Discovery of New Enamel Pellicle Proteins by Mass Spectrometry

Introduction: The acquired enamel pellicle (AEP) is well known to be a protein film with unique composition and properties. While its importance has been well recognized with respect to bacterial biofilm formation and mineralization of enamel very little information is available on its composition and structure. Objective: To use mass spectrometry to identify and characterize proteins and peptides of the human, in-vivo formed, AEP. Methods: AEP was collected using PVDF membrane saturated with sodium bicarbonate. Proteins were extracted with water, desalted, concentrated and then subjected to tryptic digestion in SDS acrylamide gel or in solution. The digests were applied to an ion trap mass spectrometer (ProteomeX LTQ, Thermo Finnigan, San Jose, CA) equipped with a nanospray reversed phase column. Results: Mass spectra were analyzed using the SEQUEST software program and m/z values were queried against human proteins in the RefSeq database. This resulted in the identification of at least 53 human proteins ranging in coverage between a low 0.3% to a high of 69.0 %. A total of 25 new AEP proteins were identified. In addition to the classical salivary pellicle precursor proteins a variety of non-exocrine proteins which derive from gingival fluid and epithelial cells were discovered. Conclusion: Mass spectrometry seems to be uniquely suited to characterize AEP protein/peptides available at ng levels. The data showed that well known acidic salivary proteins participate in pellicle formation. Unexpected is the finding that gingival fluid and epithelial cells make significant contributions to the AEP. This fact may have major functional consequences for the AEP which will have to be considered. This study was supported by NIH/NIDCR Grants DE05672, DE07652 and DE14950.