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(PP294) Understanding radiation-modulated proteins: an array based approach.
Coleman, Matthew*,1, Pearson, Francesca1, Bourguet, Feliza 1, Jones, Irene1, Wyrobek, Andrew 1, Marchetti, Francesco 1, 1 Biomedical Division, Biosciences Directorate, Livermore, CA, USA
ABSTRACT- The cellular effects of IR occur through multiple pathways involving changes in protein functionality through post-translational modifications and changes in the amounts of protein expressed. The steadily increasing sophistication in our understanding of the early biochemical responses of irradiated cells and tissues provides the opportunity for developing mechanism-based biosignatures of exposure and methodologies to validate them. Compelling breakthroughs have been made in transcriptional technologies for genome-scale analysis of cellular profiles, but less has been accomplished for the proteome. To assess current knowledge of IR effects we surveyed the literature and generated a panel of 110 radiation-sensitive proteins. Most of these studies only reported data on a few proteins. To better understand the complexity of proteomic changes in response to IR we are applying array-based technologies to look at the DNA damage response pathways. Antibody microarrays were designed using antibodies to proteins such as TP53, ATM and gamma H2AX, to simultaneously profile multiple proteins. A label-free surface plasmon resonance (SPR) technology from Biacore was used to provide sensitive kinetic data. Our pilot experiments have shown that both approaches are sensitive enough for differential profiling of H2AX protein modifications in human samples below 1 ng/ml levels. Our array-based approach will be valuable for understanding the relationship between transcriptional and proteomic responses induced by IR exposure and how differences in tissue and individual responses may relate to differential health consequences. This work was performed under the auspices of the U.S. Department of Energy by the University of California, Lawrence Livermore National Laboratory under Contract No. W-7405-Eng-48 with funding from the Laboratory Directed Research and Development and DOE Low Dose Radiation Research Programs.
Key words: proteome, arrays, antibody
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