OBJECTIVES: The aim of this study was to examine whether triple depletion of salivary-α-amylase (sAA), albumin (Alb) and immunoglobulins G (IgGs) may improve the visualization capability of proteins in two-dimensional gel electrophoresis (2-DE) of oral fluids (OF). SUBJECTS AND METHODS: Oral fluids 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 using SDS-PAGE followed by 2-DE and protein identification using ion-trap mass spectrometry (MS). RESULTS: This specific triple depletion technique unmasked spots never visualized before. A total of 36 new spots were observed after depletion (348 vs 312 before depletion). Moreover, 58 spots showed more than twofold increase intensity after depletion. In the 60-69kDa 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.
OBJECTIVES: The aim of this study was to examine whether triple depletion of salivary-α-amylase (sAA), albumin (Alb) and immunoglobulins G (IgGs) may improve the visualization capability of proteins in two-dimensional gel electrophoresis (2-DE) of oral fluids (OF). SUBJECTS AND METHODS: Oral fluids 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 using SDS-PAGE followed by 2-DE and protein identification using ion-trap mass spectrometry (MS). RESULTS: This specific triple depletion technique unmasked spots never visualized before. A total of 36 new spots were observed after depletion (348 vs 312 before depletion). Moreover, 58 spots showed more than twofold increase intensity after depletion. In the 60-69kDa 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.
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