A liquid chromatography-mass spectrometric assay for measuring activity of human 8-oxoguanine-DNA glycosylase.
A new assay for measuring glycosylase activity of human 8-oxoguanine-DNA glycosylase is described. The assay measures the amount of released 8-oxoguanine from synthetic oligonucleotides containing modified base in the middle of the sequence. After enzymatic release, the amount of base is quantified by liquid chromatography-mass spectrometry. Chromatographic separation is carried out on a reversed-phase C(18) column using 10% methanol/water. Quantitation of 8-oxoguanine is carried out by negative-ion electrospray on a single quadrupole mass spectrometer operated in selected-ion monitoring mode. The limit of quantitation was 6 nM and the assay was linear from 6 to 1000 nM. The method was evaluated by monitoring the kinetics of base excision of several substrates as well as by measuring stimulation of activity in the presence of APE1 endonuclease. The new assay provides much higher throughput compared to traditional gel-based assays, which is particularly important when large number of samples need to analyzed.
Nikolić
Dejan
D
Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 S. Wood Street, Chicago, IL 60612-7231, USA. dnikol1@uic.edu
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D045647:DNA Glycosylases / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009841:Oligonucleotides / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D009841:Oligonucleotides; C024829:8-hydroxyguanine; D006147:Guanine; D045647:DNA Glycosylases; C479058:oxoguanine glycosylase 1, human