Preparation of Bodily Fluids for 2-D PAGE

Abstract

The importance of bodily fluids 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 glyco-protein profiles (6). Highly effective depletion of albumin using monoclonal antibody selection has also been demonstrated, and coupled with protein G/IgG depletion (7).Recent research has identified 325 distinct proteins from 1800 2-D gel protein features following multi-component immunoaffinity extraction and further comprehensive chromatographic fractionation (8). Depletion of IgG by a protein G resin can also be coupled with NaCl/Ethanol precipitation to deplete albumin (9). However, depletion can cause problems in itself. The concentration of the high abundant proteins varies considerably within bodily fluids. Therefore once the samples are depleted, there is a significant change in the relative concentration of the protein constituents from the original sample. As an example in our own studies the percentage protein remaining after depletion in cerebrospinal fluid has an average of 17.9% with a standard deviation of 6.6%. This could be overcome by loading according to sample volumes rather than protein loading, which is often the norm in proteomic experiments (10). A major drawback is that the depletion not only adds further steps in sample processing, but undoubtedly alters the protein constituents within the sample as high abundant proteins bind other proteins. Consequently, the depletion may complicate quantitative comparisons and result in the loss of potential biomarkers (11).