Related Experiment Video
Updated: Dec 4, 2025
Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage
Published on: August 21, 2021
Maximized quantitative phosphoproteomics allows high confidence dissection of the DNA damage signaling network
Vitor Marcel Faca1,2, Ethan J Sanford1, Jennifer Tieu1
1Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, 14853, USA.
Abstract:
The maintenance of genomic stability relies on DNA damage sensor kinases that detect DNA lesions and phosphorylate an extensive network of substrates. The Mec1/ATR kinase is one of the primary sensor kinases responsible for orchestrating DNA damage responses. Despite the importance of Mec1/ATR, the current network of its identified substrates remains incomplete due, in part, to limitations in mass spectrometry-based quantitative phosphoproteomics. Phosphoproteomics suffers from lack of redundancy and statistical power for generating high confidence datasets, since information about phosphopeptide identity, site-localization, and quantitation must often be gleaned from a single peptide-spectrum match (PSM). Here we carefully analyzed the isotope label swapping strategy for phosphoproteomics, using data consistency among reciprocal labeling experiments as a central filtering rule for maximizing phosphopeptide identification and quantitation. We demonstrate that the approach allows drastic reduction of false positive quantitations and identifications even from phosphopeptides with a low number of spectral matches. Application of this approach identifies new Mec1/ATR-dependent signaling events, expanding our understanding of the DNA damage signaling network. Overall, the proposed quantitative phosphoproteomic approach should be generally applicable for investigating kinase signaling networks with high confidence and depth.
Insights
This study enhances DNA damage response research by improving quantitative phosphoproteomics. The new method accurately identifies more Mec1/ATR kinase substrates, expanding the known DNA damage signaling network.
Area of Science:
- Molecular Biology
- Cellular Biology
- Biochemistry
Background:
- Genomic stability is maintained by DNA damage sensor kinases that phosphorylate substrates.
- Mec1/ATR kinase is crucial for orchestrating DNA damage responses.
- Current substrate networks for Mec1/ATR are incomplete due to limitations in quantitative phosphoproteomics.
Purpose of the Study:
- To improve the identification and quantitation of phosphopeptides using mass spectrometry.
- To overcome limitations in statistical power and redundancy in phosphoproteomics datasets.
- To expand the understanding of the Mec1/ATR signaling network by identifying new substrates.
Main Methods:
- Analysis of the isotope label swapping strategy for phosphoproteomics.
- Utilizing data consistency among reciprocal labeling experiments as a filtering rule.
- Application of the refined approach to identify Mec1/ATR-dependent signaling events.
Main Results:
- The developed approach significantly reduces false positive quantitations and identifications.
- High confidence identification and quantitation of phosphopeptides, even with low spectral matches.
- Discovery of novel Mec1/ATR-dependent signaling events, thereby expanding the DNA damage signaling network.
Conclusions:
- The refined quantitative phosphoproteomic approach enhances the identification of kinase substrates.
- This method increases confidence and depth in analyzing kinase signaling networks.
- The findings contribute to a more comprehensive understanding of DNA damage response pathways.
More Related Videos
12:23Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer
Published on: August 2, 2018
10:12Author Spotlight: Quantitative Detection of DNA Protein Crosslinks and Their Post-Translational Modifications
Published on: April 21, 2023