PNNL

Abstract

The detection of low-abundance protein disease biomarkers from human blood poses significant challenges due to the high dynamic range of protein concentrations that span more than 10 orders of magnitude, as well as the extreme complexity of the serum/plasma proteome. Therefore, experimental strategies that include the removal of high-abundance proteins have been increasingly utilized in proteomic studies of serum, plasma, and other body fluids to enhance detection of low-abundance proteins and achieve broader proteome coverage. However, both the specificity and reproducibility of the high-abundance protein depletion process represent common concerns. Here, we report a detailed evaluation of the performance of two commercially available immunoaffinity subtraction systems commonly used in human serum/plasma proteome characterization by high resolution LC-MS/MS. One system uses mammalian IgG antibodies to remove six of the most abundant plasma proteins, and the other uses chicken immunoglobulin yolk (IgY) antibodies to remove twelve of the most abundant plasma proteins. Plasma samples were repeatedly processed using these two systems, and the resulting flow-through fractions and bound fractions were individually analyzed for comparison. Removal of target proteins by both immunoaffinity subtraction systems proved reproducible and efficient. Nontarget proteins, including spiked protein standards, were also observed to bind to the columns, but in a fairly reproducible manner. The results suggest that these multi-protein immunoaffinity subtraction systems are both highly effective and reproducible for removing high-abundance proteins and therefore, can be readily integrated into quantitative strategies to enhance detection of low-abundance proteins in biomarker discovery studies.