A robust, streamlined, and reproducible method for proteomic analysis of serum by delipidation, albumin and IgG depletion, and two‐dimensional gel electrophoresis

Abstract

Serum is a readily available source for diagnostic assays, but the identification of disease-specific serum biomarkers has been impeded by the dominance of human serum albumin and immunoglobulins (Igs) in the serum proteome. There is a need to reduce the technical variation in serum processing and analysis to allow for a reproducible analysis of large cohorts. To this end, we have developed a rapid and reproducible procedure for sample preparation and high-resolution two-dimensional gel electrophoresis to analyze human serum. Serum is centrifuged at high speed to remove lipids and aggregated proteins, incubated with protein G resin to remove IgG, precipitated with NaCl/ethanol to deplete albumin, and slowly resolubilized in a sodium dodecyl sulfate (SDS)/N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) (HEPES) buffer. The delipidated and IgG/albumin depleted serum proteins are focused on pH 4-7 linear large immobilized pH gradient strips, and then resolved by Bis-Tris SDS-polyacrylamide gel electrophoresis. The robustness and reproducibility of the optimized procedure was determined for three individual serum samples on three consecutive days. An image analysis of the nine silver-stained gels demonstrated that the intensity and localization of protein spots are highly reproducible. Our IgG and albumin depletion procedure will aid in screening the patient sera for normal biological variation and disease-specific biomarkers.

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].…”

Investigation of an albumin‐enriched fraction of human serum and its albuminome

“…There are two primary methods available for isolating albumin from serum: affinity-based (e.g., antibody, cibacron blue) and chemical-based methods (e.g., NaCl/EtOH [9,10] TCA/ acetone [11]). Many of the affinity-based methods have been compared and shown to effectively remove albumin [7,12,13].…”

“…Alternatively, albumin has been purified using NaCl/EtOH since the 1940s [16] and this method is routinely used for isolating pharmaceutical grade albumin. Recently, this process was optimized for the proteomics field to minimize the steps required for effective purification and removal of albumin [9], but copurification of other proteins may still be an issue.…”

Predict, prevent and personalize: Genomic and proteomic approaches to cardiovascular medicine

“…Only four proteins (albumin, transferrin, haptoglobulin and immunoglobulins) make up more than 90% of the protein mass in the blood (7). Although techniques exist for depleting these proteins from the serum (18,19), challenges remain to assure their efficiency and specificity.…”