Proteomic analysis of human plasma proteins by two-dimensional gel electrophoresis and by antibody arrays following depletion of high-abundance proteins.

ABSTRACT

Detecting proteins that are present at lower levels in human plasma, for the identification of potential disease biomarkers, is complicated by a few highly abundant proteins. One promising strategy is the removal of these abundant proteins interfering with the analysis of plasma content by proteomic techniques. This study compared three affinity-based methods to remove the most abundant proteins in human plasma. Two of them, based on antibodies, which depletes the six or the 12 most abundant proteins, demonstrated the highest efficiency in enriching less abundant plasma proteins. Comparison of two anticoagulant treatments for plasma preparation, EDTA and CTAD, showed that this treatment influenced the patterns of lower-abundance proteins visible on 2-dimensional (2-D) gels. Several staining procedures including two fluorescent dyes, Sypro Ruby and Deep Purple, were also compared with a very sensitive silver staining method for the visualization of lower-abundance proteins on 2-D gels. Furthermore, treatments of lower-abundance plasma proteins with hydroxyethyl disulfide enhanced protein sharpness and resolution. The purpose of all these systematic comparisons was to select the most reliable methods in different steps of plasma preparation and handling as well as in analysis of proteins by 2-D gels to obtain highly reproducible patterns of lower-abundance plasma proteins. Importantly, the lower-abundance plasma proteins enriched by these optimized conditions were further analyzed by antibody microarrays allowing the identification of 61 proteins using 350 antibodies directed against signalling proteins suggesting that this proteomic strategy is a valuable approach for detecting potential plasma biomarkers.