The determination of salivary biomarkers as a means of monitoring general health and for the early diagnosis of disease is of increasing interest in clinical research. Based on the linkage between salivary proteins and systemic diseases, the aim of this work was the identification of saliva proteins using proteomics. Salivary proteins were separated using two-dimensional (2-D) gel electrophoresis over a pH range between 3-10, digested, and then analyzed by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-TOF mass spectrometry (MS) and tandem mass spectrometry (MS/MS). Proteins were identified using automated MS and MS/MS data acquisition. The resulting data were searched against a protein database using an internal Mascot search routine. Ninety spots give identifications with high statistical reliability. Of the identified proteins, 11 were separated and identified in saliva for the first time using proteomics tools. Moreover, three proteins that have not been previously identified in saliva, PLUNC, cystatin A, and cystatin B were identified.

In the present study, a proteomic approach was applied to evaluate the influence of salivary protein composition on in vitro dental pellicle formation and its possible correlation with dental caries. Whole saliva, collected from caries-free and caries-susceptible subjects, was analyzed by two-dimensional electrophoresis, and protein spots were identified by mass spectrometry. Data analysis of salivary protein composition showed a statistically significant correlation between the quantity of acidic proline-rich proteins (PRPs), lipocalin, cystatin SN and cystatin S, and samples from the caries-free group of subjects [decayed, missing or filled teeth (DMFT) = 0]. Samples from subjects with a high DMFT index appear to be correlated with high levels of amylase, immunoglobulin A, and lactoferrin. In vitro pellicle-composition experiments showed the same correlations found for whole saliva. As cystatins are known physiological inhibitors of cathepsins, the higher quantities of lipocalin, and cystatins S and SN found in the samples from the caries-free subjects suggest that inhibition of proteolytic events on other salivary proteins may indirectly provide tooth protection. The correlation between higher levels of the phosphorylated acidic PRPs 1/2 with samples from the caries-free group also suggests a protective role for these proteins.

Dental caries is a complex disease, characterized by demineralization of tooth structure. With a protective role, several salivary phosphopeptides appear to be involved in remineralization processes, delaying the loss of tooth structure. In this work we have correlated peptide saliva composition with dental caries susceptibility through the analysis of saliva and hydroxyapatite-adsorbed salivary peptides samples. Saliva samples were obtained from two groups, a caries-free and a cariessusceptible group, and were analysed using HPLC-MS and a sequential extraction with 6 m of guanidine followed by tri fluoroacetate. Data analysis has allowed us to verify a strong correlation between large amounts phosphopeptides (PRP1/3, histatin 1 and statherin), and the absence of dental caries, which reinforces the importance of these peptides in the maintenance of tooth integrity. In addition, in the caries-susceptible group a high number of peptide fragments was observed, suggesting a high proteolytic activity.

Salivary peptides are involved in a wide range of functions constituting the first line of defence of oral cavity and precursors of dental pellicle formation. The presence of mucins in saliva makes difficult the analysis of the proteic content. This is due mainly to aggregation phenomenon between mucins and other high molecular weight glycoproteins and salivary proteins. Considering the importance of salivary peptides in biological functions, we have evaluated the influence of four different extraction methodologies on the separation and identification of these proteins by HPLC-MS. Based on their molecular weight, we identified a total of 22 peptides when extraction was performed using a solution of guanidine (6 m), compared with 14 peptides identified when saliva is acidified with TFA, which is an often used procedure. Our results also show the presence of mucin bind peptides, which include statherin, PRP1, PRP3, Histatin 1 and Histatin 5.

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