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Updated: Mar 26, 2026
Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis
Published on: June 19, 2018
Production, Purification, and Characterization of ¹⁵N-Labeled DNA Repair Proteins as Internal Standards for Mass
Prasad T Reddy1, Pawel Jaruga2, Bryant C Nelson3
1Institute for Bioscience and Biotechnology Research, National Institute of Standards and Technology and the University of Maryland, Rockville, Maryland, USA.
Abstract:
Oxidatively induced DNA damage is caused in living organisms by a variety of damaging agents, resulting in the formation of a multiplicity of lesions, which are mutagenic and cytotoxic. Unless repaired by DNA repair mechanisms before DNA replication, DNA lesions can lead to genomic instability, which is one of the hallmarks of cancer. Oxidatively induced DNA damage is mainly repaired by base excision repair pathway with the involvement of a plethora of proteins. Cancer tissues develop greater DNA repair capacity than normal tissues by overexpressing DNA repair proteins. Increased DNA repair in tumors that removes DNA lesions generated by therapeutic agents before they became toxic is a major mechanism in the development of therapy resistance. Evidence suggests that DNA repair capacity may be a predictive biomarker of patient response. Thus, knowledge of DNA-protein expressions in disease-free and cancerous tissues may help predict and guide development of treatments and yield the best therapeutic response. Our laboratory has developed methodologies that use mass spectrometry with isotope dilution for the measurement of expression of DNA repair proteins in human tissues and cultured cells. For this purpose, full-length (15)N-labeled analogs of a number of human DNA repair proteins have been produced and purified to be used as internal standards for positive identification and accurate quantification. This chapter describes in detail the protocols of this work. The use of (15)N-labeled proteins as internal standards for the measurement of several DNA repair proteins in vivo is also presented.
Insights
Oxidatively induced DNA damage is repaired by DNA repair proteins. Measuring these proteins in tissues can predict cancer therapy response and guide treatment strategies.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Oxidative stress causes DNA damage, leading to mutations and genomic instability, a hallmark of cancer.
- DNA repair mechanisms, particularly base excision repair, counteract DNA lesions.
- Cancer cells often exhibit enhanced DNA repair capacity, contributing to therapy resistance.
Purpose of the Study:
- To detail methodologies for measuring DNA repair protein expression in human tissues.
- To establish DNA repair capacity as a potential predictive biomarker for patient response to cancer therapies.
- To guide the development of targeted treatments for improved therapeutic outcomes.
Main Methods:
- Utilizing mass spectrometry with isotope dilution for precise quantification of DNA repair proteins.
- Producing and purifying full-length (15)N-labeled analogs of human DNA repair proteins as internal standards.
- Applying these methods for the measurement of DNA repair proteins in vivo within human tissues and cultured cells.
Main Results:
- Established protocols for the accurate measurement of DNA repair protein expression using mass spectrometry.
- Demonstrated the utility of (15)N-labeled proteins as internal standards for quantifying DNA repair proteins in vivo.
- Provided a foundation for correlating DNA repair protein levels with disease status and treatment response.
Conclusions:
- Accurate measurement of DNA repair protein expression is crucial for understanding cancer biology.
- DNA repair capacity can serve as a predictive biomarker for patient response to cancer therapies.
- Quantifying DNA repair proteins aids in predicting and optimizing cancer treatment strategies.
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