Identification and Quantification of DNA Repair Protein Apurinic/Apyrimidinic Endonuclease 1 (APE1) in Human Cells by Liquid Chromatography/Isotope-Dilution Tandem Mass Spectrometry

Abstract

Unless repaired, DNA damage can drive mutagenesis or cell death. DNA repair proteins may therefore be used as biomarkers in disease etiology or therapeutic response prediction. Thus, the accurate determination of DNA repair protein expression and genotype is of fundamental importance. Among DNA repair proteins involved in base excision repair, apurinic/apyrimidinic endonuclease 1 (APE1) is the major endonuclease in mammals and plays important roles in transcriptional regulation and modulating stress responses. Here, we present a novel approach involving LC-MS/MS with isotope-dilution to positively identify and accurately quantify APE1 in human cells and mouse tissue. A completely 15N-labeled full-length human APE1 was produced and used as an internal standard. Fourteen tryptic peptides of both human APE1 (hAPE1) and 15N-labeled hAPE1 were identified following trypsin digestion. These peptides matched the theoretical peptides expected from trypsin digestion and provided a statistically significant protein score that would unequivocally identify hAPE1. Using the developed methodology, APE1 was positively identified and quantified in nuclear and cytoplasmic extracts of multiple human cell lines and mouse liver using selected-reaction monitoring of typical mass transitions of the tryptic peptides. We also show that the methodology can be applied to the identification of hAPE1 variants found in the human population. The results describe a novel approach for the accurate measurement of wild-type and variant forms of hAPE1 in vivo, and ultimately for defining the role of this protein in disease development and treatment responses.

Addiction to MTH1 protein results in intense expression in human breast cancer tissue as measured by liquid chromatography-isotope-dilution tandem mass spectrometry

“…Fig. 7B), which is essentially identical to those previously obtained for other peptides with similar masses [37][38][39].…”

“…It is also well known that cell cultures are only one cell type, whereas tissues consist of mixtures of cell types. A similar difference in expression levels of APE1 protein was observed when its amount was measured by LC/MS-MS with isotope dilution in human cultured cells and mouse liver [39]. Again, the identification and quantification of hMTH1 in human tissues at very low levels attest to the ability of the developed methodology to accurately measure low protein levels.…”

“…However, HepG2 cells exhibited quite a low hMTH1expression compared to the other three cell lines. The levels of hMTH1 in MCF-10A, MCF-7 and HepG2 cells were ≈30-fold, 20-fold and 100-fold, respectively, lower than those of hAPE1 protein, which had been also measured by LC-MS/MS with isotope-dilution [39]. This fact indicates the ability of our methodology to measure very low amounts of proteins in cells.…”

Production, Purification, and Characterization of 15N-Labeled DNA Repair Proteins as Internal Standards for Mass Spectrometric Measurements

“…The yield of 15 N-hAPE1 from 4 L culture of minimal medium was 60 mg. Aliquots of the protein was stored at −70 °C. Figure 4, lane 6, shows the SDS–PAGE analysis and Coomassie staining of 15 N-hAPE1 and protein molecular mass standards (lane 7), indicating high purity of the labeled protein (Kirkali et al, 2013). …”

“…The application of this technique would be essential for positive identification and accurate quantification of DNA repair proteins in human tissues using proper internal standards. Our laboratory has recently developed methodologies and stable isotope-labeled standards for the measurements of DNA repair proteins using liquid chromatography–tandem mass spectrometry (LC-MS/MS) with isotope dilution (Coskun et al, 2015; Dizdaroglu, Reddy, & Jaruga, 2011; Kirkali et al, 2013; Reddy, Jaruga, Nelson, Lowenthal, & Dizdaroglu, 2011; Reddy et al, 2013). Stable isotope-labeled analogs of DNA repair proteins, which are to be used as internal standards, were produced, purified, and characterized.…”