pulp tissue [8]. First, we established that dental pulp was a source of peptide profiles identifiable by MALDI-TOF MS and MALDI- TOF MS dental pulp peptide profiling could be used for the accurate classification at modern mammalian individuals the species level. Second, we validated this method by a blind classification of modern mammalian teeth. Finally, we showed that MALDI-TOF MS analysis of dental pulp recovered from ancient buried mammalian individuals, including humans, enabled the rapid classification of these ancient individuals at the species level. Results Modern mammal database We recovered 0.024–2.148 g/L of protein (mean 6 standard deviation: 0.61960.195 g/L) from 37 dental pulp tissues of 37 modern individuals representing 13 mammalian species. Because of the sole morphological examination of teeth could be misleading, species classification was confirmed in parallel by partial sequencing of the mitochondrial cytochrome b gene used as the gold standard (Table S1) [9]. We then digested extracted proteins by trypsin and used the resulting peptide mixture as the substrate for MALDI-TOF MS analysis. Same peaks that were observed in all of ten negative controls were contaminants composed of autoproteolytic trypsin digest fragments (1,726– 2,284 Da) and keratin peaks (1,266–2,824 Da) [10,11] and some additional non-specific peaks (707.3–3,807 Da) with a signal-to- noise ratio (SNR)#3 were probably derived from the column used for the reverse-phase protein purification or from sample preparation. These peaks that we called contamination peaks were automatically eliminated from spectra obtained from dental pulp of modern mammals for generate a local modern mammal database. Spectra were derived from two to four dental pulp specimens per individual (one dental pulp per tooth). The peptide spectra had mass-to-charge ratio (m/z) of 700–4,000 Da and reproducibly yielded 11 to 42 semi-specific peaks (SEMPs) in the 1,000–3,000 Da (Table S2) that were present in all mammalian species studied. SEMPs and unique species-specific peaks (USSPs) were determined as previously published [10]. In silico analysis indicated that peptide spectra derived from the human (Homo sapiens) dental pulp contained six different collagen types. As for spectrum derived from the cow (Bos taurus), it contained 2 different collagen types. As for spectrum derived from the dog (Canis familiaris), it contained only collagen alpha-1(I) trypsin digestion products (Figure S1, Table S3). In the case of human dental pulp, peptide sequencing yielded 16 different non-keratin proteins including collagen (alpha-1 type I and alpha-2 type I) (Table S4). Applying the procedure (Materials and Methods), we then set up a 279-spectrum MALDI-TOF MS database of modern mammal dental pulp tissues to generate a modern mammal data- base. Spectra representing for each species have been deposited to Proteome Commons database https://proteomecommons.org/ [12] (Link: https://proteomecommons.org/tranche/data-downloader.jsp? h=9FYJgZ%2Bvzxy7TQtjfkLTPclKFDMB4uRYbbs9OgimjQaVCf Ay2%2FXhPKzS42hWSVoyxhTRVRMBJsbdx26afXq1trhjC7c AAAAAAAAIgw%3D%3D) (Supplementary data online). Blind classification of modern mammal individuals Protein samples from 37 modern individuals were coded, were digested by trypsin and were applied to the procedure MALDI- TOF MS. A total of 370 reproducible MALDI-TOF MS dental pulp profiles (10 per dental pulp) obtained in this time were directly compared to the local modern mammal database by using the software MALDI Biotyper 2.0 (Materials and Methods). Positive classification was not achieved in 3/37 (8.1%) dental pulp Mammal Classification by Mass-Spectrometry specimens with a protein concentration,0.1 mg/mL in the case of Cavia porcellus (2 teeth) and Rattus rattus (1 tooth), whereas 33/37 (89.1%) dental pulp specimens with a protein concentra- tion.0.1 mg/mL (P,0.05, x2 test) were accurately classified. In one Vulpes vulpes specimen, classification was of 1.436 below our classification threshold for modern mammal (2.0); in another Vulpes vulpes specimen, lack of classification was due to a low quality of the MALDI-TOF MS spectra (Table S5). Classification of ancient mammal individuals We applied the same approach to ancient teeth. By using the software MALDI Biotyper 2.0 and comparing with our modern database, we could classify 10/18 (56%) teeth collected in 8/15 (53.3%) individuals from five burial sites dating between 700 and 8,500 years ago (Table S6). In these specimens, protein concentrations of 0.2860.12 g/L yielded reproducible MALDI- TOF MS profiles that differed by 1–17 USSPs (Table S7). We did not observe contamination from one species to another one in this study. Peptide sequencing confirmed these data by giving 4 different proteins including collagen (alpha-1 type I and alpha-2 type I) in ancient human dental pulp (Table S4). Two isolated teeth collected from a first- or second-century site were identified as cow, four other teeth as dog and one additional tooth as cat. After these profiles of ancient individuals were incorporated into ancient mammal database, further comparison of teeth that had not been identified by first-round comparison with modern profiles yielded 100% identification with cow, cat, pig and human, with identification scores of$1.8. All MALDI-TOF MS results were in agreement with the observed morphological characteristics and were confirmed by cytochrome b sequencing (Figure 1, Table S8). We therefore successfully identified the remains of individuals representing four domestic mammalian species frequently en- countered in excavations. Two isolated teeth collected from an 18th-century site were identified as human, as were teeth collected from 300-year-old burial site in France. This approach also proved effective in classifying 8,500-year-old human dental pulp speci- mens collected in Syria from the deciduous teeth of two Neolithic children (Figure 2). Discussion The results herein reported establish the proof-of-concept of accurate classification of mammal individuals by MALDI-TOF MS profiling of peptides prepared from the dental pulp. We used parallel cytochrome b gene sequencing reported in literature as a gold standard in molecular classification of mammals [3,9] (Table S1) since the simple morphological examination of teeth may lack specificity. A MALDI-TOF MS database has already been published for the identification of Eucarya species based on the analysis of cultured cell lines over 34 species ranging from insects to primates [5]. Moreover, MALDI-TOF MS was recently applied to the investigation of human circulating immune cells [13]. It was, however, necessary to build a new database because we analyzed an entire organ, mixing several tissues and cell lines. Indeed, unlike DNA sequencing, MALDI-TOF MS profiling relies on post- translational modifications, which may differ with tissue specificity. Also, we trypsin-digested the protein extracted from dental pulp prior to MALDI-TOF MS analysis, meaning we analyzed peptides instead of proteins. At last, peptide sequencing data also agreed with MALDI-TOF MS data which all indicated that the ancient dental pulp contained less numerous identifiable proteins than the modern dental pulp, probably resulting from the natural decay due to digenetic alteration. These data explained the necessity to PLoS ONE | www.plosone.org 2 February 2011 | Volume 6 | Issue 2 | e17319