DIGE Analysis of Immunodepleted Plasma.

Abstract

This chapter focuses on upstream immunodepletion of high-abundance proteins from plasma samples and subsequent analysis by fluorescence two-dimensional difference gel electrophoresis (2D-DIGE). The abundances of proteins in biofluid proteomes, such as serum, plasma, saliva, and bronchoalveolar lavage fluid (BALF), can exceed ten orders of magnitude. This substantial dynamic range is problematic for the detection of medium and low-abundance proteins by 2D-DIGE analysis. To increase the detection, quantification, and identification of medium-low-abundance proteins, the targeted depletion of known abundant proteins with antibody columns has been successfully employed. From the literature, it is clear that the performance of abundant protein depletion with immunodepletion columns has been successful in broadening the coverage of the biofluid proteome and facilitating the identification of disease-specific biomarkers. The task for a successful biomarker strategy involves the combination of a reproducible and robust fractionation method, coupled with a highly accurate quantitative method, a task that is exemplified by combining both immunodepletion and 2D-DIGE together to discover significant proteins associated with the disease phenotype.