Wang Poguang, Shah Gunjan L, Landau Heather, Coulter Michael E, Walsh Christopher A, Roider Elisabeth, Kramer Caitlin S, Beuning Penny J, Giese Roger W
Department of Pharmaceutical Sciences, Northeastern University, Boston, Massachusetts 02115, United States.
Department of Medicine, Weill Cornell Medical College, New York, New York 10021, United States.
J Am Soc Mass Spectrom. 2020 Jul 9. doi: 10.1021/jasms.0c00084.
While MALDI-MS of intact genomic DNA is unheard of, actually many DNA adducts can be detected in this way under certain MALDI conditions: relatively high molar ratio of DNA nucleobases to matrix (0.01 to 0.3), hot matrix (CCA), and high laser fluence. This is because many DNA adducts create "bubbles" on dsDNA (disruption of base pairing), making it easier for these adducts as modified nucleobases to be jettisoned by the laser-derived energy of MALDI (jettison mass spectrometry or JeMS). The method also works for other nucleic acid species, namely nucleobases, nucleosides, nucleotides, and RNA. Examples of what we have detected in this way are as follows: methyladenine in DNA, 5-hydroxymethylcytosine in human brain DNA, melphalan-adenine in leukocyte DNA from patients on corresponding chemotherapy, wybutosine in tRNA, benzyl DNA adducts in cell culture treated with benzyl bromide, and various DNA adducts formed in test tube exposure experiments with calf thymus DNA. Noteworthy, in the chemotherapy study, principle component analysis of the data encourages the hypothesis that patient DNA undergoes much more damage than just melphalan adducts. Overall, our work leads to the preliminary generalization that about 5 fmol of a nucleobase deficient in base pairing, and present in a MALDI spot, will be detected by JeMS (on the equipment that we used), irrespective of the type of nucleic acid species which houses it, as long as the nucleobase is relatively basic such as A, C, or G.
虽然完整基因组DNA的基质辅助激光解吸电离质谱（MALDI-MS）闻所未闻，但实际上在某些MALDI条件下可以通过这种方式检测到许多DNA加合物：DNA核碱基与基质的摩尔比相对较高（0.01至0.3）、热基质（CCA）和高激光能量密度。这是因为许多DNA加合物会在双链DNA上产生“气泡”（碱基配对的破坏），使得这些作为修饰核碱基的加合物更容易被MALDI的激光能量抛射出去（抛射质谱法或JeMS）。该方法也适用于其他核酸种类，即核碱基、核苷、核苷酸和RNA。我们通过这种方式检测到的例子如下：DNA中的甲基腺嘌呤、人脑DNA中的5-羟甲基胞嘧啶、接受相应化疗患者白细胞DNA中的美法仑-腺嘌呤、tRNA中的怀丁苷、用苄基溴处理的细胞培养物中的苄基DNA加合物，以及在小牛胸腺DNA的试管暴露实验中形成的各种DNA加合物。值得注意的是，在化疗研究中，数据的主成分分析支持了这样一种假设，即患者的DNA所受损伤远不止美法仑加合物。总体而言，我们的工作初步归纳出，在我们使用的设备上，只要核碱基相对呈碱性，如A、C或G，无论其所在核酸种类如何，JeMS都能检测到存在于MALDI斑点中的约5飞摩尔碱基配对缺陷的核碱基。
