摘要
发展了一种超高效液相色谱-串联质谱法(UPLC-MS/MS)检测脱氧核糖核酸(DNA)分子中8-羟基脱氧鸟苷(8-OHdG)的方法。DNA分子在酶解过程中,脱氧鸟苷(dG)易被氧化形成8-OHdG,从而使得8-OHdG的检测结果不准确。通过加入甲磺酸去铁铵作为保护剂,有效地避免了酶解过程造成的dG氧化。酶解液通过超滤膜(截留相对分子质量为3 000的分子)处理,有效去除大量蛋白分子后,直接进行UPLC-MS/MS测定。采用外标法定量,在17.6～1 400fmol范围内,8-OHdG的峰面积与其物质的量具有良好的线性关系,相关系数为0.999 8。利用本方法测定了小牛胸腺DNA中8-OHdG的含量(用比值8-OHdG/106dG表示)为12.9±2.35,与前人报道的检测结果一致。本方法也可以应用于评价各种氧化因素引起的DNA氧化损伤。
An ultra high performance liquid chromatography-tandem mass spectrometry（UPLC-MS/MS） method for the analysis of biomarker 8-oxo-7,8-dihydro-2′-deoxyguanosine（8-OHdG） in deoxyribonucleic acid（DNA） was developed.The artificial oxidation of 2′-deoxyguanosine（dG） at numerous stages in the sample preparation bring a challenge to the accurate measurement of 8-OHdG in DNA.To avoid the artificial oxidation during the enzymatic digestion,desferrioxamine mesylate as a protectant was added into the mixtures.By utilizing YM-3 Centricon membrane（3 000 of relative molecular mass cut off）,excess proteins in enzymatic solutions were effectively removed,allowing direct UPLC-MS/MS analysis.The UPLC-MS/MS analysis showed a linear relationship between the peak areas and the amounts of 8-OHdG in the range of 17.6-1 400 fmol,and the correlation coefficient was 0.999 8.By using the developed method,the content of 8-OHdG in calf thymus DNA（CT DNA） was estimated about 12.9±2.35（calculated as 8-OHdG/106 dG）,which was consistent with the previous work.This method can also be applicable for the detection of 8-OHdG in DNA under various oxidative stresses.
出处
《色谱》
CAS
CSCD
北大核心
2010年第12期1123-1127,共5页
Chinese Journal of Chromatography
基金
国家“973计划”支持项目(No.2007CB407305)
关键词
超高效液相色谱-串联质谱
8-羟基脱氧鸟苷
脱氧鸟苷
脱氧核糖核酸
小牛胸腺
ultra-performance liquid chromatography-tandem mass spectrometry（UPLC-MS/MS）
8-oxo-7
8-dihydro-2′-deoxyguanosine（8-OHdG）
2′-deoxyguanosine（dG）
deoxyribonucleic acid（DNA）
calf thymus
