Depletion of major proteins to enhance detection of lower abundance proteins

The effectiveness of major protein depletion using several different methods is summarized in Fig. 1. As noted above, prior to the availability of the MARS polyclonal antibody column, the primary method for depleting major plasma or serum proteins was to use Cibacron blue for albumin and Protein A or G for IgG. As part of this study, five commercial kits were tested that used either a blue-dye alone, or in association with Protein A or G to deplete albumin and IgG. There were only minor differences among the kits (data not shown). The best of the dye-based products tested appeared to be the Proteoprep Blue Albumin Depletion kit (Sigma-Aldrich, St. Louis, MO, USA), which is shown here as representative of the dye-based/Protein G spin columns (Fig. 1A, dye-based affinity). While this method depletes considerable amounts of albumin and IgG (Fig. 1, panel A), substantial amounts of these proteins remain in the unbound fractions. In addition, there are a number of high molecular weight bands that nonspecifically bind along with the targeted proteins. In contrast, a dual antialbumin and IgG antibody column (Fig. 1B), a b-test product subsequently commercialized as the Proteoprep Immunoaffinity Albumin and IgG Depletion Kit (Sigma-Aldrich), shows more complete depletion of albumin and IgG. However, several high molecular weight proteins are evident on 1-D gels. Some, but not all of these high molecular weight bands are cross-linked or incompletely reduced albumin or IgG. Not surprisingly, the most efficient enrichment of lower abundance proteins is achieved using the MARS HPLC column. The observed protein bands in the bound fraction on 1-D gels are the six targeted proteins, with no apparent evidence of nonspecific binding, at least using this low-sensitivity, low-resolution detection method (Fig. 1, panel C). In addition, a more convenient b-test spin column version of the MARS was evaluated in preliminary experiments. Initial 1-D gel results suggested depletion similar to that obtained with the HPLC column (Fig. 1, panel D).

Fig. 1 Comparison of major protein depletion techniques. (A) Human plasma (50 pL) was processed using a representative blue-dye/Protein G based kit. While a significant amount of albumin (arrows) and IgG (solid arrowheads) is depleted from the plasma, there is nonspecific binding of other proteins. (B) Human plasma (25 pL) depleted using the prototype Proteo-prep Immunoaffinity Albumin and IgG depletion kit. Majority of albumin and IgG was successfully depleted using this column; however, other major proteins still limit sample loads and detection of low-abundance proteins, and nontargeted bands appear in the bound

Fig. 1 Comparison of major protein depletion techniques. (A) Human plasma (50 pL) was processed using a representative blue-dye/Protein G based kit. While a significant amount of albumin (arrows) and IgG (solid arrowheads) is depleted from the plasma, there is nonspecific binding of other proteins. (B) Human plasma (25 pL) depleted using the prototype Proteo-prep Immunoaffinity Albumin and IgG depletion kit. Majority of albumin and IgG was successfully depleted using this column; however, other major proteins still limit sample loads and detection of low-abundance proteins, and nontargeted bands appear in the bound fraction. (C, D) Plasma (15 and 10 pL) were separated usingthe Multiple Affinity Removal HPLC (4.6 cm x 50 mm) and spin column, respectively. Six most abundant plasma proteins were effectively removed. Albumin and IgG are indicated as above; transferrin, haptoglobin, IgA, and a-1-antitrypsin are indicated by open arrowheads. Samples were separated on 10% Bis-Tris 1-D gels and stained with colloidal CBB; all samples loaded onto the gel were volume-normalized to the undepleted sample (10 pg); P, undepleted plasma, U, unbound fraction, B, bound fraction.

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