Evaluation of protein-DNA binding affinity by electrospray ionization mass spectrometry
Evaluation of protein-DNA binding affinity by electrospray ionization mass spectrometry
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DOI：
10.1016/j.jasms.2004.09.021
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发表时间：
2005-01-01
影响因子：
3.2
通讯作者：
Nishimura, Y
中科院分区：
文献类型：
--
作者：
Akashi, S;Osawa, R;Nishimura, Y
通过电喷雾电离质谱（ESI-MS）产生的多质子化分子的碰撞诱导解离（CID）来评估转录因子c-Myb的DNA结合结构域（DBD）与几种双链DNA（dsDNA）之间的复合物的结合亲和力。在溶液中制备c-Myb DBD和dsDNA的复合物并通过ESI-MS进行分析。在ESI质谱中清楚地观察到高亲和力复合物的多质子化分子，具有特定序列的c-Myb DBD和dsDNA。络合物的质子化分子在气相中相当稳定，并且当样品在10 mM乙酸铵中制备时，即使在第一真空室源中施加高锥电压也不容易解离。对于在具有较高浓度乙酸铵的缓冲液（例如500 mM乙酸铵）中制备的样品，蛋白质-dsDNA复合物可以随着锥电压的增加而容易地解离，得到游离c-Myb DBD的多质子化分子和一些DNA片段。对c-Myb DBD和22-mer dsDNA之间的七种复合物进行了系统的CID实验，其具有不同的溶液-Kd值，范围为10（-9）M至10（-7）M。对于每个复合物，绘制作为锥电压的函数的解离曲线，并计算50%复合物解离时的锥电压（V-50%）。因此，在溶液中络合物形成中，V-50%和相对结合自由能变化（Δ Δ G）之间获得正相关性。这表明EST-CID实验可以提供基于适当校准的蛋白质-DNA复合物的稳定性的定量评估。(C)2004年美国质谱学会。
Binding affinity of complexes between a DNA-binding domain (DBD) of a transcription factor, c-Myb, and several double-stranded DNA (dsDNA) were evaluated by collision-induced dissociation (CID) of the multiply protonated molecules generated by electrospray ionization mass spectrometry (ESI-MS). Complexes of the c-Myb DBD and dsDNA were prepared in solution and analyzed by ESI-MS. Multiply protonated molecules of a high-affinity complex, the c-Myb DBD and dsDNA with a specific sequence, were clearly observed in ESI mass spectrum. Protonated molecules of the complex were quite stable in the gas-phase, and not easily dissociated even if high cone voltage was applied in the first vacuum chamber source when the sample was prepared in 10 mM ammonium acetate. As for the sample prepared in buffer with higher concentration of ammonium acetate, such as 500 mM ammonium acetate, protein-dsDNA complexes could easily be dissociated with an increase in the cone voltage, giving multiply protonated molecules of free c-Myb DBD and some DNA fragments. Systematic CID experiments were carried out on seven complexes between the c-Myb DBD and 22-mer dsDNA with different solution-Kd values in the range of 10(-9) M to 10(-7) M. For each complex dissociation curve as a function of cone voltage was plotted, and the cone voltage where 50% of the complex was dissociated (V-50%) was calculated. Consequently, positive correlation was obtained between V-50% and relative binding free energy change (DeltaDeltaG) in complex formation in solution. This suggests that EST-CID experiments can provide quantitative evaluation of the stability of protein-DNA complexes based on proper calibration. (C) 2004 American Society for Mass Spectrometry.