Measurement of 8-hydroxy-2′-deoxyguanosine in DNA by high-performance liquid chromatography-mass spectrometry:: comparison with measurement by gas chromatography-mass spectrometry
Measurement of 8-hydroxy-2′-deoxyguanosine in DNA by high-performance liquid chromatography-mass spectrometry:: comparison with measurement by gas chromatography-mass spectrometry
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DOI：
10.1093/nar/29.3.e12
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发表时间：
2001-02-01
影响因子：
14.9
通讯作者：
Rodriguez, H
中科院分区：
文献类型：
--
作者：
Dizdaroglu, M;Jaruga, P;Rodriguez, H
研究了用高效液相色谱/质谱仪(LC/MS)测定DNA中8-羟基-2‘-脱氧鸟苷(8-OH-dGuo)。建立了脱氧核糖核酸酶I、磷酸二酯酶I、磷酸二酯酶II和碱性磷酸酶联合酶解DNA中8-OH-dGuo的LC分离方法。采用大气压电离-电喷雾联用技术进行了质谱学测量。以稳定同位素标记的8-羟基-dGuo类似物为内标，用同位素稀释质谱仪(IDMS)进行定量。结果表明，选择离子监测(SIM)LC/IDMS能很好地用于背景水平和损伤DNA中8-OH-dGuo的鉴定和定量。LC/IDMS-SIM的灵敏度水平与以前报道的LC-串联质谱仪(LC/MS/MS)相当。结果发现，使用低至2微克的DNA量，每10(6)个DNA碱基可以检测到大约5个损伤。结果还表明，这种损伤可能以每10(6)个DNA碱基1个损伤的水平进行量化，或者当使用更多DNA时甚至更低。每次注射使用多达50毫克的DNA，而不会对测量产生不利影响。选择离子监测的气相色谱/同位素稀释质谱(GC/IDMS-SIM)也被用来检测DNA中的这种化合物，这些化合物通过酸性水解法或用大肠杆菌FPG蛋白水解法从DNA中去除。LC/IDMS-SIM和GC/IDMS-SIM获得的本底水平基本相同。小牛胸腺DNA和从培养的HeLa细胞中提取的DNA用于这一目的。这表明这两种技术在DNA中8-OH-dGuo的测量方面可以提供相似的结果。用LC/IDMS-SIM和GC/IDMS-SIM对不同辐射剂量的电离辐射损伤缓冲水溶液中的DNA进行了分析。同样，这两种技术也得到了类似的结果。GC/MS-SIM对7，8-二氢-8-氧鸟嘌呤的灵敏度也远高于LC/MS-SIM和LC/MS/MS对8-OH-dGuo的灵敏度。结果表明，LC/IDMS-SIM法非常适合于DNA中8-OH-dGuo的灵敏、准确的测定，而LC/IDMS-SIM法和GC/IDMS-SIM法可以得到类似的结果。
Measurement of 8-hydroxy-2'-deoxyguanosine (8-OH-dGuo) in DNA by high-performance liquid chromatography/mass spectrometry (LC/MS) was studied. A methodology was developed for separation by LC of 8-OH-dGuo from intact and modified nucleosides in DNA hydrolyzed by a combination of four enzymes: DNase I, phosphodiesterases I and II and alkaline phosphatase. The atmospheric pressure ionization-electrospray process was used for mass spectral measurements. A stable isotope-labeled analog of 8-OH-dGuo was used as an internal standard for quantification by isotope-dilution MS (IDMS). Results showed that LC/IDMS with selected ion-monitoring (SIM) is well suited for identification and quantification of 8-OH-dGuo in DNA at background levels and in damaged DNA. The sensitivity level of LC/IDMS-SIM was found to be comparable to that reported previously using LC-tandem MS (LC/MS/MS). It was found that approximately five lesions per 10(6) DNA bases can be detected using amounts of DNA as low as 2 mu g. The results also suggest that this lesion may be quantified in DNA at levels of one lesion per 10(6) DNA bases, or even lower, when more DNA is used. Up to 50 mu g of DNA per injection were used without adversely affecting the measurements. Gas chromatography/isotope-dilution MS with selected-ion monitoring (GC/IDMS-SIM) was also used to measure this compound in DNA following its removal from DNA by acidic hydrolysis or by hydrolysis with Escherichia coli Fpg protein. The background levels obtained by LC/IDMS-SIM and GC/IDMS-SIM were almost identical. Calf thymus DNA and DNA isolated from cultured HeLa cells were used for this purpose. This indicates that these two techniques can provide similar results in terms of the measurement of 8-OH-dGuo in DNA. In addition, DNA in buffered aqueous solution was damaged by ionizing radiation at different radiation doses and analyzed by LC/IDMS-SIM and GC/IDMS-SIM. Again, similar results were obtained by the two techniques. The sensitivity of GC/MS-SIM for 7,8-dihydro-8-oxoguanine was also examined and found to be much greater than that of LC/MS-SIM and the reported sensitivity of LC/MS/MS for 8-OH-dGuo. Taken together, the results unequivocally show that LC/IDMS-SIM is well suited for sensitive and accurate measurement of 8-OH-dGuo in DNA and that both LC/IDMS-SIM and GC/IDMS-SIM can provide similar results.