Label-free determination of protein-ligand binding constants using mass spectrometry and validation using surface plasmon resonance and isothermal titration calorimetry
Label-free determination of protein-ligand binding constants using mass spectrometry and validation using surface plasmon resonance and isothermal titration calorimetry
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DOI：
10.1002/jmr.951
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发表时间：
2009-07-01
影响因子：
2.7
通讯作者：
Zenobi, Renato
中科院分区：
文献类型：
--
作者：
Jecklin, Matthias C.;Schauer, Stefan;Zenobi, Renato
我们进行了系统的比较，三个无标记的方法定量评估的结合强度的蛋白质与小分子配体相互作用。比较了（1）纳米电喷雾电离质谱（nESI-MS）、（2）表面等离子体共振（SPR）和（3）等温滴定量热法（ITC）测定解离常数（K-D）的性能。为此目的研究的模型系统是人碳酸酐酶I（hCAI）与八种已知且充分表征的磺酰胺抑制剂（Krishnamurthy等人，2008，108：946-1051）。所选抑制剂的结合亲和力变化超过四个数量级，通过nESI-MS测定的乙氧唑胺的K-D值为5 +/-1nM，磺胺的K-D值为145.7 +/-10.0 μ M。通过所研究的三种方法测定的K-D值的一致性对于乙氧唑胺和苯磺酰胺是极好的（随实验误差变化），对于乙酰唑胺和4-羧基苯磺酰胺是良好的（变化接近一个数量级），但是对于其他的差，例如，舒必利。确定了K-D值的精度，并讨论了各种方法的优缺点。此外，我们批判性地评估了三种检查方法的测量，样品消耗，时间要求方面，并讨论了它们的局限性。版权所有（C）2009约翰威利父子有限公司
We performed a systematic comparison of three label-free methods for quantitative assessment of binding strengths of proteins interacting with small molecule ligands. The performance of (1) nanoelectrospray ionization mass spectrometry (nESI-MS), (2) surface plasmon resonance (SPR), and (3) isothermal titration calorimetry (ITC) was compared for the determination of dissociation constants (K-D). The model system studied for this purpose was the human carbonic anhydrase I (hCAI) with eight known and well characterized sulfonamide inhibitors (Krishnamurthy et at., Chem. Rev. 2008,108:946-1051). The binding affinities of the inhibitors chosen vary by more than four orders of magnitude e.g., the K-D value determined for ethoxzolamide by nESI-MS was 5 +/- 1 nM and the K-D value for sulfanilamide was 145.7 +/- 10.0 mu M. The agreement of the determined K-D values by the three methods investigated was excellent for ethoxzolamide and benzenesulfonamide (variation with experimental error), good for acetazolamide and 4-carboxybenzenesulfonamide (variation by similar to one order of magnitude), but poor for others e.g., sulpiride. The accuracies of the K-D values are determined, and advantages and drawbacks of the individual methods are discussed. Moreover, we critically evaluate the three examined methods in terms of ease of the measurement, sample consumption, time requirement, and discuss their limitations. Copyright (C) 2009 John Wiley & Sons, Ltd.