Profiling of cellular Signal Transduction for Analysis of primary Tumor Tissue

The analysis of cellular signaling cascades is a valuable approach for understanding the processes that underlie cellular functions. Profiling of central signal transduction pathways requires the detection of protein expression and activation. In recent years, mass spectrometry based analysis systems showed their power by allowing unbiased discovery approaches, whereas novel image-guided analysis systems have given detailed information on the localization of proteins. Nevertheless the Western blot is still the most widely employed approach. In hypothesis driven research, this tool gives reliable results with good sensitivity. With the large efforts that have been put into the generation of binding molecules for array-based proteomics, large numbers of antibodies are available and screening approaches that take advantage of these valuable resources are of interest.<br>Here, we present a novel approach that uses the principles of the Western blot (protein separation by SDS-PAGE; protein immobilization on a solid support; detection by specific antibodies) and combines it with a multiplexed bead array as a readout system. The system allows the generation of hundreds of bead-based Western blot equivalents from a few micrograms of protein and thereby information on the expression and modification of hundreds of proteins is obtained. This new approach combines reproducibility and good linearity with large dynamic range.<br>Taking advantage of our approach, we performed comprehensive signal transduction analyses of clinical ovarian tumor specimen. Whereas for ovarian cancer first-line platinum-based therapy reveals high response rates (75 %), as many as 70 % of patients develop platinum resistance and relapse. Mechanisms of cellular resistance are still unknown as well are predictive markers. Using our system we compared tumor specimen from relapsed to cured patients directly on the protein level. Hundreds of antibodies were employed to analyze the activation state of different signaling cascades. Data on changes in signal transduction during resistance development is shown