New techniques for the recovery of small amounts of mature enamel proteins

Currently there are no non-destructive techniques to obtain protein from the dental enamel, the most mineralized tissue in mammals and most resistant to diagenesis, which provides a window to the developing period by means of incremental markings containing proteins. To recover protein, dissolution of powdered enamel is required. Here we tested whether samples obtained by micro-etching of the enamel surface were adequate for protein analysis by MALDI-TOF/TOF mass spectrometry and identification in protein databases. The micro-etch techniques were effective in generating adequate samples for mass spectrometry (from 3 to 13.4 μm superficial enamel), being also highly conservative, since they rendered masses of enamel ranging from 0.1 to 0.4 mg. Using these techniques the separation of proteins by SDS-PAGE was not necessary, and the whole procedure was easier. Results showed successful identification of specific enamel proteins after whole crown superficial etching with 11% EDTA in the case of immature porcine samples, and with 10% HCl in the case of mature human enamel. X- and Y-isoforms of amelogenin, ameloblastin, and enamelin peptides were identified. The new techniques described here allowed the successful recovery of enamel proteins, opening new avenues for the use of enamel protein information in fossil/archeological material, where sometimes little protein is left.