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Measurement of DNA Repair Protein Apurinic/Apyrimidinic Endonuclease 1 (APE1) in Human Tissues by Liquid Chromatography/Tandem Mass Spectrometry with Isotope Dilution

Published

Author(s)

Pawel Jaruga, Guldal Kirkali

Abstract

DNA repair proteins may be used as biomarkers in disease etiology and therapy. Thus, the accurate determination of DNA repair protein expression and genotype in human tissues is of fundamental importance. Apurinic/apyrimidinic endonuclease 1 (APE1) is a DNA repair protein and plays other important roles in transcriptional regulation and modulating stress responses. Increased levels of APE1 in cancer have been reported. However, available methods for measuring APE1 levels are indirect and not quantitative. Methods: We developed an approach using liquid chromatography/tandem mass spectrometry with isotope dilution to accurately measure APE1 levels. A completely 15N-labeled full-length human APE1 was produced and used as an internal standard. Fourteen tryptic peptides of both human APE1 (hAPE1) and 15N-labeled hAPE1 were identified following trypsin digestion. These peptides matched the theoretical peptides expected from trypsin digestion and provided a statistically significant protein score that would unequivocally identify hAPE1. Preliminary Data: Using the developed methodology, APE1 was positively identified and quantified in nuclear and cytoplasmic extracts of multiple human cell lines and mouse liver using selected-reaction monitoring of typical mass transitions of the tryptic peptides. We also show that the methodology can be applied to the identification of hAPE1 variants found in the human population. We applied this methodology to measure APE1 levels in disease-free and cancerous human breast tissues. Extreme expression of APE1 in malignant tumors was observed, suggesting that breast cancer cells may require APE1 for survival. Accurate measurement of hAPE1 may be essential for the development of novel treatment strategies and APE1 inhibitors as anticancer drugs. Novel aspect: The results describe a novel approach for the accurate measurement of APE1 in human normal and cancer tissues using LC-MS/MS
Proceedings Title
64th ASMS Conference on Mass Spectrometry and Allied Topics
Conference Dates
June 5-9, 2016
Conference Location
San Antonio, TX
Conference Title
Proceedings of the 64th ASMS Conference on Mass Spectrometry and Allied Topics

Keywords

apurinic/apyrimidinic endonuclease, liquid chromatography/tandem mass spectrometry with isotope

Citation

Jaruga, P. and Kirkali, G. (2016), Measurement of DNA Repair Protein Apurinic/Apyrimidinic Endonuclease 1 (APE1) in Human Tissues by Liquid Chromatography/Tandem Mass Spectrometry with Isotope Dilution, 64th ASMS Conference on Mass Spectrometry and Allied Topics, San Antonio, TX (Accessed June 24, 2024)

Issues

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Created June 5, 2016, Updated January 27, 2020