Paulovich - DDR_assay_panel_radiation_response

DDR panel radiation response

  • Organism: Homo sapiens
  • Instrument: 6500 QTRAP
  • SpikeIn: No
We developed and fully analytically characterized a 62-plex assay quantifying protein expression and post-translational modification (phosphorylation and ubiquitination) following induction of DNA damage. The multiplexed assay was applied in proof-of-principle studies to quantify signaling following genotoxic stress (ionizing radiation and 4-nitroquinoline 1-oxide) exposure in immortalized cell lines and primary human cells. The effects of genomic variants and pharmacologic kinase inhibition (Atm/Atr) were profiled using the assay. The study shows the utility of using a quantitative multiplexed assay for studying cellular signaling dynamics, and the potential application to studies on inter-individual variation in the radiation response.
Experiment Description
Lymphoblast cell lines derived from an ataxia telangiectasia (A-T) patient (ATM -/-) and from a healthy control (ATM +/+) were used as a model system for profiling the effects of genomic mutation and pharmacological kinase inhibition on kinase activity following DNA damage by ionizing radiation. Using the 62-plex assay, we profiled the time-course (1, 6, and 24 hours post-IR) of cell signaling (i.e. pharmacodynamic profile) in cells treated with 5 Gy ionizing radiation. An aliquot of ATM+/+ cells were also treated with the ATM kinase inhibitor KU-55933 prior to exposure to 5 Gy ionizing radiation. LCLs treated with 4-nitroquinoline 1-oxide (4NQO) were used to mimic the effects of ultraviolet radiation. Human LCLs were harvested after 2 hours incubation with 2.5 uM 4NQO. Finally, to demonstrate the potential for applying the assay to primary human cells, we measured the DDR in peripheral blood mononuclear cells (PBMCs) exposed to ionizing radiation.
Created on 10/17/17 10:17 AM
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