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Radiat Res. 2018 May;189(5):505-518. doi: 10.1667/RR14963.1. Epub 2018 Feb 23.

A Multiplexed Mass Spectrometry-Based Assay for Robust Quantification of Phosphosignaling in Response to DNA Damage.

Author information

a   Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washingon.
b   Antibody Characterization Laboratory, Leidos Biochemical Research, Inc., Frederick National Laboratory for Cancer Research ATRF, Frederick, Maryland.
c   Office of Cancer Clinical Proteomics Research, National Cancer Institute, Bethesda, Maryland.
d   School of Public Health, University of Washington, Seattle, Washington.
e   Department of Genetics and Genomic Sciences, Icahn Institute of Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, New York.


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.

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