Differences among techniques for high‐abundant protein depletion

Abstract

The need to identify protein or peptide biomarkers via readily available biological samples like serum, plasma, or cerebrospinal fluid is often hindered by a few particular proteins present at relatively high concentrations. The ability to remove these proteins specifically, reproducibly, and with high selectivity is increasingly important in proteomic studies, and success in this procedure is leading to an ever-increasing list of lower abundant proteins being identified in these biological fluids. The current work addresses some of the potential problems in depleting proteins in typical biomarker studies, including nonspecific binding during depletion procedures and whether low molecular weight (LMW) species bind to the column in a so-called "sponge" effect caused by the ability of albumin or other high-abundant proteins to bind peptides or protein fragments. LC-MS/MS methods were applied to the comparative analysis of an IgG-based immunodepletion method and a Cibacron blue (CB)-dye-based method, for specificity of removing targeted proteins (binding fraction), as well as for assessing efficiency of target removal. This analysis was extended to examine the effects of repeated use of materials (cycles of binding and elution), in order to assess potential for carryover of one sample to the next. Capacity studies and efficiency of protein removal from the serum samples were followed for the IgG-based system using both immunochemical assays (ELISA) as well as LC-MS/MS methods. Additionally, the IgG-based system was further characterized for the removal of LMW polypeptides by nonspecific binding. We conclude that the IgG-based system provided effective removal of targeted proteins, with minimal carryover, high longevity, and minimal nonspecific binding. Significant differences are noted between the depletion techniques employed, and this should be considered based on the expectations set during experimental design.

Proteomics Analysis of Human Amniotic Fluid

“…Among various methods, LC-MS/MS allows identification of proteins in a high throughput fashion unlike the rather slow and laborious 2DE-MS/MS methods. To avoid the masking effect of high abundance proteins, methods based on dye affinity resins (Cibacron blue), Protein A/G, and IgY combinations have been tried (14,15). These approaches have two major disadvantages.…”

Human body fluid proteome analysis

“…The Cibacron blue dye method is a traditional way to deplete albumin. However, the binding of albumin to Cibacron blue dyes is nonspecific, and the sensitivity and specificity are not as effective as mAb-based immunoaffinity resin or columns [15][16][17][18]. Removal of IgG can be realized with Protein G resins or columns [15][16][17][18][19][20].…”

“…However, the binding of albumin to Cibacron blue dyes is nonspecific, and the sensitivity and specificity are not as effective as mAb-based immunoaffinity resin or columns [15][16][17][18]. Removal of IgG can be realized with Protein G resins or columns [15][16][17][18][19][20]. Comparing to Cibacron blue dye and Protein G methods, immunoaffinity depletion using multiple affinity removal columns (MARC) is more effective because it can simultaneously remove multiple abundant proteins, with minimal carryover, high longevity, and minimal nonspecific binding [16,17,21,22].…”

Comparative proteomic analysis of ductal and lobular invasive breast carcinoma

“…We have not removed plasma proteins from the samples. Many studies have used methods for precipitation, centrifugation, and affinity chromatography to remove albumin and immunoglobulin (Chen et al, 2005;Colantonio et al, 2005;Zolotarjova et al, 2005). However, these immunodepletion methods have a limited capacity to bind these profuse proteins; thus, depletion is not complete.…”