Compositional analysis of human acquired enamel pellicle by mass spectrometry

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Abstract

Relatively little is known about the formation of the acquired enamel pellicle other than that it involves the selective adsorption of specific proteins from oral fluids. Previous studies on the identification of pellicle components have relied largely on immunological or enzymatic detection and have been hampered by the fact that only minute quantities of pellicle can be removed from tooth surfaces. The present work describes an improved method of harvesting pellicle that combines mechanical and chemical removal; this approach was used to investigate systematically the desorption of in vitro pellicle components with different solutions. Eleven major in vitro pellicle proteins were identified by using a combination of electrophoretic separation and matrix-assisted laser desorption/ionization–reflectron time-of-flight mass spectrometry. A similar analysis of in vivo-formed pellicle revealed the presence of intact statherin, lysozyme, albumin and amylase. Further analysis of in vivo pellicle by liquid chromatography–electrospray ionization mass spectrometry suggested the presence of numerous low molecular-weight fragments of precursor proteins. The protein composition of in vitro whole-salivary pellicle adsorbed to hydroxyapatite and that of in vivo enamel pellicle differed for proline, the result of a reduction in the content of acidic proline-rich proteins in the in vivo samples. Unique features of the oral environment such as enzymatic activities or mineral surface properties may account for these differences between in vivo and in vitro pellicle formation.

Introduction

Upon tooth eruption, enamel surfaces become exposed to the mouth and are immediately coated by an acellular, essentially bacteria-free organic film known as the acquired enamel pellicle (Dawes et al., 1963). Histochemical staining shows that this film is largely composed of proteins of salivary origin (Meckel, 1965). The acquired enamel pellicle provides a surface to which bacteria adhere during the initial stages of dental plaque formation (Saxton, 1973). Therefore, a detailed understanding of the composition and structure of the pellicle might lead to the development of interventions that could reduce or modify bacterial colonization of tooth surfaces.

Several investigations have used in vitro pellicles formed on hydroxyapatite or enamel slabs after exposure to saliva to obtain insight into their formation and structure. These studies have identified protein phosphoryl, aspartyl and glutamyl side-chains as important mediators of adsorption to the mineral surface. Other studies of pellicle have provided insight into the adhesion of early bacterial colonizers and demonstrate an important role for pellicle in protecting the enamel against demineralization by displaying permselectivity (Zahradnik et al., 1976) and favouring subsurface remineralization (Zahradnik, 1979). The significance of results obtained with in vitro pellicle is not, however, entirely clear as the amino acid composition of in vitro pellicles differs, in some respects, from that of pellicles formed in vivo (Sonju and Rolla, 1973, Al-Hashimi and Levine, 1989, Jensen et al., 1992, Carlen et al., 1998).

Progress in the elucidation of the macromolecular composition of in vivo pellicle has been achieved by using immunological identification, which has resulted in the detection of acidic PRPs (Kousvelari et al., 1980), mucin MG1, sIgA, amylase, cystatin SA-1 (Al-Hashimi and Levine, 1989), parotid saliva agglutinin, serum albumin, mucin MG2 (Carlen et al., 1998) and carbonic anhydrase VI (Leinonen et al., 1999). Amylase, lysozyme (Orstavik and Kraus, 1974), and glucosyltransferase (Scheie et al., 1987) activity has also been identified in pellicle samples. However, the quantitative interrelations of these components, and how they are structurally organized on the tooth surface, is unknown.

We have now used mass spectrometry to identify differences between in vitro and in vivo pellicles. In-gel digestion of electrophoretically separated proteins was used to identify the major proteins adsorbed to hydroxyapatite from whole saliva. To compare this finding with that for protein adsorbed to enamel in vivo, a non-abrasive method of collection involving the mechanicochemical removal of pellicle samples was developed. A combination of matrix-assisted laser desorption/ionization (MALDI)–reflectron time-of-flight (TOF) and liquid chromatographic–electrospray ionization (LC–ESI) mass spectral analysis was used to identify protein components of in vivo pellicle samples (Perkins et al., 1993). The electrophoretically separated proteins of in vivo pellicle were compared to those of whole-salivary hydroxyapatite-adsorbed in vitro pellicle.

Section snippets

Materials and methods

Pellicle was collected from six male and six female, healthy, non-medicated individuals, ranging in age from 20 to 31 years. They had good oral hygiene, firm gingiva and no overt signs of gingivitis or caries. The protocol was approved by the Institutional Review Board of Boston University Medical Center and informed consent was obtained from each participant.

In vitro pellicles

The capacity of different solvents to dissociate protein from whole-saliva supernatant adsorbed in vitro to hydroxyapatite was measured in terms of total protein released, as shown in Table 1. It is evident that the solution containing 0.1% SDS removed close to 80% whereas H2O had a negligible effect on protein desorption. NH4HCO3 and NaHCO3 released 70 and 50%, respectively, of the total protein at a concentration of 0.5 M. To gain insight into qualitative differences between these solutions

Discussion

The procedure for in vivo collection of pellicle is very important to the outcome of efforts to characterize its protein composition. Both plaque and pellicle can be removed by mechanical tooth cleaning with a rubber cup and pumice (Sonju and Rolla, 1973). After 2 h, the newly formed pellicle is still essentially free of colonizing bacteria (Sonju and Rolla, 1973) and this provides an opportunity to harvest pellicle free of plaque contamination. Pellicle has traditionally been collected by

Acknowledgements

This work was supported in part by NIH grants DE 05672, DE 07652, RR10888.

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