Mammal Classification by Mass-Spectrometry Figure 1. The dendrogram obtained by the software Maldi Biotyper 2.0 (Bruker Daltonics) after peptide spectral analysis is congruent with the one derived from cytochrome b sequencing by the software Tree View (Free down load from the Internet site: http://darwin.zoology.gla.ac.uk/,rpage/treeviewx/download.html). The images indicate the mammalian species (left) from which the ancient teeth were identified (right). doi:10.1371/journal.pone.0017319.g001 complement the database of modern specimens by a database of ancient specimens in order to increase the ratio of classification of the ancient specimens. We developed a simple protocol, with protein preparation being limited mainly to dialysis; indeed, we did not aim to identify unique proteins but instead to analyze a complex peptidic profile comprising several dozens of picks. Such identifying peptidic profiles were reproducible for any individual, and it was reproducible from one individual to another of the same species. Moreover, we observed that three USSPs herein reported have been already described in the case of human, rabbit and cow [7], thus validating our approach. This approach simplifies and strengthens previous approaches that rely on the analysis of a single soluble protein [10]. Testing of keratinous tissues (95% of the total material in feathers and down is composed of a-keratin) yielded four SEMPs and three USSPs for human hair, compared to 40 SEMPs and 17 USSPs from human dental pulp [10]. All MALDI-TOF MS results were in agreement with the observed morphological characteristics and were confirmed by cytochrome b sequencing (Figure 1, Table S1, Table S5, Table S6, Table S8). We therefore successfully classified the remains of individuals representing four domestic mammalian species fre- quently encountered in excavations. In addition, MALDI-TOF MS analysis of keratin recovered from the clothing of a 5,300- year-old Tyrolean mummy, which was remarkably preserved due to natural freezing in the Alps, revealed that the material originated from sheep and cattle [14]. The results presented here establish the proof-of-concept that MALDI-TOF MS peptide profiling of dental pulp can accurately classify ancient mammals at the species level. The finding that wild boars and pigs, which are members of the same genetic species, had 5 and 2 USSPs from a total of 11 and 12 SEMPs, respectively, illustrates the discrimina- tory power of MALDI-TOF MS peptide profiling (Figure 3). Ancient mammals were previously identified by analyzing dried muscle and skin from well-preserved museum specimens, hair shafts from permafrost-preserved animals, and bones and teeth of buried animals [15–18]. These tissues are directly exposed to the environment; contamination and degradation of surface proteins have been observed [19]. Reduced numbers of USSPs have been identified in tinted or bleached hair samples, and new peaks in these specimens have also been observed [10]. In contrast, dental pulp was our material of choice and this material has many advantages in studying ancient samples [20]. The tooth cavity protects the dental pulp from potential contamination and thermal degradation [21]. Moreover, teeth are the only body parts that remain after the bones have disintegrated. For instance, only teeth remained in the Early Upper Paleolithic site of Kostenski in Eastern Europe, which proved vital in understanding the dispersal of modern humans even though morphological identification was impossible [22]. In PLoS ONE | www.plosone.org 3 February 2011 | Volume 6 | Issue 2 | e17319