Determination of ligand-protein dissociation constants by electrospray mass spectrometry-based diffusion measurements
Determination of ligand-protein dissociation constants by electrospray mass spectrometry-based diffusion measurements
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DOI：
10.1021/ac049344o
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发表时间：
2004-12-01
影响因子：
7.4
通讯作者：
Konermann, L
中科院分区：
文献类型：
--
作者：
Clark, SM;Konermann, L
提出了一种定量配体-蛋白质相互作用的新方法。电喷雾电离质谱 (ESI-MS) 用于监测非共价配体在其蛋白质受体存在时的扩散行为。这些数据允许确定溶液中游离配体的分数，从而可以计算相应的解离常数。开发了一组条件，为此类测定提供了“允许的浓度范围”。该方法通过应用于两种不同的抑制剂-酶系统进行测试。测得的苯甲脒-胰蛋白酶和 N,N',N"-三乙酰壳三糖-溶菌酶的解离常数分别为 (50 +/- 10) 和 (6 +/- 1) mM。这两个结果与之前文献中的数据非常一致。与传统的基于 ESI-MS 的方法相比，本工作中使用的方法不依赖于特定溶液类型的保存 气相中的非共价相互作用。结果表明，即使 ESI-MS 中游离蛋白与配体结合蛋白的离子丰度比不能反映溶液中相应的浓度比，该方法也可以准确测定解离常数。
A novel approach for the quantification of ligand-protein interactions is presented. Electrospray ionization mass spectrometry (ESI-MS) is used to monitor the diffusion behavior of noncovalent ligands in the presence of their protein receptors. These data allow the fraction of free ligand in solution to be determined, such that the corresponding dissociation constants can be calculated. A set of conditions is developed that provides an "allowable range" of concentrations for this type of assay. The method is tested by applying it to two different inhibitor-enzyme systems. The dissociation constants measured for benzamidine-trypsin and for N,N',N"-triacetylchitotriose-lysozyme are (50 +/- 10) and (6 +/- 1) mM, respectively. Both of these results are in good agreement with previous data from the literature. In contrast to traditional ESI-MSbased methods, the approach used in this work does not rely on the preservation of specific solution-type noncovalent interactions in the gas phase. It is shown that this method allows an accurate determination of dissociation constants, even in cases in which the ion abundance ratio of free to ligand-bound protein in ESI-MS does not reflect the corresponding concentration ratio in solution.