Introduction: Understanding the composition and structure of the enamel pellicle (EP) has been a major goal in oral biology. The minute quantities of the in vivo formed EP that can be harvested from tooth surfaces and the low resolution and sensitivity of techniques used in the past have prevented major inroads into the analysis of the composition of the EP. Objectives: The present study explores the use of reversed phase liquid chromatography/tandem mass spectroscopy for analysis of EP proteins digested with trypsin. Methods: EP was collected using PVDF membranes saturated with sodium bicarbonate. Proteins were extracted with water and the extraction solution was desalted and concentrated. Aliquots were either subjected to trypsin digestion in SDS gel containing all EP proteins or digestion directly in solution containing 4 M urea and 0.1 % RapiGest detergent (Millipore, Bedford, MA). The resulting digests were analyzed by HPLC-MS/MS and computer-based data analysis using a variety of known databases. Results: At least 53 protein species were found based on the identification of two or more different peptides or one peptide of which the parent protein had been previously confirmed in salivary proteomes. The theoretical isoelectric points of these EP proteins fell mostly with the acidic range indicating that they exhibit negative charges at neutral pH. An additional group of 116 protein species was identified based on the presence of only one peptide. This group contained 23 proteins not originating from mammalian cells but from various oral microbial species. Conclusion: Among 192 proteins identified 53 proteins appear to be true EP proteins. The MS approaches used open new avenues for the characterization of EP components facilitating structural and functional studies. This study was supported by NIH/NIDCR Grants DE05672, DE07652 and DE14950.

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