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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1 May 2018
Research Article|
February 23 2018
A Multiplexed Mass Spectrometry-Based Assay for Robust Quantification of Phosphosignaling in Response to DNA Damage
Jeffrey R. Whiteaker;
Jeffrey R. Whiteaker
aClinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washingon
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Lei Zhao;
Lei Zhao
aClinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washingon
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Rick Saul;
Rick Saul
bAntibody Characterization Laboratory, Leidos Biochemical Research, Inc., Frederick National Laboratory for Cancer Research ATRF, Frederick, Maryland
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Jan A. Kaczmarczyk;
Jan A. Kaczmarczyk
bAntibody Characterization Laboratory, Leidos Biochemical Research, Inc., Frederick National Laboratory for Cancer Research ATRF, Frederick, Maryland
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Regine M. Schoenherr;
Regine M. Schoenherr
aClinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washingon
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Heather D. Moore;
Heather D. Moore
aClinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washingon
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Corey Jones-Weinert;
Corey Jones-Weinert
aClinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washingon
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Richard G. Ivey;
Richard G. Ivey
aClinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washingon
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Chenwei Lin;
Chenwei Lin
aClinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washingon
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Tara Hiltke;
Tara Hiltke
cOffice of Cancer Clinical Proteomics Research, National Cancer Institute, Bethesda, Maryland
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Kerryn W. Reding;
Kerryn W. Reding
aClinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washingon
dSchool of Public Health, University of Washington, Seattle, Washington
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Gordon Whiteley;
Gordon Whiteley
bAntibody Characterization Laboratory, Leidos Biochemical Research, Inc., Frederick National Laboratory for Cancer Research ATRF, Frederick, Maryland
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Pei Wang;
Pei Wang
eDepartment of Genetics and Genomic Sciences, Icahn Institute of Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, New York
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Amanda G. Paulovich
Amanda G. Paulovich
1
aClinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washingon
1Address for correspondence: Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N. E2-154, Seattle, WA 98109-1024; email: apaulovi@fredhutch.org
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Radiat Res (2018) 189 (5): 505–518.
Article history
Received:
October 23 2017
Accepted:
January 22 2018
Citation
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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