Abstract 618: Identification and quantification of human DNA repair protein NEIL1 by liquid chromatography/isotope-dilution tandem mass spectrometry as a potential cancer biomarker.

Abstract

Abstract Accumulated evidence points to DNA repair capacity as an important therapeutic factor in cancer in predicting patient response to DNA-damaging agents such as chemotherapeutic drugs and ionizing radiation. Recent findings suggest that some types of malignant tumors possess increased DNA repair capacity that may affect the therapy and outcome of cancer. Thus, the knowledge of the over-expression or under-expression levels of DNA repair proteins in tumors and disease-free tissues will help develop and guide treatment strategies that will likely lead to the best treatment results for patients. In this context, DNA repair proteins are becoming predictive, prognostic and therapeutic factors in cancer, and also promising drug targets for cancer treatment as DNA repair inhibitors are being developed to increase the efficacy of cancer therapy. We developed assays for the mass spectrometric measurement in tissues of the human DNA repair protein NEIL1 (hNEIL1), which is involved in base excision and nucleotide excision repair pathways of oxidatively induced DNA damage. There is strong evidence for a critical role of NEIL1 in maintaining the genetic stability and in prevention of diseases such as cancer and metabolic syndrome. We applied liquid chromatography/isotope-dilution tandem mass spectrometry (LC-MS/MS), using the fully 15N-labeled analogue of hNEIL1 (15N-hNEIL1) as an internal standard, which we produced, purified and characterized. Both hNEIL1 and 15N-hNEIL1 were hydrolyzed with trypsin. Eighteen tryptic peptides of each protein were identified by LC-MS/MS on the basis of their full-scan mass spectra. These peptides matched the theoretical peptides expected from trypsin hydrolysis of hNEIL1, and provided a statistically significant protein score that would unequivocally identify hNEIL1. The product ion spectra of the tryptic peptides of both proteins were recorded and the characteristic product ions were defined. Selected-reaction monitoring was used to analyze mixtures of hNEIL1 and 15N-hNEIL1 on the basis of product ions. We also showed the detection of these proteins following their separation by gel electrophoresis and in-gel trypsin digestion. Our results suggest that the assays developed would be highly suitable for the positive identification and accurate quantification of hNEIL1 in tissues in vivo as a potential cancer biomarker. . Citation Format: Miral Dizdaroglu. Identification and quantification of human DNA repair protein NEIL1 by liquid chromatography/isotope-dilution tandem mass spectrometry as a potential cancer biomarker. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 618. doi:10.1158/1538-7445.AM2013-618