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

Radiation Research
Jeffrey R WhiteakerAmanda G Paulovich

Abstract

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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Citations

Feb 1, 2019·Expert Review of Proteomics·Amanda LorentzianPhilipp F Lange
Nov 30, 2018·Nature Reviews. Clinical Oncology·Bing ZhangAmanda G Paulovich
Nov 27, 2020·Cell·Francesca PetraliaUNKNOWN Clinical Proteomic Tumor Analysis Consortium
Sep 2, 2021·Expert Review of Proteomics·Jacqueline S Gerritsen, Forest M White
Sep 28, 2021·Molecular Systems Biology·Hasmik KeshishianSteven A Carr

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Datasets Mentioned

BETA
GM07057
GM01526

Methods Mentioned

BETA
Protein Assay
chip
Assay
blood draws
biopsies
ubiquitination

Software Mentioned

Panorama
Skyline
lmer
Nextgen

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