title: Stable isotope-labeling of DNA repair proteins, and their purification and characterization.
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. 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., (15)N-labeled Escherichia coli formamidopyrimidine DNA glycosylase (Fpg) and (15)N-labeled 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 (15)N-labeled proteins and showed that the (15)N-labeling of both proteins was more than 99.7%. We also measured the DNA glycosylase activities using gas chromatography/mass spectrometry with isotope-dilution. The enzymic activities of both (15)N-labeled Fpg and (15)N-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 analogs of other DNA repair proteins for mass spectrometric measurements of these proteins as disease biomarkers.
authors.affiliation: Biochemical Science Division, National Institute of Standards and Technology, Gaithersburg, MD, USA. prasad.reddy@nist.gov
authors.forename: Prasad T
authors.initials: PT
authors.lastname: Reddy
authors.forename: Pawel
authors.initials: P
authors.lastname: Jaruga
authors.forename: Bryant C
authors.initials: BC
authors.lastname: Nelson
authors.forename: Mark
authors.initials: M
authors.lastname: Lowenthal
authors.forename: Miral
authors.initials: M
authors.lastname: Dizdaroglu
mesh_terms: D045647:DNA Glycosylases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004260:DNA Repair; D043382:DNA-Formamidopyrimidine Glycosylase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D007700:Kinetics; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D045647:DNA Glycosylases; C479058:oxoguanine glycosylase 1, human; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli