Stable isotope-labeling of DNA repair proteins, and their purification and characterization
M Miral Dizdar, Prasad T. Reddy, Pawel Jaruga, Bryant C. Nelson
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. However, suitable internal standards must be used to ensure the precision and accuracy of measurements. An ideal internal standard in this case would be a stable isotope-labeled analog of the analyte protein. In the present work, we over-expressed, purified and characterized two stable isotope-labeled DNA glycosylases, i.e., 15N-labeled E. coli formamidopyrimidine DNA glycosylase (Fpg) and human 8-oxoguanine-DNA glycosylase (hOGG1). DNA glycosylases are involved in the first step of the base excision repair of oxidatively induced DNA damage by removing modified DNA bases. The measurement by MALDI ToF mass spectrometry of the molecular mass and isotopic purity proved the identity of the 15N-labeled proteins and showed that the 15N-labeling of both proteins was more than 99%. We also measured the DNA glycosylase activities using gas chromatography/mass spectrometry with isotope-dilution. The enzymic activities of both 15N-labeled Fpg and 15N-labeled hOGG1 were essentially identical to those of their respective unlabeled counterparts, ascertaining that the labeling did not perturb their catalytic sites. The procedures described in this work may be used for obtaining stable isotope-labeled analogues of other DNA repair proteins for mass spectrometric measurements of these proteins as disease biomarkers.
, Reddy, P.
, Jaruga, P.
and Nelson, B.
Stable isotope-labeling of DNA repair proteins, and their purification and characterization, Protein Expression and Purification, [online], https://doi.org/10.1016/j.pep.2011.02.011
(Accessed September 27, 2022)