Determination of apurinic/apyrimidinic lesions in DNA with high-performance liquid chromatography and tandem mass spectrometry.
Determination of apurinic/apyrimidinic lesions in DNA with high-performance liquid chromatography and tandem mass spectrometry.
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DOI：
10.1021/tx0502589
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发表时间：
2006-01
影响因子：
4.1
通讯作者：
K. Roberts;Justin A Sobrino;Julie Payton;Lavinnia B Mason;R. Turesky
中科院分区：
文献类型：
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作者：
K. Roberts;Justin A Sobrino;Julie Payton;Lavinnia B Mason;R. Turesky
已经开发了一种新的方法来准确地测量脱嘌呤和脱嘧啶（AP）DNA损伤位点，这是DNA中由氧化应激或致癌物加合物引起的核碱基丢失所形成的损伤。如果AP位点未修复（或修复不当），这些位点可能导致DNA突变，最终可能导致癌症的形成。因此，AP位点的检测可以提供对化学致癌物和氧化应激的暴露和易感性的有用指标。AP检测目前通过使用醛反应性探针的免疫检测方法来完成[中村，J.，步行者，V.E.，厄普顿，P. B.，Chiang，S.-是的，Kow，Y. W.，和Swenberg，J. A.（1998）Cancer Res.58，222-225; Atamna，H.，张岛，和艾姆斯，B. N.等人（2000）Proc. Acad. Sci. U.S.A.97，686-691];然而，这些方法缺乏明确鉴定AP位点所需的特异性。因此，我们已经开发了一种准确的方法，基于质谱检测AP位点的AP DNA已预标记O-4-硝基苄基羟胺（NBHA）。一旦标记并且一旦过量的标记剂已经被去除，就可以完成DNA到单体亚基的酶促消化，然后用高效液相色谱耦合电喷雾电离串联质谱（HPLC-ESI-MS/MS）进行分离和检测。优化和验证的实验程序和检测限已建立使用模型DNA寡聚体（11聚体）含有尿嘧啶。用尿嘧啶糖基化酶酶促去除尿嘧啶在单链和双链DNA中产生明确定义的AP位点。添加NBHA标记寡聚体中的AP位点，产生标记的11聚体。使用负离子化模式的HPLC-ESI-MS/MS监测并确认NBHA与AP寡聚体的结合。NBHA标记的寡聚体经历内切核酸酶和外切核酸酶消化至5 '-脱氧核糖一磷酸（5'-dRp）水平，从而释放游离的5 '-dRp-NBHA。通过固相萃取部分纯化5 '-dRp-NBHA产物，并通过LC-MS/MS使用去质子化分子的几个跃迁（[M-H]-，m/z 363）和同位素标记的5'-dRp-NBHA作为内标进行定量。用5 '，3'-二磷酸脱氧核糖和热/酸处理的小牛胸腺DNA进行的进一步实验显示出相似的标记、消化和检测结果。初步结果显示，100 μ g DNA的定量限为100 fmol（每10（7）个碱基有3个脱碱基位点）。
A new method has been developed to accurately measure apurinic and apyrimidinic (AP) DNA damage sites, which are lesions in DNA formed by loss of a nucleobase from oxidative stress or carcinogen adducts. If AP sites are left unrepaired (or if improperly repaired), these sites can lead to DNA mutations that may ultimately result in the formation of cancer. Hence, detection of AP sites may provide a useful indicator of exposure and susceptibility to chemical carcinogens and oxidative stress. AP detection is currently accomplished by immunodetection methods using an aldehyde reactive probe [Nakamura, J., Walker, V. E., Upton, P. B., Chiang, S.-Y., Kow, Y. W., and Swenberg, J. A. (1998) Cancer Res. 58, 222-225; Atamna, H., Cheung, I., and Ames, B. N. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 686-691]; however, these approaches lack the specificity required for unequivocal identification of the AP site. Therefore, we have developed an accurate method based on mass spectrometry detection of AP sites from AP DNA that have been prelabeled with O-4-nitrobenzylhydroxylamine (NBHA). Once labeled and once the excess labeling agent has been removed, enzymatic digestion of DNA to monomeric subunits can be accomplished, followed by isolation and detection with high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Optimization and validation of the experimental procedures and detection limits have been established using a model DNA oligomer (11-mer) containing uracil. Enzymatic removal of uracil with uracil glycosylase generates well-defined AP sites in both single- and double-stranded DNA. The addition of NBHA labels the AP site in the oligomer, creating a labeled 11-mer. HPLC-ESI-MS/MS in the negative ionization mode was used to monitor and confirm binding of NBHA to the AP oligomer. The NBHA-tagged oligomer underwent endo- and exonuclease digestion to the 5'-deoxyribose monophosphate (5'-dRp) level, thereby releasing free 5'-dRp-NBHA. The 5'-dRp-NBHA product was partially purified by solid phase extraction and quantified by LC-MS/MS using several transitions of the deprotonated molecule ([M - H]- at m/z 363) and isotopically labeled 5'-dRp-NBHA as an internal standard. Further experiments with 5',3'-bisphosphate-deoxyribose and heat/acid-treated calf thymus DNA showed similar labeling, digestion, and detection results. Initial results show a quantification limit with 100 mug of DNA to be 100 fmol (three abasic sites per 10(7) bases).