Comparison of Diverse High-Abundance Protein Depletion Strategies in Oral-Fluids

Oral fluids (OF) have been suggested as a source of biomarkers for oral and systemic health, 6but as with other bio-fluids, the presence of high-abundance proteins interferes with the detection of lower-abundance biomarkers. Objectives: to compare the performance of four depletion treatments: triple depletion (TD) of amylases, albumins and immunoglobulin G; multiple depletion (MD) of amylases and a panel of 20 proteins, a combination of the two (EMD) and combinatorial peptide ligand library based depletion termed CPLL and to implement this methodology on burning mouth syndrome (BMS)' as a case study. Results: TD, MD, EMD and CPLL removed 76%, 83%, 85% and 94% of total proteins, respectively, coupled with increased low abundance protein detection and narrowed dynamic range. 2-DE revealed that all depletion pretreatments successfully clarified areas hampered by high-abundance proteins; however, EMD and CPLL exposed the highest number of proteins. Quantitative MS of EMD samples relative to none treated samples indicated that most of downregulated proteins (>90%) were EMD target proteins. Quantitative MS analysis of BMS OF  after EMD showed 100 BMS  unique proteins. Conclusion: a multiple step EMD and CPLL depletion approaches bring about the highest number of protein detection ability and the best hampered-area clearance, as demonstrated in BMS OF followed by EMD. As CPLL requires at least 10 fold more protein starting material, we suggest EMD pretreatment as a new detection tool in instances of low protein starting material