Comparison of diverse affinity based high-abundance protein depletion strategies for improved bio-marker discovery in oral fluids

Oral fluids (OF) have been suggested as a source of biomarkers for oral and systemic health, but as with other bio-fluids, the presence of high-abundance proteins interferes with the detection of lower-abundance biomarkers. Here, we compared 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. 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. In 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 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.

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► We evaluated and compared four OF protein depletion approaches.
► All depletion treatments (TD, MD, CPLL and EMD) successfully cleared hampered areas.
► All treatments increased the visibility of low abundance proteins.
► EMD exposed highest number of spots in instances of low protein starting material.
► Quantitative MS showed 32 over expressed proteins (> 2 fold) following EMD.