Koomen J M, Russell D H
Laboratory for Biological Mass Spectrometry, Chemistry Department, Texas A&M University, College Station, Texas 77843, USA.
J Mass Spectrom. 2000 Aug;35(8):1025-34. doi: 10.1002/1096-9888(200008)35:8<1025::AID-JMS30>3.0.CO;2-9.
The changes in the ion signals in the isotope cluster, mass resolution, signal-to-noise ratio and mass accuracy for matrix-assisted laser desorption/ionization (MALDI) of DNA oligonucleotides (dGGATC, dCAGCt, and dAACCGTT) and their fragment ions were evaluated, and these data were compared with those obtained using 3-hydroxypicolinic acid. Mass spectra obtained by using 2,5-dihydroxybenzoic acid (2,5-DHB) appear to have differences from the theoretical isotopic clusters, which arise by reductive hydrogenation producing a second peak at the M + 2 isotope of the native oligonucleotide. Based on the patterns of the isotopic envelope observed in the in-source decay fragments, we propose that cytosine is the site of reduction. We do not find evidence of reduction of oligonucleotides, viz. dTGGGGTT, that do not contain cytosine; however, 2'-deoxycytidine and 2'-deoxycytidine-5'-monophosphate undergo reductive hydrogenation. Several experiments were carried out in an effort to determine whether the reductive hydrogenation occurs during sample preparation or as a result of laser irradiation. The results of these experiments suggest that it occurs during sample preparation. The relative intensities of ion signals corresponding to the reduced base can be altered by using different matrix additives (aminonaphthalenes) or a different substrate (copper). Also, the oxidized form of 2,5-DHB is trapped by reaction with the side chain of cysteine in glutathione, providing evidence that the reaction occurs in solution as the matrix crystallizes.
评估了DNA寡核苷酸（dGGATC、dCAGCt和dAACCGTT）及其碎片离子在基质辅助激光解吸/电离（MALDI）过程中同位素簇中的离子信号、质量分辨率、信噪比和质量准确度的变化，并将这些数据与使用3-羟基吡啶甲酸获得的数据进行了比较。使用2,5-二羟基苯甲酸（2,5-DHB）获得的质谱似乎与理论同位素簇存在差异，这是由于还原氢化在天然寡核苷酸的M + 2同位素处产生了第二个峰。基于在源内衰变碎片中观察到的同位素包络模式，我们提出胞嘧啶是还原位点。我们没有发现不含胞嘧啶的寡核苷酸（即dTGGGGTT）发生还原的证据；然而，2'-脱氧胞苷和2'-脱氧胞苷-5'-单磷酸会发生还原氢化。进行了几项实验以确定还原氢化是在样品制备过程中发生还是激光照射的结果。这些实验的结果表明它发生在样品制备过程中。通过使用不同的基质添加剂（氨基萘）或不同的底物（铜），可以改变与还原碱基相对应的离子信号的相对强度。此外，2,5-DHB的氧化形式通过与谷胱甘肽中半胱氨酸的侧链反应而被捕获，这证明该反应在基质结晶时在溶液中发生。
