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

[Image: see text] 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.