An approach to remove albumin for the proteomic analysis of low abundance biomarkers in human serum

Abstract

Proteomic technologies are being used to discover and identify disease-associated biomarkers. The application of these technologies in the search for potential diagnostic/prognostic biomarkers in the serum of patients has been limited by the presence of highly abundant albumin and immunoglobulins that constitute approximately 60-97% of the total serum proteins. The purpose of the study was to evaluate whether treatment of human serum with Affi-Gel Blue alone or in combination with Protein A (Aurum serum protein mini kit, Bio-Rad) before two-dimensional gel electrophoresis (2-DE) analysis removed high abundance proteins to allow the visualization of low abundant proteins. Serum samples were treated with either Affi-Gel Blue or Aurum kit and then subjected to 2-DE using 11 cm, pH 4-7 isoelectric focussing strips for the first dimension and 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis for second dimension. Protein spots were visualized using a fluorescent protein dye (SYPRO Ruby, Bio-Rad). Comparison between treatment methods showed significant removal of albumin by both Affi-Gel Blue and Aurum kit and considerable differences in the protein profile of the gels after each treatment. Direct comparison between treatments revealed twenty-eight protein spots unique to Affi-Gel Blue while only two spots were unique after Aurum kit treatment. Unique spots in Affi-Gel Blue and Aurum kit treated serum were not visualized in untreated serum. Sixteen hours of Affi-Gel Blue treatment resulted in enhanced visualization of fifty-three protein spots by two-fold, thirty-one by five-fold, twelve by ten-fold and six by twenty-fold. In parallel after Aurum kit treatment two-, five-, ten- and twenty-fold enhancements of thirty, thirteen, eight and five protein spots, respectively, were observed. The pattern of increased visualization of protein spots with both treatment methods was similar. In conclusion, treatment of serum samples with Affi-Gel Blue or Aurum kit before 2-DE analysis can be used to remove high abundance proteins in order to increase the detection sensitivity of proteins present in low abundance.

Proteomic-based identification of haptoglobin-1 precursor as a novel circulating biomarker of ovarian cancer

“…Recently, we have demonstrated that the use of AffiGel Blue and Aurum kit (Bio Rad Laboratories, USA) results in the removal of highly abundance albumin and simultaneously enhances the detection of several low abundance proteins (Ahmed et al, 2003). In this study, we used columns containing a mixture of Affi-Gel Blue and protein A (5 : 1) that is equally effective in removing high abundance albumin.…”

“…The spin columns containing a mixture of Affi-Gel Blue and Protein A selectively bind and remove albumin and immunoglobulin (Ahmed et al, 2003). The spin columns were washed twice with 1 ml of binding buffer (20 mM phosphate buffer, pH 7.0) by centrifugation for 20 s at 1000 g. In all, 50 ml of serum was added to 150 ml of binding buffer and mixed by vortex and loaded on the spin columns.…”

Human body fluid proteome analysis

“…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].…”

“…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].…”

Mature Erythrocyte Surface Antigen Protein Identified in the Serum of Plasmodium falciparum-Infected Patients

“…4 In combination with mass spectrometry techniques, 2-DE is important in proteomics research for the discovery and identification of disease-associated markers. 5 In this study, we investigated the presence of P. falciparum proteins in the serum of malaria patients with the aim of identifying circulating proteins that may be useful as markers for diagnosis. Serum samples were depleted and analyzed using two approaches: direct mass spectrometry analysis and 2-DE followed by western blot and mass spectrometry.…”