Improved visualization of oral fluid proteome after triple depletion treatment

INTODUCTION: Proteomic characterization of oral fluids (OF) for the identification of disease-specific biomarkers is guaranteed to be an easy to use and powerful diagnostic tool for defining the onset, progression and prognosis of human systemic diseases and in particular oral diseases. Salivary amylase, Albumin and IgGs are considered as high abundance proteins in the saliva. As such, they hamper the detection of low abundant proteins appearing in disease states and therefore should be removed. The removal of high abundant proteins has been claimed as one of the "critical challenges" in the salivary proteomics' field. OBJECTIVES: To analyze human OF protein composition following the removal of salivary amylase, albumin and IgGs by a triple depletion method. SUBJECTS and METHODS: OF from healthy volunteers were subjected sequentially to sAA removing device followed by application to an Alb and IgG immunoaffinity column (triple depletion). The depleted OF samples were analyzed by SDS-PAGE followed by 2-DE and protein identification by ion-trap mass-spectrometry (MS). RESULTS: This specific triple depletion technique unmasked spots never visualized before. 36 new spots were observed after depletion (348 vs. 312 before depletion). 58 spots showed more than 2-fold increase intensity after depletion. In the 60 kDa-69kDa area the depletion procedure unmasked 14 proteins including HSP70, LTA4H, L-Plastin, Desmoplakin that are known to be involved in disease pathogenesis. CONCLUSION: The ability to selectively remove and elute the most abundant OF proteins visualized on the 2-DE represents an important step in the characterization of human OF. The better visualization and gel resolution achieved will improve quantification abilities in 2-DE and in tag-MS leading to better identification of disease-specific biomarkers. We further analyzed the eluted Alb and IgGs isoforms suggesting a new methodology venue for OF.