Evaluation of highabundance protein removal using 2DE

More detailed comparisons of the alternative depletion methods were conducted using 2-D gels (Fig. 2). The incomplete depletion of albumin and IgG using the dye-based spin column was confirmed by analysis of the flow-through fraction at two different loads (Fig. 2, top panels). Even at low loads (100 pg) both "depleted" proteins could be readily detected. Nonetheless, as a result of the reduced levels of these two most abundant proteins, sample loads could be increased up to five-fold compared with unfractionated serum before excessive horizontal streaking was observed with a high-sensitivity silver stain. The more efficient removal of albumin and IgG with the two-protein antibody affinity spin column was readily evident

Fig. 2 Evaluation of major protein depletion on 2D gels. Top panels, separation of human plasma before and after depletion using a dye affinity method shows incomplete removal of albumin (indicated with arrow) and IgG (ellipses) in the depleted fractions. Dual-antibody depletion shows slightly better removal ofthe two proteins from a serum sample ofthe same blood donor (middle panels). Bottom panels, separation of

Fig. 2 Evaluation of major protein depletion on 2D gels. Top panels, separation of human plasma before and after depletion using a dye affinity method shows incomplete removal of albumin (indicated with arrow) and IgG (ellipses) in the depleted fractions. Dual-antibody depletion shows slightly better removal ofthe two proteins from a serum sample ofthe same blood donor (middle panels). Bottom panels, separation of plasma on the MARS HPLC column (albumin indicated with arrow; the other five proteins, including major proteolytic products and aggregates are enclosed in ellipses). Top-6 depletion effectively removes ~85% of total protein content (based on manufacturer product claims as well as BCA assay results), allowing 10-to 20-fold higher protein loads, at which point other abundant proteins become problematic.

when depleted fractions from 100 and 500 mg of serum were analyzed (Fig. 2, middle panels). Specifically, the unbound fraction from the albumin/IgG immu-noaffinity resin resulted in less horizontal streaking at a five-fold increased load, and reduced amounts of albumin and IgG were observed compared with the dye-based method. Finally, as expected, depletion of six abundant proteins on the

MARS HPLC column instead of only two proteins resulted in further improvements in protein loading capacity on 2-D gels (Fig. 2, bottom panels). Under these conditions, the HPLC column removed about 85% of the total protein in human serum or plasma samples and this reduced complexity enabled 10- to 20-fold higher loads of depleted serum or plasma (1.0-2.0mg) on silver-stained gels compared with unfractionated samples (about 100 mg). In addition, 2-D gels of pools of the bound fraction from replicate runs ofthe same sample are essentially identical, indicating that the same set of proteins are continually removed (data not shown).

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