Abstract
Large scale comparative evaluation of protein expression requires miniaturized techniques to provide sensitive and accurate measurements of the abundance of molecules present as individual and/or assembled protein complexes in cells. The principle of competition between target molecules for binding to arrayed antibodies has recently been proposed to assess differential expression of numerous proteins with one-color or two-color fluorescence detection methods. To establish the limiting factors and to validate the use of alternative detection for protein profiling, we performed competitive binding assays under different conditions. A model experimental protocol was developed whereby the competitive displacement of multi-subunit bacterial RNA polymerase and/or its subunits was evaluated through binding to subunit-specific immobilized monoclonal antibodies. We show that the difference in physico-chemical properties of unlabeled and labeled molecules significantly affects the performance of one-color detection, whereas epitope inaccessibility in the protein complex can prohibit the assessment of competition by both detection methods. Our data also demonstrate that antibody cross-reactivity, target protein truncation and abundance, as well as the cellular compartment of origin are major factors that affect protein profiling on antibody arrays. The experimental conditions established for prokaryotic proteins were adopted to compare protein profiles in the breast tumor-derived cell lines MDA MB-231 and SKBR3. Competitive displacement was detected and confirmed for a number of proteins using both detection methods; however, we show that overall the two-color method is better suited for accurate expression profile evaluation of a large, complex set of proteins. Antibody array data confirm the functional linkage between the ErbB2 receptor and AP-2 transcription factors in these cell lines and highlight unexpected differences in G1 cyclin expression.
Highlights
Large scale comparative evaluation of protein expression requires miniaturized techniques to provide sensitive and accurate measurements of the abundance of molecules present as individual and/or assembled protein complexes in cells
Epitopes of the E. coli RNA polymerase ␣, , and Ј subunits were used to follow the displacement of the purified ␣RNAP protein or the whole RNAP protein complex or the overexpressed proteins in crude extracts obtained from IPTG-induced cells
Western blotting of total protein from MDA MB-231 and SKBR3 cells confirmed that cyclin D1 and AP-2␥ were overexpressed in SKBR3 cells, whereas c-Jun and JNK1/2 were at a lower abundance in these cells
Summary
Affinity Purification of Tagged Proteins—The plasmid pET rpoA-his carrying the E. coli gene rpoA coding for ␣RNAP with a C-terminal His tag has been described previously [21]. After washing in PBS/0.05% Tween 20, the membrane was incubated for 1 h with Alexa Fluor 680 goat anti-mouse IgG secondary antibody or IRDye 800-conjugated affinity-purified goat anti-rabbit IgG (LiCOR Biosciences). In two-color detection, antibody arrays were incubated with an equal mixture of samples labeled with IRDye 800CW and Alexa Fluor 680 under the conditions described above and washed using a Protein Array Workstation (PerkinElmer Life Sciences). Normalized ratio (NMRAT) values outside the interval of 0.72–1.28 were considered differentially expressed with 95% statistical confidence when analyzing E. coli cell extracts in imitation experiments. NMRAT values outside the interval of 0.83–1.17 and 0.73–1.27 were considered differentially expressed with, respectively, 70 and 90% statistical confidence when analyzing total protein lysates of breast cancer MDA MB-231 and SKBR3 cell lines. To analyze proteins derived from the nuclear subfraction of the same cell lines, NMRAT values outside the intervals 0.82–1.18 and 0.71–1.29 were considered differentially expressed with, respectively, 70 and 90% statistical confidence
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