France). The sequences were analyzed using the ABI PRISM DNA Sequencing Analysis Software version 3.0 (Applied BioSys- tems) and compared to sequences available in the GenBank database using the BLAST algorithm (http://www.ncbi.nlm.nih. gov/blast/Blast.cgi). Sample preparation for MALDI-TOF peptide profiling Total protein was extracted by incubating the dental pulp with 1 mL of 500 mM EDTA, pH 8.0, with agitation at room temperature for 24 hours. The samples were then sonicated five times for 1 min each and centrifuged at 17,900 x g for 40 min at room temperature. This was followed by dialysis overnight in 2 L of a solution containing 50 mM Tris-HCl, pH 8.0, and 150 mM NaCl. Protein concentration was determined using the Bradford protein quantification protocol. For MALDI-TOF analysis, 100 mL of the protein solution was digested overnight with 100 mL tryptic solution (12.5 ng/ mL trypsin, 50 mM NH4HCO3) at 30uC. Negative controls without specimen samples were run in parallel to test for contaminant peptides. The trypsin-digested peptides were purified with C18 ZipTips (Millipore, Billerica, MA, USA) following the manufacturer’s instructions. An aliquot of 3 mL of the solution was spotted from the ZipTipC18 onto the MTP AnchorChip 384 TF MALDI steel plate. After each spot had dried, 1 mL of a-cyano-4- hydroxycinnamic acid matrix solution was applied on top of the sample. The samples were analyzed in an Autoflex II (MALDI-TOF, Bruker Daltonics GmbH, Leipzig, Germany) in reflectron mode. The mass list was derived automatically using FlexAnalysis software (Bruker Daltonics) with the following criteria: (i) SNR$ 3, (ii) mass detection ranging from 700 to 4,000 Da and (iii) mass tolerance set to60.5 Da. Calibration spectra were produced using the ‘‘user- defined’’ function of the FlexAnalysis software, set at 300 ppm. For each MALDI-TOF profile, we calculated the quality score by analyzing peaks between 900 and 3,000 Da. For each spectrum, we noted the number of peaks and the maximum value of the SNR. Two balanced parameters were taken into account to determine quality scores: (i) the number of peaks with a balance of 0 for n values#8, 1 for 8,n#27, 2 for 27,n #43, 3 for 43,n#78, 4 for 78,n# 86, 5 for 86,n#110, and 6 for n.110; and (ii) the maximal SNR with a balance of 0 for SNmax values#6.8, 1 for 6.8,SNmax#26.8, 2 for 26.8,SNmax#52.6, 3 for 52.6,SNmax#208.2, 4 for 208.2,SNmax#398.6, 5 for 398.6,SNmax#1,092.2, and 6 for SNmax.1,092.2. The quality scores = the value of the number of peaks + the value of the maximal SNR. MALDI-TOF MS classification We observed contamination peaks in the negative controls, which were automatically removed from the spectra using the FlexAnalysis software (Bruker Daltonics). Manual annotation was necessary to eliminate some of the contaminant peaks that were not automatically discarded. A peptide spectrum database was then generated by analyzing dental pulp collected from 13 mammalian species, including human. An established public database for identifying the peptide spectra of the mammalian species used in our study could not be located. For each mammalian species, we analyzed at least two teeth collected from a minimum of two individuals. For each dental pulp specimen, two independent spots were analyzed with five independent acquisi- tions per spot, for a total of 10 spectra per dental pulp specimen. From 370 spectra (37610 = 370), we selected the 279 spectra exhibiting quality scores superior 2 to contribute our modern mammal database by using the software Maldi Biotyper 2.0 (Bruker Daltonics). We analyzed peptides with molecular weights between 700 and 4,000 Da, resulting in a total of 42 to 207 peaks per specimen. We further established an in-silico trypsin digestion Mammal Classification by Mass-Spectrometry pattern of collagen molecules by retrieving collagen molecule protein sequences in http://www.uniprot.org/uniprot/P08123 and using the BioTools software for restriction pattern. For blindly classification, we coded the protein samples from the same 37 teeth and applied the same approach MALDI-TOF MS. But for the blindly identification, we didn’t need to remove the contamination peaks as we did for contributing the local modern database reference. 370 spectra from 37 coded protein samples were directly applied into the software MALDI Biotyper 2.0 for classification. A specimen was identified at the species level based on the highest identification score$2.000 for modern mammal. In the case of ancient mammal, we decreased the classification score threshold because of the degradation of protein with time. MALDI Biotyper procedure of mammal species classification The procedure of mammal species classification by using MALDI Biotyper 2.0 (Bruker Daltonics) was applied from the previously publication [31]. However, in this study, we edited processing methods suitable to our work: we analyzed from 900 to 3,000 Da (Figure S2). Peptide sequencing Proteins extracted from one modern and two ancient human dental pulp specimens were separated by 1 D gel electrophoresis, bands were excised from the gel and digested with trypsin as described [32]. Tryptic peptides were analyzed by MS/MS on a nanoelectrospray ionization quadrupole TOF hybrid mass spec- trometer (Q-TOF Ultima; Waters Micromass) coupled with a nano-HPLC (Cap-LC; Waters). The peptide and fragment ion masses obtained were matched automatically to proteins in a non- redundant database NCBI NR (version 20101105, taxonomy Homo sapiens, 232,072 sequences) using the Mascot version 2.3 MS/MS ions search algorithm (http://www.matrixscience. com). To validate protein identification, only matches with individual ion scores above 20 were considered. Supporting Information Figure S1 In silico analysis of peptide spectrum derived from the human (Homosapiens) dental pulp. (DOC) Figure S2 Workflow of mammal individual classification at the species level using dental pulp MALDI-TOF MS peptide profiling. (DOC) Table S2 Observed mass to change ratio of semi-specific peaks (SEMPs) (1000–3000 Da) from 13 mammal species. (DOC) Table S3 The results of in-silico analysis of peptide spectra derived from the modern human (Homo sapiens) dental pulp, from the modern cow (Bos taurus) and the modern dog (Canis familiaris) dental pulp. (DOC) Table S4 Peptide sequencing of human dental pulp: ‘‘+’’ denotes the presence of the protein; ‘‘-’’denotes the absence of the protein. (DOC) Table S1 study. (DOC) List of the modern dental pulp specimens used in the PLoS ONE | www.plosone.org 6 February 2011 | Volume 6 | Issue 2 | e17319