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Molecular & Cellular Proteomics 4:605-617, 2005.
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Competition on Nitrocellulose-immobilized Antibody Arrays

From Bacterial Protein Binding Assay to Protein Profiling in Breast Cancer Cells^*

Garabet Yeretssian, Michèle Lecocq, Guillaume Lebon, Helen C. Hurst^¶ and Vehary Sakanyan^,,||

From the Biotechnologie, Biocatalyse, Biorégulation, Unité Mixte de Recherche 6204, Centre National de la Recherche Scientifique, Université de Nantes, 2 rue de la Houssinière, 44322 Nantes, France; ProtNeteomix, Université de Nantes, 2 rue de la Houssinière, 44322 Nantes, France; and ^¶ Cancer Research UK Molecular Oncology Unit, Barts’ and The London School of Medicine, London EC1M 6BQ, United Kingdom

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.

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