Serum or Plasma Sample Preparation for Two-Dimensional Gel Electrophoresis
The importance of serum/plasma as a source of clinically relevant biomarkers/surrogate markers of human disease has increased significantly over the last decade (1,2), and modern proteomic methods have evolved and been adapted to meet the demand. The specific challenges facing serum analysis include the wide dynamic range in the concentration of individual components and the tremendous number of potential variants of glycosylated proteins (3). The most dominant plasma proteins, albumin and immunoglobulin (Ig)G, typically comprise up to 70% of the plasma proteome in abundance. To enable the majority of the remaining, far less abundant proteins to be better visualized by two-dimensional gel electrophoresis (2-DE), these two proteins must first be removed, or at least depleted in relative concentration. There are a number of currently available commercial products from a range of suppliers that enable albumin depletion by chemical affinity, exploiting the remarkable albumin-binding ability of structures closely related to the reactive dye molecule Cibacron blue 3GA (4), and the IgG binding properties of protein G (5). The blue dye has been shown to have a special affinity for proteins containing the dinucleotide fold, a structural feature that is common to several classes of proteins (4). Albumin can be separated from other plasma proteins using lectin affinity, as it is not normally glycosylated, while the majority of classical plasma proteins are. This approach allows both enrichment of lower-abundance proteins, and the study of differences in glycoprotein profiles (6).
KeywordsNeuraminic Acid Magnesium Acetate Plasma Proteome Lectin Affinity Steady Flow Rate