 2 h, 65 °C, ultrasonication.
Figure 4
Figure 4
Total abundance of all the food-derived proteins in the various samples as extracted by the DES and GuHCl-based extraction methods.
Figure 5
Figure 5
Bar charts comparing the protein scores of the proteins with the highest Mascot score in the six samples (each in triplicate) with SwissProt as the reference database. i and ii indicate that different proteins had the highest score in the GuHCl and the DES extraction. A, B, and C indicate the three subsamples sampled from each cooking pot.
Figure 6
Figure 6
Net protein abundance (non-normalized, expressed as log 10 abundance) in the various samples determined using Proteome Discoverer with the custom database as the reference database.
Figure 7
Figure 7
Representative tandem spectra of peptides identified to be from G. gallus identified in samples 3A and 4A. The top row contains peptides identified as belonging to myosin heavy chain, skeletal muscle, and the bottom row peptides belonging to F-actin capping protein.
Figure 8
Figure 8
Scatter plots showing the number of sequences marked U (as determined by Mascot) against the total number of sequences. The species of the proteins with the highest number of sequences marked U have been mentioned. Each dot represents a protein (Custom database).
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