Meta-proteomic analysis of the Shandrin mammoth by EVA technology and high-resolution mass spectrometry: what is its gut microbiota telling us?

Abstract

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 > .

Deep Time Paleoproteomics: Looking Forward

“…The field of paleoproteomics (here defined as the characterization of proteins from archeological and paleontological tissues using mass spectrometry [MS]) has grown exponentially since the first application of matrix-assisted laser desorption ionization (MALDI) MS to mammal remains 800–450 000 years old in 2000 . In the 22 years that followed, a variety of mass spectrometry-based methods have been used to investigate the preserved proteomic content of a diverse array of biological tissues (e.g., bones, teeth, baleen, turtle shell, mummified tissues), − objects (e.g., paintings, ethnologic objects, potsherds, parchment), − and species (e.g., mammoth, moa, giant beaver, whales, sea turtles). ,,,,,− However, the vast majority of these studies have focused on relatively young (<100 thousand years old) paleontological and archeological materials and remains. In this perspective, we discuss the development and progress of “deep time paleoproteomics (DTPp)”, here defined as MS characterization of material older than ∼1 million years (1 Ma).…”

Meta-proteomic analysis of two mammoth’s trunks by EVA technology and high-resolution mass spectrometry for an indirect picture of their habitat and the characterization of the collagen type I, alpha-1 and alpha-2 sequence

“…Altogether, these results supported the hypothesis of a different North Siberia habitat during the Late Pleistocene. Moreover, the composition of microorganism-related peptides found in the trunks surface reflected that observed in the Shandrin mammoth gut (Cucina et al 2021 ), with a predominance of Proteobacteria, followed by Firmicutes, probably attributed to the presence of grass more than tree shrub. Interestingly, in the trunks surface the presence of Firmicutes, and particularly of Bacilli, seemed to be higher.…”

“…Finally, taking into account the inestimable value of archaeological samples, minimally destructive or non-destructive sampling techniques are needed. One of the more promising non-invasive techniques, known under the acronym EVA (ethylene–vinyl acetate impregnated with hydrophilic and hydrophobic resins) was introduced by Manfredi et al 2017 and recently applied in our lab to a gut tissue sample of a woolly mammoth (i.e., the Shandrin’s mammoth) (Cucina et al 2021 ). In particular, by coupling the EVA technology, a shotgun approach, and the use of high-resolution mass spectrometry it was possible to perform a meta-proteomic analysis which allowed us to get insight into the gut microbiota composition and turned out to be related to the diet of the Shandrin mammoth and its habitat.…”

Assessing contemporary Arctic habitat availability for a woolly mammoth proxy

“…A combination of independent lines of evidence including palynological (pollen) analyses of gut content 28 , multiproxy analyses of dung from the lower intestine (including microscopic, chemical, and molecular techniques such as gas chromatography/mass spectrometry, thermally assisted hydrolysis and methylation, and DNA sequencing) 29 , visual analyses of gut content 30 , fatty acid profiling of fatty tissues 31 , DNA sequencing / metabarcoding of gut and coprolite samples 32 , meta-proteomic analyses by shotgun mass spectrometry of gut tissue samples 33 , DNA metabarcoding, palynological and macrofossil analyses of coprolite (feces) samples 34 , and isotopic analyses of tooth enamel 35 , 36 , suggest that woolly mammoths consumed primarily forbs, graminoids (grasses and sedges), and shrubs, supplementing their diet with trees mosses and even lichen and green algae (as detailed in Supplementary Table 1 ).…”