Profiling receptor tyrosine kinase activation by using Ab microarrays

Abstract

Signal transduction in mammalian cells is mediated by complex networks of interacting proteins. Understanding these networks at a circuit level requires devices to measure the amounts and activities of multiple proteins in a rapid and accurate manner. Ab microarrays have previously been applied to the quantification of labeled recombinant proteins and proteins in serum. The development of methods to analyze intracellular signaling molecules on microarrays would make Ab arrays widely useful in systems biology. Here we describe the fabrication of multiplex Ab arrays sensitive to the amounts and modification states of signal transduction proteins in crude cell lysates and the integration of these arrays with 96-well microtiter plate technology to create microarrays in microplates. We apply the Ab arrays to monitoring the activation, uptake, and signaling of ErbB receptor tyrosine kinases in human tumor cell lines. Data obtained from multicolor ratiometric microarrays correlate well with data obtained by using traditional approaches, but the arrays are faster and simpler to use. The integration of microplate and microarray methods for crude cell lysates should make it possible to identify and analyze small molecule inhibitors of signal transduction processes with unprecedented speed and precision. We demonstrate the future potential of this approach by characterizing the action of the epidermal growth factor receptor inhibitor PD153035 on cells by using Ab arrays; direct scale-up to array-based screening in 96-and 384-well plates should allow small molecules to be identified with specific inhibitory profiles against a signaling network.T he signal transduction systems that control cellular physiology are comprised of biochemical networks with shared components, common inputs, and overlapping outputs. Understanding how signals flow through these pathways, how the pathways vary among cell types, and how normal and diseased tissues differ requires information on signaling networks as a whole rather than simply on one or two components. To make network (or systems) biology possible, we need devices that can probe the activities of signaling proteins in a parallel and reliable manner. We envision these as a biological analog of the multiprobe ''bed of nails'' testers that are a mainstay of the electronics industry. Bed of nails testers can monitor printed circuit boards at enough locations to fully trace and test a circuit.In this paper we describe the development of an Ab microarray integrated with 96-well microtiter plates that can quantify the amounts and modification states of ErbB receptors in crude cell lysates. Ab microarrays are an extension of DNA microarrays. In both cases, ratiometric comparisons derived from differentially labeled control and experimental samples are an effective way to standardize measurements among and within experiments (1). Ab arrays have the potential to reveal the amounts and modification states of proteins and also, when integrated with fractionation steps, subcellular protein compartm...

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