Proteomics identified from 70 to 300 proteins of different organismal origin in foodcrusts from Friesack 4 pottery (S1 File). To facilitate the interpretation, proteins considered as a common background were grouped together and remaining protein hits were grouped in four categories according to their organismal origin (Table 1, S1 File): 1. microbial, 2. plant, 3. animals and 4. fish. Next, we calculated deamidation rates in detected glutamine-contained peptides (S1 File). Deamidation of asparagine (N) and particularly of slowly converting glutamine (Q) is an indicator of protein aging and degradation [46, 47]. Pronounced deamidation of these residues was reported for diverse archaeological materials analysed by mass spectrometry [16–20, 48–50]. Deamidation rate of glutamine whose slower turnover is compatible with the time span of archaeological samples was also suggested as an useful marker for identification of outliers and distinguishing contaminants [18]. We calculated deamidation rate of glutamine in reference human keratins and compared it with other proteins identified in archaeological samples.

Plant proteins were identified in crusts #3078 and #3251. They originated from seeds of a plant of Triticeae family, green of a plant from Lamiaceae family, phloem of squash Cucurbita maxima and birch pollen ( Table 1 , S1 File ). Although some of these proteins could be associated with foods, particularly seeds from Triticeae plant, deamidation rate of glutamine residues in correspondent peptides was also the same as for keratins. We assumed that these proteins together with a few insect proteins detected in #3078 belong to modern environmental contaminants.

Microbial proteins: the bulk of remaining proteins (except for crust #3078) has microbial origin ( Table 1 , S1 File ) and includes species typical for the natural microorganismal community of a peat bog. Most of identified microbial proteins were matched to Streptomyces spp.—a bacteria which is common to humid soils and might be involved in lysis of cellulose [ 53 , 54 ]. Other detected microbial proteins belong to bacteria inhabiting meromictic freshwater lakes as well as fungal proteins from plant-associated genera Fusarium and from plant pathogens occurring in microbiome of peat bog [ 55 ]. Though, no Aspergillus spp which are typical for destructive microflora on archaeological artefacts [ 56 ] were detected. Deamidation rate of asparagine and glutamine residues in microbial proteins was the same as in background keratins.

Bovine milk proteins were detected in all Friesack foodcrusts ( Table 1 ). In total, 26 peptides matching various bovine milk proteins contained glutamine residues. Considering that none of glutamine or asparagine residues in peptides matched to milk proteins was deamidated ( S1 File ), although they are observed intensively modified in ancient dairies [ 16 ], and milk proteins were also detected in control samples (pottery glue and void SDS gels), they were considered contemporary contaminants.

Common protein background comprised human keratins together with several skin and saliva proteins introduced during sample handling, the majority of them are listed in available predefined contaminants database for proteomics (for example, for MaxQuant software at http://www.coxdocs.org ). Conserved proteins whose organismal origin cannot be determined because they are sharing identical matching peptides, e.g. histones or actin, and laboratory standard (hen egg ovalbumin) [ 51 ] were also included in this group. Because the fragments of the vessel #3258 T were glued together before sampling the foodcrust, we also analysed a sample of the pottery adhesive and added identified proteins to the background list ( S1 File ). Interestingly, minute amount of silk protein sericin (gi 112984400, B.mori) which is used as a moisturizing agent in modern skin and hair cosmetics (up to 20% w/w in some cream formulations) [ 52 ] was identified in the glue sample. We then calculated relative deamidation rate in glutamine-containing peptides matched to five most abundant keratins present in each sample ( S1 File ). We selected only fully tryptic peptide comprising single glutamine residue. Deamidated and unmodified forms of a peptide can be clearly distinguished by LC-MS/MS analysis: they have different m/z and eluted at different retention time ( S3 Fig ). Altogether, 34 unique keratin peptides comprising single glutamine residue were detected in both native and deamidated forms in all analysed samples ( Table 2 , S1 File ). Average deamidation rate for all these peptides was 0.4% and did not exceed 2.2% which corroborates with estimations for bulk deamidation reported for keratins [ 18 ]. Relative deamidation rate in asparagine peptides was at the similar range (2%, calculated for 36 individual sequences).

Two core collagens—alpha1(I) and alpha2(I)—matching altogether 25 peptides were identified in foodcrusts #3078 and #3251.1 and, in trace amounts, also in the control sample (pottery adhesive) (Tables 1 and 3 ). Whereas the majority of collagen-matched peptides was identical to several animal collagens, a few of them in #3078 and control sample were unique for protein entries from Bovidae (acc no P02453 and P02465) and in crust #3251.1—from pig (acc no A0A1S7J210 and A0A1S7J1Y9). Notably, asparagine and glutamine residues in collagen peptides were partially deamidated only in crust sample 3251.1. Composition, species specificity and deamidation of detected peptides ( Table 3 ) suggested that foodcrusts #3078 and #3251.1 comprised modern Bovidae collagen as a contaminant. In addition, the foodcrust #3251.1 also included minute amount of, likely ancient, pig collagen.

Fish proteins in the foodcrust #3258, their properties and species attribution.

