Serum proteins may often serve as indicators of disease and is a rich source for biomarker discovery. However, the large dynamic range of proteins in serum makes the analysis very challenging because high-abundant proteins tend to mask those of lower abundance. A prefractionation step, such as depletion of a few high-abundant proteins before protein profiling, can assist in the discovery and detection of less abundant proteins that may prove to be informative biomarkers. In the present study, five different depletion columns were investigated considering efficiency, specificity, and reproducibility. Our research included quantitative determination of total protein, albumin, and immunoglobulin G (IgG) concentrations, one- and two-dimensional gels and mass spectrometric analysis of the serum samples before and after the depletion step. Our results showed that all five depletion columns tested removed albumin and IgG with high efficiency. We found that based on reproducibility and binding specificity, the Multiple Affinity Removal Column that removed a total of six high-abundant proteins (albumin, IgG, antitrypsin, IgA, transferring, and haptoglobin) offered the most promising depletion approach. Among the disposable (single-use) products, the ProteoExtract Albumin/IgG Removal kit displayed the best results. Depleted serum from the Multiple Affinity Removal column was further evaluated by 2-D gel electrophoresis (2-DE) analysis, and the results indicated increased resolution and improved intensity of low-abundant proteins in a reproducible fashion. Our study provides a comprehensive investigation of commercially available depletion columns and will be of high importance for future proteomic studies on serum samples.

Comparison of different depletion strategies for improved resolution in proteomic analysis of human serum samples.

Systematic detection of low-abundance proteins in human blood that may be putative disease biomarkers is complicated by an extremely wide range of protein abundances. Hence, depletion of major proteins is one potential strategy for enhancing detection sensitivity in serum or plasma. This study compared a recently commercialized HPLC column containing antibodies to six of the most abundant blood proteins ("Top-6 depletion") with either older Cibacron blue/Protein A or G depletion methods or no depletion. In addition, a prototype spin column version of the HPLC column and an alternative prototype two antibody spin column were evaluated. The HPLC polyclonal antibody column and its spin column version are very promising methods for substantially simplifying human serum or plasma samples. These columns show the lowest nonspecific binding of the depletion methods tested. In contrast other affinity methods, particularly dye-based resins, yielded many proteins in the bound fractions in addition to the targeted proteins. Depletion of six abundant proteins removed about 85% of the total protein from human serum or plasma, and this enabled 10- to 20-fold higher amounts of depleted serum or plasma samples to be applied to 2-D gels or alternative protein profiling methods such as protein array pixelation. However, the number of new spots detected on 2-D gels was modest, and most newly visualized spots were minor forms of relatively abundant proteins. The inability to detect low-abundance proteins near expected 2-D staining limits was probably due to both the highly heterogeneous nature of most plasma or serum proteins and masking of many low-abundance proteins by the next series of most abundant proteins. Hence, non2-D methods such as protein array pixelation are more promising strategies for detecting lower abundance proteins after depleting the six abundant proteins.

Depletion of multiple high-abundance proteins improves protein profiling capacities of human serum and plasma.

Proteomics is the large-scale study of the structure and function of proteins in complex biological sample. Such an approach has the potential value to understand the complex nature of the organism. Current proteomic tools allow large-scale, high-throughput analyses for the detection, identification, and functional investigation of proteome. Advances in protein fractionation and labeling techniques have improved protein identification to include the least abundant proteins. In addition, proteomics has been complemented by the analysis of posttranslational modifications and techniques for the quantitative comparison of different proteomes. However, the major limitation of proteomic investigations remains the complexity of biological structures and physiological processes, rendering the path of exploration paved with various difficulties and pitfalls. The quantity of data that is acquired with new techniques places new challenges on data processing and analysis. This article provides a brief overview of currently available proteomic techniques and their applications, followed by detailed description of advantages and technical challenges. Some solutions to circumvent technical difficulties are proposed.

Proteomics: challenges, techniques and possibilities to overcome biological sample complexity

Immunoaffinity depletion with antibodies to the top 7 or top 14 high-abundance plasma proteins is used to enhance detection of lower abundance proteins in both shotgun and targeted proteomic analyses. We evaluated the effects of top 7/top 14 immunodepletion on the shotgun proteomic analysis of human plasma. Our goal was to evaluate the impact of immunodepletion on detection of proteins across detectable ranges of abundance. The depletion columns afforded highly repeatable and efficient plasma protein fractionation. Relatively few nontargeted proteins were captured by the depletion columns. Analyses of unfractionated and immunodepleted plasma by peptide isoelectric focusing (IEF), followed by liquid chromatography−tandem mass spectrometry (LC−MS/MS), demonstrated enrichment of nontargeted plasma proteins by an average of 4-fold, as assessed by MS/MS spectral counting. Either top 7 or top 14 immunodepletion resulted in a 25% increase in identified proteins compared to unfractionated plasma. Although 23 low-...

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A Keizer-Gunnink

Papers

Sample preparation of human serum for the analysis of tumor markers : Comparison of different approaches for albumin and gamma-globulin depletion