Comparison of High Abundance Protein Depletion Techniques

To identify candidate biomarkers present in human plasma at moderate to low abundance, high abundance proteins (HAPs) must first be removed. Several methods have been described for HAP removal, including antibody affinity, peptide affinity, and ion exchange. The goal of this study was to compare one popular antibody-based affinity method with a newer peptide-based affinity method for HAP removal from human plasma. Two proteomic workflows were used. The first employed separation HAP-depleted eluates on a one-dimensional PAGE gel, followed by in-gel digestion of lane segments and reversed-phase phase LC-ESI-MS-MS (“GeLC-MS”). The second workflow employed tryptic digestion of HAP-depleted eluates followed by nano-reversed phase HPLC and MALDI-TOF-TOF analysis of chromatographic fractions. This study is the first direct comparison of an antibody affinity-based depletion method and a peptide affinity-based depletion method looking at the entire proteomics workflow as used in biomarker discovery programs