Leave no stone unturned:Exploring the metaproteome of beerstone for the identification of archaeological beer production

In archaeological contexts, identifying processes of beer production and consumption has contributed to our understanding of agriculture, labor mobilization, economic surplus, feasting, gender dynamics, social structure, tribute, community, identity and politics. Nevertheless, in the absence of pictorial representations and characteristic objects, beer brewing is difficult to identify in the archaeological record, and molecular methods are often limited by constraints of preservation and specificity. A potential target for studies of ancient beer production are residues formed during brewing activity, including beerstone, a calcium oxalate residue. Here, we apply shotgun proteomics analyses to a sample of modern beerstone to explore this residue's capacity as a marker for beer in archaeological contexts. The beerstone proteome was compared to the protein profiles of ungerminated and germinated barley to identify key proteins indicative of malted grains which may be encased by the residue. Proteins matching to barley grain (Hordeum vulgare) and Baker's/Brewer's yeast (Saccharomyces cerevisiae) were successfully identified in the beerstone. In particular, we identified hordeins, lipid transfer proteins, trypsin/α-amylase inhibitors, and protein Z, which are barley proteins abundant in proteomic characterisations of beer. In comparison to ungerminated and germinated barley grains, we find that beerstone preserves only a subset of the barley proteome, with the residue being more reflective of the final brewing product than of earlier brewing steps such as malting. Overall, we demonstrate that beerstone has potential to entrap and preserve proteins reflective of the beer-making process and identify proteins that we might anticipate in future archaeological analyses.