Classification of Ancient Mammal Individuals Using Dental Pulp MALDI-TOF MS Peptide Profiling Thi-Nguyen-Ny Tran 1 , Ge´ rard Aboudharam1, Armelle Gardeisen 2 , Bernard Davoust3, Jean-Pierre Bocquet-Appel4, Christophe Flaudrops1, Maya Belghazi5, Didier Raoult1, Michel Drancourt1* 1 Unite´ de recherche sur les maladies infectieuses et tropicales e´mergentes (URMITE), UMR CNRS 6236 IRD 198, IFR48, Faculte´ de me´decine, Universite´ de la Me´diterrane´e, Marseille, France, 2 UMR 5140-CNRS, Universite´ Montpellier III, Montpellier, France, 3 Direction re´gionale du service de sante´ des arme´es de Toulon, Toulon, France, 4 Ecole Pratique des Hautes Etudes and CNRS UPR 2147, Paris, France, 5 Centre d’Analyses Prote´omiques de Marseille (CAPM), IFR Jean Roche, Faculte´ de Me´decine - Secteur Nord, Universite´ de la Me´diterrane´e, Marseille, France Abstract Background: The classification of ancient animal corpses at the species level remains a challenging task for forensic scientists and anthropologists. Severe damage and mixed, tiny pieces originating from several skeletons may render morphological classification virtually impossible. Standard approaches are based on sequencing mitochondrial and nuclear targets. Methodology/Principal Findings: We present a method that can accurately classify mammalian species using dental pulp and mass spectrometry peptide profiling. Our work was organized into three successive steps. First, after extracting proteins from the dental pulp collected from 37 modern individuals representing 13 mammalian species, trypsin-digested peptides were used for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis. The resulting peptide profiles accurately classified every individual at the species level in agreement with parallel cytochrome b gene sequencing gold standard. Second, using a 279–modern spectrum database, we blindly classified 33 of 37 teeth collected in 37 modern individuals (89.1%). Third, we classified 10 of 18 teeth (56%) collected in 15 ancient individuals representing five mammal species including human, from five burial sites dating back 8,500 years. Further comparison with an upgraded database comprising ancient specimen profiles yielded 100% classification in ancient teeth. Peptide sequencing yield 4 and 16 different non-keratin proteins including collagen (alpha-1 type I and alpha-2 type I) in human ancient and modern dental pulp, respectively. Conclusions/Significance: Mass spectrometry peptide profiling of the dental pulp is a new approach that can be added to the arsenal of species classification tools for forensics and anthropology as a complementary method to DNA sequencing. The dental pulp is a new source for collagen and other proteins for the species classification of modern and ancient mammal individuals. Citation: Tran T-N-N, Aboudharam G, Gardeisen A, Davoust B, Bocquet-Appel J-P, et al. (2011) Classification of Ancient Mammal Individuals Using Dental Pulp MALDI-TOF MS Peptide Profiling. PLoS ONE 6(2): e17319. doi:10.1371/journal.pone.0017319 Editor: M. Thomas Gilbert, Natural History Museum of Denmark, Denmark Received August 18, 2010; Accepted January 29, 2011; Published February 25, 2011 Copyright: 2011 Tran et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: TNNT, DR and MD declare that they are owners of a pending patent related to MALDI-TOF identification of animals. This does not alter our adherence to all the PLoS ONE policies on sharing data and materials. The other authors declare no competing interests. * E-mail: Michel.Drancourt@univmed.fr Introduction Classifying the remains of animals at the species level is an important task in various fields of research, such as zooarcheology, anthropology and related applied sciences, including forensic sciences and the surveillance of wildlife trade and endangered species [1–3]. This could, however be a challenging task, as species classification based on morphological characteristics lacks speci- ficity and is of little use with fragmented or severely damaged material, such as a mixture of small skeletal pieces from several individuals [2]. Sequencing of specific genes and other metage- nomic approaches using next-generation DNA sequencing tech- nology are available and allow the classification of animal species. However, these approaches depend on the presence of amplifiable nucleic acids and time-consuming PCR-based methods that are prone to in-laboratory contamination [4]. Recent work has demonstrated that protein profiles obtained after matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of cultured cells can accurately determine the species origin of the cell line [5,6]. This approach is very promising, as MALDI-TOF MS is an easy-to-run and rapid method of analysis. Also, classification of animals has been done by MALDI-TOF MS of bone collagen [7]. Based on these seminal demonstrations, we hypothesized that MALDI-TOF MS could further classify mammal species by analyzing dental pulp tissue, i.e., a mixture of cell lines, instead of a unique cell line or protein. To demonstrate that it was possible to classify animal remains, including ancient remains, at the species level starting from the dental pulp by using MALDI-TOF MS technology, we carried out a study into three successive sections combining MALDI-TOF MS expertise [5] with the experimental recovery expertise of dental PLoS ONE | www.plosone.org 1 February 2011 | Volume 6 | Issue 2 | e17319