Meta-proteomic Analysis of the Shandrin Mammoth by EVA Technology and High-Resolution Mass Spectrometry: What is Its Gut Microbiota Telling Us?

During the last decade, paleoproteomics allowed us to open a direct window into the biological past, improving our understanding of the phylogenetic relationships of extant and extinct species, past human diseases, and reconstruction of the human diet. In particular, meta-proteomic studies, mainly carried out on ancient human dental calculus, provided insights into past oral microbial communities and ancient diets. On the contrary, very few investigations regard the analysis of ancient gut microbiota, which may enable a greater understanding of how microorganisms and their hosts have co-evolved and spread under the influence of changing diet practices and habitat. In this respect, this paper reports the results of the first-ever meta-proteomic analysis carried out on a gut tissue sample some 40,000 years old. Proteins were extracted by applying EVA (ethylene-vinyl acetate) films to the surface of the gut sample of a woolly mammoth (Mammuthus primigenus), discovered in 1972 close to the Shandrin River (Yakutia, Russia), and then investigated via a shotgun MS-based approach. Proteomic and peptidomic analysis allowed in-depth exploration of its meta-proteome composition. The results were validated through the level of deamidation and other diagenetic chemical modifications of the sample peptides, which were used to discriminate the "original" endogenous peptides from contaminant ones. Overall, the results of the meta-proteomic analysis here reported agreeing with the previous paleobotanical studies and with the reconstructed habitat of the Shandrin mammoth and provided insight into its diet. The data have been deposited to the ProteomeXchange with identifier < PXD025518 > .