Identification of Protein Components in Human Acquired Enamel Pellicle and Whole Saliva Using Novel Proteomics Approaches

Abstract

Precursor proteins of the acquired enamel pellicle derive from glandular and non-glandular secretions, which are components of whole saliva. The purpose of this investigation was to gain further insights into the characteristics of proteins in whole saliva and in vivo formed pellicle components. To maximize separation and resolution using only micro-amounts of protein, a two-dimensional gel electrophoresis system was employed. Protein samples from parotid secretion, submandibular/sublingual secretion, whole saliva, and pellicle were subjected to isoelectric focusing followed by SDS-PAGE. Selected protein spots were excised, subjected to "in-gel" trypsin digestion, and examined by mass spectrometry (MS). The data generated, including peptide maps and tandem MS spectra, were analyzed using protein data base searches. Components identified in whole saliva include cystatins (SA-III, SA, and SN), statherin, albumin, amylase, and calgranulin A. Components identified in pellicle included histatins, lysozyme, statherin, cytokeratins, and calgranulin B. The results showed that whole saliva and pellicle have more complex protein patterns than those of glandular secretions. There are some similarities and also distinct differences between the patterns of proteins present in whole saliva and pellicle. MS approaches allowed identification of not only well characterized salivary proteins but also novel proteins not previously identified in pellicle.Human teeth are exposed to whole saliva (WS), 1 consisting mainly of secretions derived from three pairs of major salivary glands, which comprise parotid, submandibular, and sublingual glands. Protein components that have been identified in all of the major glandular secretions are proline-rich proteins (acidic, basic, and glycosylated families), amylase, statherin, histatins, lysozyme, lactoferrin, lactoperoxidase, and secretory IgA (1-10), whereas cystatins and mucins have been identified in submandibular/sublingual secretions (9, 11-13). However, detailed understanding of the protein composition in WS is still limited because of the lack of knowledge about proteins in other contributors to whole saliva such as secretions from minor salivary glands and gingival crevicular fluid. In addition, little is known about modifications that occur on proteins during or after secretion into the oral cavity.The acquired enamel pellicle (EP) is a protein film thought to result from the selective adsorption of precursor proteins present in WS onto tooth surfaces. Because of its intimate contact with enamel surfaces, the EP plays an important role in maintaining tooth integrity by controlling the mineral solution dynamics of enamel. At its interface with the oral environment, the EP exerts selectivity on bacterial attachment and is involved in the initial stages of plaque formation (14). Because of the limiting amount of proteins that can be harvested from EP formed in vivo, previous investigations have utilized sensitive but indirect approaches such as enzymatic assays and immunologic detectio...

Complexity of the human whole saliva proteome

“…On this point of view, our results amplify the most recent saliva proteomic characterization (7,9,20) and do not support the previous conclusion that major proteins, such as α-amylase and albumin, display little if any modification (21).…”

“…Saliva is usually taken as a complex medium, that was recently characterized by proteomic approach (7,9,20,21), allowing the cumulated identification of up to 35 accessions.…”

“…A main feature raising from the 320 protein spots that were characterized in these works (7,9,20,21), is that the saliva proteome contains a relatively low number of different accessions, but, conversely, spread out over a large number of protein spots (Table II).…”

“…However, very recently, three works introduced a proteomic technology, combining resolutive two-dimensional electrophoresis and mass spectrometry, to map whole saliva proteins. Accordingly, the saliva proteome was firstly shown to display a relatively simple pattern with 7 accessions identified in 10 protein spots (21). By opposition, in two other works, a much more complex situation was described (7,20).…”

On-plate digestion using a commercial microfraction collector for nano-HPLC matrix-assisted laser desorption/ionization tandem time-of-flight protein analysis

“…Pellicle formation is considered a dynamic process influenced by several factors inherent to the individual subject, such as circadian cycle, oral microflora, proteolytic activity, physical and chemical properties of the tooth, as well as, the location of the teeth in the mouth (6). Several proteins have been identified as major components of enamel pellicle, such as proline-rich proteins (PRPs), statherin, amylase, albumin, S-IgA, cystatins, lysozyme, carbonic anhydrase, lactoferrin and histatins (8)(9)(10)(11). The small amount of sample obtained from each individual, associated with its biological importance, makes the characterization of acquired enamel pellicle proteins an analytical challenge.…”