Mammal Classification by Mass-Spectrometry Figure 3. MALDI-TOF MS peptide profiling of proteins extracted from dental pulp. Pseudo-gel displays (Gel View, Bruker Daltonics) of the peptide spectra obtained with wild boar (a) and pig (b) specimens were used as the reference peptide. The mass-to-charge ratio (m/z) of each peptide is indicated on the x-axis, and the relative intensity of each peak is shown on the y-axis. Blue bars correspond to peaks from pig specimens, red bars correspond to peaks from wild boar specimens, and green bars represent peaks shared by both. doi:10.1371/journal.pone.0017319.g003 written consent from the patients (Table S1). This study was approved by the Ethic Committee, Institute Fe´de´ratif de Recherche 48, Marseille, France. Ancient individuals were excavated from five archeological sites and initially classified by morphological criteria. The classified species included roman pig, cow and dog from the Lattes site (first or second century), France; cat from a 13th- or 14th-century site in Lille, France; and human teeth from a 6,500 BC site in Syria, a first- to third-century site in the Roman catacombs, Italy, and an 18th-century site in Douai, France. (Table S8). The criteria for the ancient teeth selected were used from previous publication [20]. Nucleotide sequence–based classification To confirm the classification of the collected samples, DNA was extracted from dental pulp, and a 307-bp partial cytochrome b sequence was amplified using primers L14841 and H15149 for modern samples [30]. Partial amplification (149 bp) of cytochrome b was performed in ancient samples using primers N2-cyt b F (5’-GTAGAATGAATCTGAGGCGG-3’) and N2-cyt b R (5’- CCTGTAGGGTTGTTGGATCC-3’). DNA concentration was measured with a NanoDrop 1000 (Thermo Fisher Scientific, Courtaboeuf, France). Amplifications were performed in a 2720 Thermal Cycler (Applied Biosystems, Courtaboeuf, France) under the following conditions: activation for 10 min at 95uC, followed by 40 cycles of a 30-s denaturation step at 95uC, a 45-s hybridization step at 57uC and a 90-s elongation step at 72uC, with a final 7-min extension step at 72uC. The PCR mixtures consisted of 2.5 mL 10X PCR buffer, 2.5 mL dNTPs (2 mM each of dATP, dCTP, dGTP, dTTP), 0.8 mL MgCl2 (25 mM), 0.5 mL each primer (10 pmoL/ mL), 1.25 U HotStarTaq polymerase (Qiagen), 13.5 mL H2O, 0.5 ml BSA, and 4 mL template DNA. As a negative control, 4 mL distilled water was used in lieu of template DNA. PCR products were separated by electrophoresis for 26 min at 100 V in a 2% agarose gel with 0.5X TBE. PCR products were sequenced using a Big Dye Terminator kit and an ABI PRISM 3130 Genetic Analyzer (Applied BioSystems, Courtaboeuf, PLoS ONE | www.plosone.org 5 February 2011 | Volume 6 | Issue 2 | e17319