A lack of analytically robust and multiplexed assays has hampered studies of the large, branched phosphosignaling network responsive to DNA damage. To address this need, we developed and fully analytically characterized a 62-plex assay quantifying protein expression and post-translational modification (phosphorylation and ubiquitination) after induction of DNA damage. The linear range was over 3 orders of magnitude, the median inter-assay variability was 10% CV and the vast majority (∼85%) of assays were stable after extended storage. The multiplexed assay was applied in proof-of-principle studies to quantify signaling after exposure to genotoxic stress (ionizing radiation and 4-nitroquinoline 1-oxide) in immortalized cell lines and primary human cells. The effects of genomic variants and pharmacologic kinase inhibition (ATM/ATR) were profiled using the assay. This study demonstrates the utility of a quantitative multiplexed assay for studying cellular signaling dynamics, and the potential application to studies on inter-individual variation in the radiation response.
A Multiplexed Mass Spectrometry-Based Assay for Robust Quantification of Phosphosignaling in Response to DNA Damage
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Jeffrey R. Whiteaker, Lei Zhao, Rick Saul, Jan A. Kaczmarczyk, Regine M. Schoenherr, Heather D. Moore, Corey Jones-Weinert, Richard G. Ivey, Chenwei Lin, Tara Hiltke, Kerryn W. Reding, Gordon Whiteley, Pei Wang, Amanda G. Paulovich; A Multiplexed Mass Spectrometry-Based Assay for Robust Quantification of Phosphosignaling in Response to DNA Damage. Radiat Res 1 May 2018; 189 (5): 505–518. doi: https://doi.org/10.1667/RR14963.1
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