Wortmann Arno, Jecklin Matthias C, Touboul David, Badertscher Martin, Zenobi Renato
Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland.
J Mass Spectrom. 2008 May;43(5):600-8. doi: 10.1002/jms.1355.
We describe an approach for the determination of binding constants for protein-ligand complexes with electrospray ionization mass spectrometry, based on the observation of unbound ligands competing for binding to a protein target. For the first time, dissociation constants lower than picomolar could be determined with good accuracy by electrospray ionization mass spectrometry. The presented methodology relies only on the determination of signal intensity ratios for free ligands in the low mass region. Therefore, all the advantages of measuring low masses with mass spectrometry, such as high resolution are preserved. By using a reference ligand with known binding affinity, the affinity of a second ligand can be determined. Since no noncovalently bound species are observed, assumptions about response factors are not necessary. The method is validated with ligands binding to avidin and applied to ligands binding to p38 mitogen-activated protein kinase.
我们描述了一种用电喷雾电离质谱法测定蛋白质-配体复合物结合常数的方法，该方法基于观察未结合的配体竞争与蛋白质靶点结合的情况。首次通过电喷雾电离质谱法能够以良好的准确度测定低于皮摩尔的解离常数。所提出的方法仅依赖于低质量区域中游离配体信号强度比的测定。因此，保留了质谱法测量低质量物质的所有优点，如高分辨率。通过使用具有已知结合亲和力的参考配体，可以确定第二种配体的亲和力。由于未观察到非共价结合的物种，因此无需对响应因子进行假设。该方法已用与抗生物素蛋白结合的配体进行了验证，并应用于与p38丝裂原活化蛋白激酶结合的配体。
