Unless repaired, DNA damage can drive mutagenesis or cell death. DNA repair proteins may therefore be used as biomarkers in disease detection or therapeutic response estimation. Thus, the accurate measurement of DNA repair protein expression is of fundamental importance. We describe a novel approach involving LC-MS/MS with isotope-dilution to positively identify and accurately quantify apurinic/apyrimidinic endonuclease 1 (APE1) in human cells and mouse tissue. An 15N-labeled full-length hAPE1 was produced and used as an internal standard. Fourteen tryptic peptides of both hAPE1 and 15N-hAPE1 were identified following trypsin digestion. APE1 was identified and quantified in nuclear and cytoplasmic extracts of multiple human cell lines and mouse liver using selected-reaction monitoring of mass transitions of tryptic peptides. The results describe a new method for accurate measurement of human APE1 in vivo and represent a critical step towards defining the role of this protein in disease processes and as a biomarker in various disorders, such as cancer.
Pub Type: Journals
Cancer biomarkers, DNA damage and repair, AP Endonuclease, Mass spectrometry, Proteomics