Rosu Frederic, Gabelica Valérie, Houssier Claude, De Pauw Edwin
Biospectroscopy Laboratory, University of Liège, Chemistry Institute, Bâtiment B6c, B-4000 Liège, Belgium.
Nucleic Acids Res. 2002 Aug 15;30(16):e82. doi: 10.1093/nar/gnf081.
Electrospray mass spectrometry was evaluated regarding the reliability of the determination of the stoichiometries and equilibrium association constants from single spectra. Complexes between minor groove binders (Hoechst 33258, Hoechst 33342, DAPI, netropsin and berenil) and 12mer oligonucleotide duplexes with a central sequence (A/T)4 flanked by G/C base pairs were chosen as model systems. To validate the electrospray ionization mass spectrometry (ESI-MS) method, comparisons were made with circular dichroism and fluorescence spectroscopy measurements. ESI-MS allowed the detection of minor (2 drug + DNA) species for Hoechst 33258, Hoechst 33342, DAPI and berenil with duplex d(GGGG(A/T)4GGGG). d(CCCC(A/T)4CCCC), which were undetectable with the other techniques. Assuming that the duplexes and the complexes have the same electrospray response factors, the equilbrium association constants of the 1:1 and 2:1 complexes were determined by ESI-MS, and the values show a good quantitative agreement with fluorescence determined constants for Hoechst 33258 and Hoechst 33342. It is also shown that ESI-MS can quickly give reliable information on the A/T sequence selectivity of a drug: the signal of a complex is directly related to the affinity of the drug for that particular duplex. The potential of ESI-MS as a qualitative and quantitative affinity screening method is emphasized.
针对从单张光谱图确定化学计量比和平衡缔合常数的可靠性,对电喷雾质谱法进行了评估。选择了小沟结合剂(Hoechst 33258、Hoechst 33342、DAPI、纺锤菌素和贝尼尔)与具有由G/C碱基对侧翼的中心序列(A/T)4的12聚体寡核苷酸双链体之间的复合物作为模型系统。为了验证电喷雾电离质谱法(ESI-MS),将其与圆二色光谱和荧光光谱测量结果进行了比较。ESI-MS能够检测到Hoechst 33258、Hoechst 33342、DAPI和贝尼尔与双链体d(GGGG(A/T)4GGGG). d(CCCC(A/T)4CCCC)形成的次要(2药物 + DNA)物种,而其他技术无法检测到这些物种。假设双链体和复合物具有相同的电喷雾响应因子,通过ESI-MS确定了1:1和2:1复合物的平衡缔合常数,其值与Hoechst 33258和Hoechst 33342的荧光测定常数在定量上显示出良好的一致性。还表明ESI-MS可以快速给出关于药物A/T序列选择性的可靠信息:复合物的信号与药物对该特定双链体的亲和力直接相关。强调了ESI-MS作为定性和定量亲和力筛选方法的潜力。