Toward Defining the Human Parotid Gland Salivary Proteome and Peptidome: Identification and Characterization Using 2D SDS−PAGE, Ultrafiltration, HPLC, and Mass Spectrometry †

Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California, United States
Biochemistry (Impact Factor: 3.02). 04/2005; 44(8):2885-99. DOI: 10.1021/bi048176r
Source: PubMed


Saliva plays many biological roles, from lubrication and digestion to regulating bacterial and leukocyte adhesion. To understand the functions of individual components and families of molecules, it is important to identify as many salivary proteins as possible. Toward this goal, we used a proteomic approach as the first step in a global analysis of this important body fluid. We collected parotid saliva as the ductal secretion from three human donors and separated the protein components by two-dimensional SDS-polyacrylamide gel electrophoresis (2D SDS-PAGE). Proteins in gel spots were identified by peptide mass fingerprinting, and the results were confirmed by tandem mass spectrometry of selected peptides. Complementing this approach we used ultrafiltration to prepare a low-molecular-weight fraction of parotid saliva, which was analyzed directly or after reversed phase high-performance liquid chromatography separation by using mass spectrometric approaches. MS analyses of 2D SDS-PAGE spots revealed known components of saliva, including cystatins, histatins, lysozyme, and isoforms and/or fragments of alpha-amylase, albumin, and proline-rich proteins. We also discovered novel proteins, such as several isoforms of Zn-alpha-2-glycoprotein and secretory actin-binding protein. MS analyses of the ultrafiltrate showed that the low-molecular-weight fraction of parotid saliva was peptide-rich, with novel fragments of proline-rich proteins and histatins in abundance. Experiments using Candida albicans as the test organism showed that at least one of the novel peptides had antifungal activity. Our results show that saliva is a rich source of proteins and peptides that are potential diagnostic and therapeutic targets.

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    • "binding calcium and hydroxyapatite [36], and proline-rich proteins, such as IB-5 characterized by the repetitive sequence -KPQGPPPP-, seem involved in dietary tannin binding [37]. Hundreds of smallmedium sized proline-rich peptides showing highly overlapping frames and similar sequences are generated by the pre-and post-secretory events occurring at the expenses of the salivary basic proline-rich proteins [15] [16] [17], but their function is almost still unknown. Among these, a 20 residues peptide (p1932, NH 2 -GPPPQGGNKPQGPPPPGKPQ) was already patented by some of authors of this study for its strong anti-viral activity [PCT/IB2012/050415]. "
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    ABSTRACT: Saliva contains hundreds of small proline-rich peptides most of which derive from the post-translational and post-secretory processing of the acidic and basic salivary proline-rich proteins. Among these peptides we found that a 20 residues proline-rich peptide (p1932), commonly present in human saliva and patented for its antiviral activity, was internalized within cells of the oral mucosa. The cell-penetrating properties of p1932 have been studied in a primary gingival fibroblasts cell line and in a squamous cancer cell line, and compared to its retro-inverso form. We observed by mass-spectrometry, flow cytometry and confocal microscopy that both peptides were internalized in the two cell lines on a time scale of minutes, being the natural form more efficient than the retro-inverso one. The cytosolic localization was dependent on the cell type: both peptide forms were able to localize within nuclei of tumoral cells, but not in the nuclei of gingival fibroblasts. The uptake was shown to be dependent on the culture conditions used: peptide internalization was indeed effective in a complete medium than in a serum-free one allowing the hypothesis that the internalization could be dependent on the cell cycle. Both peptides were internalized likely by a lipid raft-mediated endocytosis mechanism as suggested by the reduced uptake in the presence of methyl-ß-cyclodextrin. These results suggest that the natural peptide may play a role within the cells of the oral mucosa after its secretion and subsequent internalization. Furthermore, lack of cytotoxicity of both peptide forms, highlights their possible application as novel drug delivery agents. Copyright © 2015. Published by Elsevier B.V.
    Biochimica et Biophysica Acta 08/2015; DOI:10.1016/j.bbamem.2015.08.019 · 4.66 Impact Factor
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    • "Integrated platforms have been used to provide fast and reproducible analyses. Despite significant developments in analytical technologies for biofluid analyses, the identification of biomarkers remains a challenge (Bergandi et al. 2007; Hardt et al. 2005). "
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    ABSTRACT: A metabolomic approach was used to analyze endogenous metabolites and to correlate with a specific biological state. The analysis of salivary metabolites is a growing area of investigation with potential for basic and clinical applications. Analyses of children's saliva in dif-ferent dentitions and with or without caries could poten-tially reveal a specific profile related to oral disease risk. Nuclear Magnetic Resonance (NMR) is well suited for mixture analysis followed by Principal Component Anal-ysis combined with Linear Regression (PCA-LR) statistics and was used to identify differences in the salivary metabolites. The classificatory analysis was performed using PCA-LR based on 1,000 cross-validation bootstrap runs from both classifiers in order to increase the data information from a small sample size. The PCA-LR pre-sented a statistically good classificatory performance for children with and without caries with an accuracy of 90.11 % (P \ 0.001), 89.61 % sensitivity (P \ 0.001), and 90.82 % specificity (P \ 0.001). Children with caries lesions presented higher levels of several metabolites, including lactate, fatty acid, acetate and n-butyrate. Saliva from subjects with different dentition stages was also analyzed. Although the salivary samples were poorly classified, permanent dentition presented increased levels of acetate, saccharides and propionate. The NMR data and PCA-LR were able to classify saliva from children with or without caries, with performance indexes comparable to the partial least-squares regression discriminant analysis (PLS-DA) results also performed. Our data also showed similar salivary metabolite profiles for healthy subjects despite the differences in their oral hygiene habits, socio-economic status and food intake.
    Metabolomics 01/2013; 9(3):657-666. · 3.86 Impact Factor
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    • "As IR affects protein production, we examined its global changing effect on the saliva proteome by comparing it to non-irradiated animals and to previous reported proteomic maps [31], [32], [33], [34]. Proteomic analysis of oral fluid (whole saliva) protein composition represents the summed effect of the IR treatment on all glands, not only on the SSGs. "
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    ABSTRACT: Salivary glands (SGs) are irreversibly damaged by irradiation (IR) treatment in head and neck cancer patients. Here, we used an animal irradiation model to investigate and define the molecular mechanisms affecting SGs following IR, focusing on saliva proteome and global transcription profile of submandibular salivary gland (SSG) tissue. We show that saliva secretion was gradually reduced to 50% of its initial level 12 weeks post-IR. Saliva protein composition was further examined by proteomic analysis following mass spectrometry (MS) analysis that revealed proteins with reduced expression originating from SSGs and proteins with increased expression derived from the serum, both indicating salivary tissue damage. To examine alterations in mRNA expression levels microarray analysis was performed. We found significant alterations in 95 genes, including cell-cycle arrest genes, SG functional genes and a DNA repair gene. Tissue damage was seen by confocal immunofluorescence of α-amylase and c-Kit that showed an increase and decrease, respectively, in protein expression. This was coherent with real-time PCR results. This data indicates that IR damages the SSG cells' ability to produce and secrete saliva and proteins, and maintain the physiological barrier between serum and saliva. The damage does not heal due to cell-cycle arrest, which prevents tissue regeneration. Taken together, our results reveal a new insight into IR pathobiology.
    PLoS ONE 07/2012; 7(7):e40636. DOI:10.1371/journal.pone.0040636 · 3.23 Impact Factor
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