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

Year 2022, Volume: 6 Issue: 3, 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.

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