Roberts Kenneth P, Sobrino Justin A, Payton Julie, Mason Lavinnia B, Turesky Robert J
Department of Chemistry and Biochemistry, The University of Tulsa, Tulsa, Oklahoma 74104, USA.
Chem Res Toxicol. 2006 Feb;19(2):300-9. doi: 10.1021/tx0502589.
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).
一种新方法已被开发出来，用于精确测量脱嘌呤和脱嘧啶（AP）DNA损伤位点，这些位点是DNA中的损伤，由氧化应激或致癌物加合物导致的核碱基丢失所形成。如果AP位点未得到修复（或修复不当），这些位点会导致DNA突变，最终可能导致癌症的形成。因此，检测AP位点可能为接触化学致癌物和氧化应激以及易感性提供一个有用的指标。目前，AP检测是通过使用醛反应探针的免疫检测方法来完成的[中村，J.，沃克，V. E.，厄普顿，P. B.，蒋，S.-Y.，科，Y. W.，和斯文伯格，J. A.（1998年）《癌症研究》58卷，222 - 225页；阿塔姆纳，H.，张，I.，和艾姆斯，B. N.（2000年）《美国国家科学院院刊》97卷，686 - 691页]；然而，这些方法缺乏明确鉴定AP位点所需的特异性。因此，我们开发了一种基于质谱检测的精确方法，用于检测用O - 4 - 硝基苄基羟胺（NBHA）预标记的AP DNA中的AP位点。一旦标记且去除过量的标记剂后，可将DNA酶解为单体亚基，随后通过与电喷雾电离串联质谱联用的高效液相色谱（HPLC - ESI - MS/MS）进行分离和检测。使用含有尿嘧啶的模型DNA寡聚物（11聚体）建立了实验程序的优化和验证以及检测限。用尿嘧啶糖苷酶酶促去除尿嘧啶会在单链和双链DNA中产生明确的AP位点。添加NBHA会标记寡聚物中的AP位点，形成标记的11聚体。采用负电离模式的HPLC - ESI - MS/MS来监测和确认NBHA与AP寡聚物的结合。带有NBHA标记的寡聚物经过内切酶和外切酶消化至5'-脱氧核糖单磷酸（5'-dRp）水平，从而释放出游离的5'-dRp - NBHA。5'-dRp - NBHA产物通过固相萃取进行部分纯化，并使用去质子化分子（m/z 363处的[M - H]-）的几个跃迁以及同位素标记的5'-dRp - NBHA作为内标，通过LC - MS/MS进行定量。对5',3'-双磷酸脱氧核糖和热/酸处理的小牛胸腺DNA进行的进一步实验显示了类似的标记、消化和检测结果。初步结果表明，对于100μg DNA，定量限为100 fmol（每10⁷个碱基中有三个无碱基位点）。
