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. 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

Jeffrey R Whiteaker  1 , Lei Zhao  1 , Rick Saul  2 , Jan A Kaczmarczyk  2 , Regine M Schoenherr  1 , Heather D Moore  1 , Corey Jones-Weinert  1 , Richard G Ivey  1 , Chenwei Lin  1 , Tara Hiltke  3 , Kerryn W Reding  1   4 , Gordon Whiteley  2 , Pei Wang  5 , Amanda G Paulovich  1
Affiliations

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

Jeffrey R Whiteaker et al. Radiat Res. 2018 May.

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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Figures

FIG. 1
FIG. 1
Overview of the immuno-MRM assay workflow. Proteins are extracted from the biological sample using procedures amenable to downstream mass spectrometry. The protein lysates are then enzymatically digested, typically using trypsin, and stable isotope-labeled standard (i.e., heavy) peptides corresponding to each analyte are added at known quantities to act as internal standards. The analyte peptides, along with their stable-isotope analogs, are enriched from the complex sample using anti-peptide antibodies coupled to magnetic beads. Measurement of the enriched analytes and standards is performed by liquid chromatography (LC) coupled to quantitative mass spectrometry (MS) using a targeted technique called multiple reaction monitoring (MRM). The peak area ratio (light endogenous analyte peptide to heavy peptide) is used for quantification. Specificity is confirmed by equivalent retention time and relative transition areas for light (endogenous) and heavy (standard) peptides.
FIG. 2
FIG. 2
Use of the multiplexed assay to quantify radiation-induced changes in cellular signaling due to genomic mutations and/or pharmacologic inhibition of kinases. Human lymphoblast cell lines derived from wild-type (ATM+/+, GM07057) and ATM-deficient (ATM−/−, GM01526) patients were harvested in a time course (1, 6, 24 h) after perturbation with 5 Gy irradiation. Cultures of ATM+/+ cells were additionally treated with ATM kinase inhibitor KU-55933 (or control vehicle, DMSO). Panel A: Heatmap for the mean peak area ratio from three biological replicates normalized across samples for each peptide. Analytes detected at all timepoints above the LLOQ are plotted. Group “1”: analytes with decreased response in the presence of the inhibitor compared to the genomic variant. Group “2”: analytes with increased expression levels in the genetic variant (ATM−/−) compared to wild-type cells treated in the presence of kinase inhibitor. Panel B: The peak area ratio (light:heavy) for selected nonphosphorylated and phosphorylated peptides in the DDR panel with changes greater than twofold (P < 0.05). Error bars are the standard deviation of three biological replicates. The LLOQ is indicated by the gray dotted line.
FIG. 3
FIG. 3
Use of the multiplexed assay to quantify the response to 4-nitroquinoline 1-oxide (4NQO). Human lymphoblast cell lines were treated with 4NQO, and the DDR was profiled with the 62-plex immuno-MRM assay in biological triplicate. Control samples received no treatment, 4NQO-treated samples were harvested after 2 h. Samples labeled “AZD6738” refer to treatment in the presence of the ATR kinase inhibitor, and samples labeled “DMSO” contain vehicle only. Panel A: Heatmap showing the response to DNA damage induced by 4-nitroquinoline 1-oxide and the effects of AZD6738. Analytes detected above the LLOQ in all conditions are plotted in the heatmap. Panel B: The peak area ratio (light:heavy) for control (green), 4NQO (orange), 4NQO with vehicle (tan) and 4NQO with inhibitor (black) treatments. Error bars are the standard deviation of three biological replicates. The LLOQ is indicated by the gray dotted line.
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