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

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

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