Proteomic Characterization of Human Saliva after Alpha Amylase Removal

Proteomic characterization of human whole saliva 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. The high abundance of proteins, mainly alpha amylase, hampers the detection of low abundant proteins appearing in the disease state and therefore should be removed. This has been claimed as one of the "critical challenges" in this field. Objectives: To analyze human whole saliva protein composition following the removal of alpha amylase by an affinity adsorption device. Methods: Whole saliva from four healthy volunteers and Sjogren syndrome patients was collected and subjected to affinity based amylase removing device (provisional patent application 60915204). The filtrate was analyzed by activity assay, protein composition by SDS-PAGE, 2-DE and identified by Mass spectrometry (MS). Results were reported only when p<0.005 using t-test. The captured amylase was eluted from the device and analyzed by SDS-PAGE and MS. Results: Depleted whole saliva was analyzed by SDS-PAGE showing at least six fold removal efficiency and by an alpha amylase activity assay showing 97% reduced activity. 2-DE analysis showed the selective removal of alpha amylase and consequently increased gel resolution. MS identification of protein spots in the 60 kDa area revealed 13 spots which were masked before alpha amylase removal. Specificity was proven by elution of the captured amylase followed by SDS-PAGE and MS. Alpha amylase content was 86% from total protein content. Examining depleted whole saliva from Sjogren patients demonstrated new protein spots suggesting a new area in the salivary proteomic map to be studied. Conclusions: Treatment of human whole saliva with an alpha amylase removing device increases both gel resolution and enabled a higher protein sample for analysis.