Identification and Quantification of Apurinic/Apyrimidinic Endonuclease 1 in Human Peripheral Blood Leukocytes by Liquid Chromatography/Isotope-Dilution High Resolution Mass Spectrometry

Year 2022, , 851 - 857, 29.09.2022

Gamze Tuna

https://doi.org/10.30621/jbachs.1120569

Abstract

Background Increasing evidence in recent years highlights the predictive and prognostic importance of the expression of DNA repair proteins in cancer treatment. Generally, western-blotting or immunohistochemical staining methods are often used to determine the expression of DNA repair proteins. These methods might cause misleading results such as binding to nonspecific molecules by cross-reaction or false negativity as a result of the inability of antibodies to bind; absolute quantitations of proteins can not be performed.
Purpose In this study, an analytical measurement technique was developed for human apurinic/apyrimidinic endonuclease 1 (hAPE1) protein for identification and absolute quantification in human leukocyte sample using high resolution mass spectrometry (HR-MS) with the targeted proteomics based approach.
Methods Sample preparation was performed by using density gradient centrifugation and total protein extraction cartridges. hAPE1 was analyzed by liquid chromatography isotope-dilution-HR-MS (LC-HR-MS). A fully 15N-labeled analogue of hAPE1 was used for the quantitative measurements.
Results Six peptides were identified, which matched to a subset of the theoretically predicted tryptic peptides of hAPE1. Mass accuracy was calculated as <1.8 ppm. The amount of hAPE1 protein was calculated as 0.07 ng hAPE1/μg protein in the leukocyte sample.
Conclusion The absolute quantification of APE1 protein performed within the scope of this study is expected to be used for the follow-up of the prognosis, response to treatment and survival rates of various cancer patients.

Keywords

DNA repair protein, hAPE1, high resolution mass spectrometry, leukocyte, protein quantification, targeted proteomics

Supporting Institution

Bulunmamaktadır.

Project Number

Bulunmamaktadır.

Thanks

The author would like to extend special thanks to Dr. Miral Dizdaroğlu and Dr. Pawel Jaruga of the National Institute of Standards and Technology, Gaithersburg, MD, USA for providing stable isotope-labeled internal standard.

References

• Ghosal G, Chen J. DNA damage tolerance: A double-edged sword guarding the genome. Vol. 2, Translational Cancer Research. 2013.
• Loeb LA. Human cancers express mutator phenotypes: Origin, consequences and targeting. Vol. 11, Nature Reviews Cancer. 2011.
• Kelley MR, Fishel ML. Overview of DNA repair pathways, current targets, and clinical trials bench to clinic. In: DNA Repair in Cancer Therapy: Molecular Targets and Clinical Applications: Second Edition. 2016.
• Raffoul JJ, Heydari AR, Hillman GG. DNA repair and cancer therapy: Targeting APE1/Ref-1 using dietary agents. Journal of Oncology. 2012.
• Tell G, Fantini D, Quadrifoglio F. Understanding different functions of mammalian AP endonuclease (APE1) as a promising tool for cancer treatment. Vol. 67, Cellular and Molecular Life Sciences. 2010.
• Vascotto C, Poletto M, Tell G. Understanding the basics for translating the base excision repair pathway from benchtop to bedside in cancer treatment. In: DNA Repair in Cancer Therapy: Molecular Targets and Clinical Applications: Second Edition. 2016.
• Fung H, Demple B. A vital role for Ape1/Ref1 protein in repairing spontaneous DNA damage in human cells. Molecular Cell. 2005;17(3).
• Jiang A, Gao H, Kelley MR, Qiao X. Inhibition of APE1/Ref-1 redox activity with APX3330 blocks retinal angiogenesis in vitro and in vivo. Vision Research. 2011;51(1).
• Coskun E, Tuna G, Jaruga P, Tona A, Erdem O, Dizdaroglu M. Identification and quantification of DNA repair protein poly(ADP ribose) polymerase 1 (PARP1) in human tissues and cultured cells by liquid chromatography/isotope-dilution tandem mass spectrometry. DNA Repair. 2019;75.
• Kirkali G, Jaruga P, Reddy PT, Tona A, Nelson BC, Li M, et al. Identification and Quantification of DNA Repair Protein Apurinic/Apyrimidinic Endonuclease 1 (APE1) in Human Cells by Liquid Chromatography/Isotope-Dilution Tandem Mass Spectrometry. PLoS ONE. 2013;8(7).
• Shin JH, Choi S, Lee YR, Park MS, Na YG, Irani K, et al. APE1/ref-1 as a serological biomarker for the detection of bladder cancer. Cancer Research and Treatment. 2015;47(4).
• Tudek B. Base excision repair modulation as a risk factor for human cancers. Vol. 28, Molecular Aspects of Medicine. 2007.
• Coskun E, Jaruga P, Reddy PT, Dizdaroglu M. Extreme Expression of DNA Repair Protein Apurinic/Apyrimidinic Endonuclease 1 (APE1) in Human Breast Cancer As Measured by Liquid Chromatography and Isotope Dilution Tandem Mass Spectrometry. Biochemistry. 2015;54(38).