Two fish proteins, vitellogenin and parvalbumin, were matched with seven and three peptides respectively, and were only identified in the fooodcrust from the base shard of the #3258 vessel (Tables 1 and 4). Four out of seven vitellogenin peptides were unique for Cypriniformes order including common carp Cyprinus carpio and the remaining three were present in multiple vitellogenin sequences from various Actinopterygii (Table 4). Detected parvalbumin peptides belong to a conserved part of the sequence and are shared between many fish species. MS BLAST sequence similarity search matched them all together to a single sequence from common carp (Cypriniformes) as the top hit (Table 4, Fig 3). The results suggested that the foodcrust #3258 comprised protein material from a Cypriniformes. There are no evidence that fish proteins are a part of the sediment at the site: samples 3258 and 3251.1 which were recovered in the striking distance from each other (thin layers 4 and 5 of the test trench, Fig 1) revealed common environmental background largely consisting of Streptomyces spp. whereas fish proteins were identified exclusively in #3258.

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larger image TIFF original image Download: Fig 3. Parvalbumin peptides detected in #3258 basal crust are matched to C. carpio sequence by MS BLAST. “Query” includes sequences of peptides identified in the sample; “Sbjct”—sequences of corresponding peptides matched to C.carpio protein (acc no KTF75949.1). https://doi.org/10.1371/journal.pone.0206483.g003

Vitellogenins are the precursors of yolk proteins which make up the bulk of egg in fish [57]. They are synthetized in female liver as preproproteins and then delivered by bloodstream to ovaries where they are processed and accumulated. Ovaries are most enriched with vitellogenin compared to other organs of the fish body: its content exceeds 2.5pmol/μg of total protein [58]. The amount of vitellogenins in fish liver and in blood is 500 folds lower than in ovaries [58] and their expression in other tissues, e.g. skin or muscles, is below 10% of that in liver [59]. Vitellogenins were not detected by proteomics in fresh carp liver and muscles [60, 61]. Taking together, vitellogenins could be considered as marker protein specific for fish eggs. No major proteins from tissues, in which vitellogenins could be also present (e.g. blood hemoglobin or muscle myosin), were detected in crust #3258. Therefore, we assumed that one of the ingredients of the meal in #3258 was fish caviar or whole roe. To further test this we analysed fresh carp roe sac (unseparated eggs and skin) (S1 File). As anticipated, vitellogenins constituted the bulk of proteins in roe. More than 40% of the total of 65 000 MS/MS spectra matched vitellogenins sequences.

Parvalbumins are small proteins with remarkable chemical and thermal stability and high solubility in water that are ubiquitously expressed within the fish body [62]. Two parvalbumins were also detected in trace amounts in fresh carp roe (S1 File). Thus, one of plausible explanations of why parvalbumin was present in the foodcrust #3258 might be that it originated from roe sac together with vitellogenins. Parvalbumins are enriched in white (non-fatty) muscles of fishes [63] from which they can be readily extracted by boiling [64]. Thus, the presence of parvalbumins might also indicate that the composition of ancient meal #3258 included fish broth. To test this hypothesis we prepared fish broth and analysed it by proteomics. Indeed, four proteins of parvalbumin family were found among 339 proteins detected in the broth and one of them (acc no ACH71041.1, matched with 300 spectra) was among top ten most abundant proteins in the sample (S1 File).

Remarkably, in contrast to other proteins identified in Friesack samples, all glutamine residues in peptides matched to vitellogenin and parvalbumin in #3258 were detected strongly deamidated (Fig 4, Table 4). Deamidation rate of the same peptides in fresh roe did not exceed background level of 1% indicating that their deamidation does not naturally occur in living fish (Table 2, S1 File).

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larger image TIFF original image Download: Fig 4. Pronounced deamidation of the glutamine residue (Q) in fish vitellogenin from foodcrust #3258. Upper panel: Fragmentation spectrum of vitellogenin peptide LELEVQVGPR (m/z 570.32, z +2) identified in fresh carp roe. In foodcrust #3258, 88% of glutamine residue in this peptide was converted into glutamic acid (E); mirrored image of fragmentation spectrum of peptide LELEVEVGPR detected in #3258 is shown at the lower panel. Masses of fragment ions including Q or E at the position six are shown in bold. https://doi.org/10.1371/journal.pone.0206483.g004

Could sample #3258 be occasionally contaminated with thermally processed fish during handling? To exclude this possibility we calculated glutamine deamidation rate in 12 most abundant proteins identified in the fish broth (Table 2, S1 File). These proteins matched 65 tryptic peptides comprising one glutamine residue which were detected partially deamidated, 34 of them belonged to soluble fraction of collagens. Each protein was presented by more than one glutamine-contained peptide. Average deamidation rate was ca 5% and, importantly, it did not significantly vary between proteins. An exception was a single collagen peptide QGPGGPVGER that was fully deamidated. However, the deamidation rates in other peptides belonging to the same protein did not differ from the average; moreover the peptide was not detected in oxoproline-containing form and therefore it seems likely that here the quantitative estimates are biased by incomplete detection of its relevant molecular forms. Taking together, pronounced glutamine deamidation of fish proteins in residue #3258 cannot be solely explained by food cooking and we concluded that both vitellogenin and parvalbumin proteins were ancient.