James Martosella

Multi-component immunoaffinity subtraction and reversed-phase chromatography of human serum.

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. Fig. 1 Composition of proteins in human serum. The protein composition is schematically depicted based on mass abundance in normal human serum. The six high-abundant proteins removed by the immunoaffinity column comprise approx 85% of the total protein mass in human serum. Herein we describe immunoaffinity depletion combined with reversed-phase separation modes to reduce the sample complexity of human serum. We selectively immunodepleted six of the most abundant proteins from human serum, then employed gradient elution reversed-phase (RP) HPLC to fractionate the remaining serum proteins. The workflow shown in (Fig. 2) was optimized to process immunodepleted flow-through serum samples directly to a RP column with minimal sample handling. The RP operational conditions permitted robust and repeatable separations and have been optimized specifically for immunodepleted serum samples.