Abstract 3891: Identification and quantification of DNA repair proteins by liquid chromatography/isotope-dilution tandem mass spectrometry using 15N-labeled whole proteins as internal standards

Abstract

Abstract Reduced DNA repair capacity is associated with increased risk for a variety of disease processes including carcinogenesis. Thus, DNA repair proteins have the potential to be used as important predictive, prognostic and therapeutic biomarkers in cancer and other diseases. The measurement of the expression level of these enzymes may be an excellent tool for this purpose. Mass spectrometry is becoming the technique of choice for the identification and quantification of proteins. We applied liquid chromatography/ isotope-dilution tandem mass spectrometry (LC-MS/MS) for the identification and quantification of DNA repair proteins human 8-oxoguanine-DNA glycosylase (hOGG1) and E. coli formamidopyrimidine DNA glycosylase (Fpg), which are involved in base-excision repair of DNA damage. We over-expressed, purified and characterized 15N-labeled analogues of these proteins to be used as internal standards. 15N-labeled whole proteins are ideal internal standards to ensure the accuracy of quantification of proteins by mass spectrometry. DNA glycosylase activities of 15N-labeled hOGG1 and 15N-labeled Fpg were determined and found to be essentially identical to those of their respective unlabeled counterparts, ascertaining that the 15N-labeling did not perturb their catalytic sites. hOGG1, Fpg and their 15N-labeled analogues were digested with trypsin and analyzed by LC-MS/MS. A large number of tryptic peptides of both proteins were identified on the basis of their full-scan mass spectra. These peptides matched the theoretical peptide fragments expected from the trypsin digestion and provided statistically significant protein scores that would unequivocally identify these proteins. We also recorded the product ion spectra (MS/MS spectra) of the tryptic peptides and defined the characteristic product ions. Mixtures of the analyte proteins and their 15N-labeled analogues as internal standards were analyzed by selected-reaction monitoring (SRM) on the basis of the previously identified product ions in the MS/MS spectra. The experimental conditions for these analyses were optimized. The methodology was successfully applied to the measurement of human OGG1 in human cells and Fpg in E.coli. The results obtained in this work suggest that the methodology developed would be highly suitable for the positive identification and accurate quantification of DNA repair proteins in vivo as potential biomarkers for cancer and other diseases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3891. doi:10.1158/1538-7445.AM2011-3891