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Medieval Villagers' Diets, Dental Health Analyzed Through Metaproteomic Profiling

NEW YORK (GenomeWeb) – A team led by researchers at the University of Copenhagen has performed a metaproteomic analysis of dental plaque from 21 medieval villagers found at a Danish gravesite.

For the study, which was published this week in Nature Communications, the scientists used mass spectrometry to identify 3,671 proteins across 22 samples collected from the 21 individuals, including proteins from 220 bacterial species.

By profiling the human and bacterial proteins they detected, the researchers were able to separate the villagers into two groups — one predisposed to relatively good oral health and one more susceptible to oral disease. These groupings, the authors wrote, were "not apparent from the bioarcheological analysis, illustrating that quantitative metaproteomics has the potential to provide additional levels of molecular information about the oral health status of individuals from archeological contexts."

The study also identified components of the villagers' diets, including oats and cow and goat milk.

The 21 study subjects were part of a medieval Danish community founded around 1150 CE. All had evidence of periodontitis as determined by use of the Bradford classification system.

To analyze the subjects' dental metaproteomes, the researchers collected plaque from their teeth and ran it using nanoLC coupled to a Thermo Fisher Scientific Q-Exactive HF mass spec. They then searched mass spectra against a database combining the standard SwissProt database and the Human Oral Microbiome Database. They also analyzed the plaque of modern healthy volunteers using the same workflow.

Excluding proteins found only in the modern samples, the researchers identified the 3,671 proteins, with 85 percent to 95 percent being bacterial, 4 percent to 14 percent being human, and less than 1 percent coming from other sources (primarily food and non-bacterial prokaryotes).

They then took protein quantitation data from all proteins detected in at least half of the medieval samples and used unsupervised hierarchical clustering to look at differences between the various individuals. This analysis distinguished between the modern and medieval samples and between two groups with the larger medieval sample set.

Further analysis of the two medieval groups found that one group, called G1, exhibited higher levels of bacterial virulence factors as well as higher levels of proteins from bacterial species linked to periodontal disease. The second group, termed G2, also contained proteins from pathogenic species but also had higher levels of commensal bacteria, which are characteristic of a healthy oral microbiome.

Analysis of the 205 human proteins identified in the medieval samples identified 13 proteins that were more abundant in G2, including a number of neutrophil-specific proteins that were also observed in the healthy modern samples. This could point to "the first line of response to bacterial infection" and "a possible normal oral immune response," the authors wrote.

The researchers also tested a mass spec approach using offline fractionation and isobaric tagging to improve depth of coverage. Taking five of the archeological samples and labeling them using TMT isobaric tags, they split the pooled sample into 12 fractions, which they then analyzed using short LC-MS/MS runs that were equivalent in run time to an experiment running the five samples individually without fractionation.

That approach identified 3,359 proteins across the five samples, compared to 2,609 proteins identified running the five unfractionated samples individually.

Use of TMT labeling also minimized the number of missing quantitative values across samples, "making quantitative comparison between all samples possible," the authors wrote, suggesting that the approach has great potential "for future quantitative proteomics analyses of archeological remains."