Serum analysis represents an extreme challenge because of the dynamic range of the proteins of interest, and the high structural complexity of the constituent proteins. High-abundant proteins such as albumin, IgG, transferrin, haptoglobin, IgA and alpha1-anti-trypsin represent up to 85% of the total protein mass in serum (Fig. 1). These major protein constituents interfere with identification and characterization of important moderate- and low-abundant proteins by limiting the dynamic range of mass spectral and electrophoretic analysis. During protein isolation, separation, and analysis, these six proteins often mask the detection of the more important low-abundant proteins that are of high interest as biomarkers of disease or drug targets. In one- and two-dimensional gel electrophoresis (1DGE and 2DGE) for example, the spots or bands because of these six highly abundant proteins, as well as their fragments, often overlap or completely mask large regions of the gel, making detection of the myriad low-abundant proteins very difficult, if not impossible. Moreover, proteomic analysis methods commonly include an electrophoretic or chromatographic separation step which, of course, has a finite mass loading tolerance. The presence of a large quantity of high-abundant proteins limits the mass load of targeted proteins that can be initially sampled by these separation methods and thus requires the need for multidimensional separation techniques to reduce sample complexity.

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