Comparative Proteomic Analysis of Human Oral Fluids According to Gender

Background: Oral fluids emerge as a promising tool for the purposes of disease diagnostics and therapy monitoring. The advantages of oral fluids include simple and non-invasive method, with low costs and high compliance of subjects' cooperation. Objectives: To map and detect proteins from human oral fluids using 2-D gels and mass-spectrometry, and to identify and verify differences in protein expression according to gender nature. Methods: Unstimulated whole saliva samples were collected from 6 healthy volunteers ranging ages between 20 and 30 (3 males and 3 females). Proteins were extracted and separated by 2-D SDS-PAGE using pH range of 3-10 followed by 9.5-16.5% gradient gel. Thereafter, the gels were visualized by ammonical silver stain or Coomassie Brilliant blue stain. Gels were digitalized and were processed using PDQuest software (BioRad, USA) in order to compare the samples between male and female groups. Selected protein spots were excised from gel and were analyzed by electrospray ionization - tandem mass spectrometry (ESI-MS/MS). Obtained data were searched against the NCBI non-redundant protein database using Mascot software (Matrix-Science USA). Results: Selected spots, representing 36 proteins complexes that were visualized on the 2-D map, were excised from gels and analyzed by mass-spectrometry. 16 upregulated protein complexes were detected (Ten upregulated protein spots in the female group and 6 upregulated proteins in the male group). These spots are presently being in the process of identification by mass-spectrometry analysis. Conclusion: Our work demonstrates physiological protein composition differences according to gender within the oral fluids of healthy subjects. These differences may be relevant as essential salivary biomarkers in health and disease.