Identification and quantification of DNA repair protein apurinic/apyrimidinic endonuclease 1 (APE1) in human cells by liquid chromatography/isotope-dilution tandem mass spectrometry.
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 (15)N-labeled full-length human APE1 was produced and used as an internal standard. Fourteen tryptic peptides of both human APE1 (hAPE1) and (15)N-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.
Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg, Maryland, United States of America.
Güldal
G
Kirkali
Pawel
P
Jaruga
Prasad T
PT
Reddy
Alessandro
A
Tona
Bryant C
BC
Nelson
Mengxia
M
Li
David M
DM
Wilson
Miral
M
Dizdaroglu
D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D004260:DNA Repair / Q000502:physiology; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D051379:Mice; D053719:Tandem Mass Spectrometry / Q000379:methods*
C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase