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Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry
11:54

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

Published on: March 23, 2020

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Targeted mass spectrometry enables robust quantification of FANCD2 mono-ubiquitination in response to DNA damage

Jeffrey R Whiteaker1, Lei Zhao1, Richard G Ivey1

  • 1Clinical Research Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, WA, United States.

DNA Repair
|April 2, 2018
PubMed

Insights

A new assay accurately measures FANCD2 protein forms crucial for DNA repair. This method aids in diagnosing Fanconi Anemia (FA) and predicting cancer treatment response to DNA crosslinking agents.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • The Fanconi anemia (FA) pathway is critical for DNA repair, particularly for inter-strand crosslinks.
  • Monoubiquitination of FANCD2 protein is a central event in the FA pathway, dependent on an upstream core complex.
  • Accurate quantification of FANCD2 proteoforms is essential for understanding FA pathway function.

Purpose of the Study:

  • To develop and characterize a novel assay for quantifying unmodified and monoubiquitinated FANCD2 proteoforms.
  • To validate the assay's analytical performance for reliable measurement.
  • To demonstrate the assay's utility in clinical and research settings.

Main Methods:

  • Development of a novel peptide immunoaffinity enrichment assay.
  • Targeted multiple reaction monitoring mass spectrometry (immuno-MRM) for quantification.
  • Analytical characterization including linear range and repeatability assessment.

Main Results:

  • The immuno-MRM assay exhibits a linear range over 3 orders of magnitude with repeatability <16% CV.
  • Quantification of FANCD2 proteoforms was demonstrated in cell lines, patient cells, and human breast cancer tissue.
  • Successful detection of endogenous FANCD2 monoubiquitination was achieved.

Conclusions:

  • The developed immuno-MRM assay is a robust tool for measuring FANCD2 proteoforms.
  • This assay offers a potential functional diagnostic for Fanconi Anemia patients with FA pathway defects.
  • The assay may predict patient sensitivity to DNA crosslinking agents in various cancers.
Keywords:
DNA damage responseFanconi anemiaImmuno-MRMMultiple reaction monitoring

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