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.
Highlights
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
We have addressed complications that arise from the presence of multiple components in an apparent single electrophoretic band by achieving better resolution of the protein mixture through the use of two-dimensional electrophoresis (2-DE)
Data generated in these experiments using 2-DE provided qualitative comparison of the proteins present in enamel pellicle (EP), WS, and major glandular secretions, as well as definitive identification of selected proteins/peptides
Summary
Human Subjects—Healthy non-medicated male and female volunteers, ranging in age from 20 to 60 years, were selected. To recover pellicle proteins from PVDF membranes, 1 ml of distilled water was added to each tube and extraction of pellicle was carried out by vortexing the sample for 30 s followed by sonication (Branson Cleaning Equipment Co., Shelton, CT) for 5 min in an ice bath at 4 °C. Pellicle samples were desalted using sequential dilutioncentrifugation steps in an Amicon microcentrifuge device (Millipore) with a molecular mass cut-off of 3000 Da. Desalted pellicle samples were analyzed using a micro-BCA protein assay to determine protein concentration. 50 l of PS, SMSL, WS, or pooled EP samples containing 100 g of proteins was mixed with 300 l of IEF rehydration buffer in a focusing tray upon which a 17-cm-long, pre-made IPG strip was added. The validity of protein matches was confirmed by either additional tandem data or a careful examination of all MS data available for that particular sample, and total coverage for each result was calculated
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