AbstractThe recent paleoproteomic studies, including paleo-metaproteomic analyses, improved our understanding of the dietary of ancient populations, the characterization of past human diseases, the reconstruction of the habitat of ancient species, but also provided new insights into the phylogenetic relationships between extant and extinct species. In this respect, the present work reports the results of the metaproteomic analysis performed on the middle part of a trunk, and on the portion of a trunk tip tissue of two different woolly mammoths some 30,000 years old. In particular, proteins were extracted by applying EVA (Ethylene–Vinyl Acetate studded with hydrophilic and hydrophobic resins) films to the surface of these tissues belonging to two Mammuthus primigenus specimens, discovered in two regions located in the Russian Far East, and then investigated via a shotgun MS-based approach. This approach allowed to obtain two interesting results: (i) an indirect description of the habitat of these two mammoths, and (ii) an improved characterization of the collagen type I, alpha-1 and alpha-2 chains (col1a1 and col1a2). Sequence characterization of the col1a1 and col1a2 highlighted some differences between M. primigenius and other Proboscidea together with the identification of three (two for col1a1, and one for col1a2) potentially diagnostic amino acidic mutations that could be used to reliably distinguish the Mammuthus primigenius with respect to the other two genera of elephantids (i.e., Elephas and Loxodonta), and the extinct American mastodon (i.e., Mammut americanum). 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. The data have been deposited to the ProteomeXchange with identifier < PXD029558 > .