September 1, 2008 (Vol. 28, No. 15)
Roberto Campos-Gonzalez Ph.D.
Kathy Brumbaugh Ph.D.
Greta Wegner Ph.D.
Monitoring Protein Levels and Post-Translational Modifications
Antibody arrays are solid matrix assays designed to simultaneously monitor changes in the expression or post-translational modification of multiple cellular protein targets. The multiplexing capabilities are possible due to the “printing” of individual antibodies at precise and defined positions (or coordinates) on the matrix. These arrays offer a rapid and economical way to identify pathways or molecules of interest before targeting them for further study using more focused techniques.
The small footprint of the array combined with a large collection of antibodies also requires a relatively small sample size. In addition, the use of well-characterized antibodies eliminates the need for further target characterization (e.g., mass spectrometry) to identify the signal in the array. The supporting matrix in the antibody array can be made of different materials such as derivatized glass, nitrocellulose, and synthetic polymers. All of these substrates offer advantages and disadvantages depending on the detection methodology used.
The sandwich configuration, a detection methodology adapted from the classic sandwich ELISA, is a proven technique that utilizes a pair of carefully selected antibodies, both recognizing the same molecule. One antibody captures the molecule of interest, while the other is labeled and acts as a means of detection (Figure 1). Because two antibodies must recognize each molecule, the potential for cross-reactivity between any given individual antibody is greatly reduced.
In a sandwich antibody array, the capture antibody is printed on the matrix and binds the target of interest in the sample. This, in effect, acts as an affinity-enrichment step. The detection antibody, coupled to a readout system, confirms the levels of the targeted protein in the array. By analogy, the sandwich antibody array method works like a rapid in situ immunoprecipitation/Western blot, with the obvious added advantage of rapidly assessing many proteins simultaneously.
A main limitation in the development of sandwich antibody arrays is the availability of high-affinity antibody pairs that are fully characterized and optimized for simultaneous use in an array format. Careful screening and validation of many antibody pairs is required to ensure compatibility, specificity, and reproducibility.
Profiling with Macroarrays
Proteome Profiler Antibody Arrays from R&D Systems are macroarrays that use the sandwich antibody configuration. These arrays can routinely detect protein targets in the low picogram per milliliter range. All matched antibody pairs are screened from a myriad of potential combinations until the best pair is identified and optimized for detecting the native protein. Each capture antibody is spotted in duplicate onto a nitrocellulose membrane and chemiluminescence is utilized as the reporter system. In comparison to the microarray format that often requires the use of slide readers or CCD cameras for analysis, the macroarray requires no specialized equipment, allowing it to be used by virtually any laboratory without any additional investment in equipment.
Currently, Proteome Profiler Arrays are capable of measuring up to 59 proteins simultaneously, although additional assays capable of assessing more analytes continue to be developed. The available arrays include assays to monitor the relative levels of molecules associated with key research areas, including angiogenesis, intracellular signaling, apoptosis, and the immune response.
Multiple factors contribute to the angiogenesis process including soluble growth and differentiation molecules, extracellular matrix components, proteases, membrane-bound receptors, and intracellular signaling molecules. The factors involved can vary between cancer types, and the antibody array provides a useful tool for assessing those differences (Figure 2).
The Proteome Profiler Angiogenesis Antibody Array is designed to simultaneously monitor the expression of over 50 different proteins. This collection of targets includes both secreted and intracellular proteins, allowing researchers to evaluate many of the mechanisms and pathways underlying vascular growth.
Kinase cascades can be complex and overlapping, and the integration of many intracellular signals is associated with a biological response. Because of the development of antibodies that recognize phospho-specific sites, intracellular antibody arrays are a useful tool for indicating which proteins might be involved in a given cellular process.
R&D Systems offers several antibody arrays designed to assess the relative phosphorylation of a range of intracellular molecules including those downstream of cytokines, growth factors, cell stress, and cell-to-cell contact. For instance, the response to oxidative stress in Jurkat T cells is accompanied by the site-specific phosphorylation of multiple intracellular substrates including members of the MAP Kinase family, PLC-g, CREB, and AMPKa (Figure 3).
A defective apoptotic system can lead to developmental abnormalities, suppression of immune function, or the inhibition of mechanisms intended to control unregulated cell growth. There are several different pathways that lead to apoptosis including growth factor withdrawal, stimulation by members of the tumor necrosis factor (TNF) superfamily, or through the activity of cytotoxic cells of the immune system. The intracellular factors involved may be context-dependent, differing based on the cell type or the initiating stimulus.
The Proteome Profiler Apoptosis Antibody Array can be used to simultaneously assess 35 key players involved in apoptosis. These include members of the Bcl-2 and TNF superfamilies, caspases, heat shock proteins, tumor suppressors, and several mitochondrial proteins.
For instance, treatment of MCF-7 cells (human breast adenocarcinoma) with the topoisomerase I inhibitor, camptothecin, leads to an increase in the levels of phospho-p53 at three specific residues (Figure 4). The effect is suppressed by pre-treatment with caffeine, an inhibitor of ATM and ATR kinases.
Sandwich antibody arrays offer a sensitive and rapid approach to monitor changes in protein levels and post-translational modifications. In macroarray format they can be used at modest expense, requiring no specialized equipment, and bypassing the need for individual immunoprecipitation/Western blots. The use of antibody arrays as proteomics tools in the areas of biomarker discovery and validation is steadily on the rise.
Concomitantly, they have been used in clinical research to monitor the unique expression patterns of biomarkers found in tissues, serum, and other biological fluids. Increasing the content and the variety of antibody arrays available for basic research will undoubtedly provide a faster path to a more comprehensive understanding of both normal and diseased cells.
Roberto Campos-Gonzalez, Ph.D. (Roberto.Campos@rndsystems.com), is director of the intracellular department, Kathy Brumbaugh, Ph.D., is an intracellular department manager, and Dave Finkel, Greta Wegner, Ph.D., and Jackie Ernst are members of the array group at R&D Systems.