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首页> 外文期刊>Journal of the American Society for Mass Spectrometry >Which electrospray-based ionization method best reflects protein-ligand interactions found in solution? A comparison of ESI, nanoESI, and ESSI for the determination of dissociation constants with mass spectrometry
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Which electrospray-based ionization method best reflects protein-ligand interactions found in solution? A comparison of ESI, nanoESI, and ESSI for the determination of dissociation constants with mass spectrometry

机译：哪种基于电喷雾的电离方法最能反映溶液中的蛋白质-配体相互作用？ ESI，nanoESI和ESSI在质谱测定解离常数方面的比较

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摘要

We present a comparison of three different electrospray-based ionization techniques for the investigation of noncovalent complexes with mass spectrometry. The features and characteristics of standard electrospray ionization (ESI), chip-based nanoESI, and electrosonic spray ionization (ESSI) mounted onto a hybrid quadrupole time-of-flight mass spectrometer were compared in their performance to determine the dissociation constant (K,) of the model system hen egg white lysozyme (HEWL) binding to N,N',N ''-triacetylchitotriose (NAG(3)). The best K-D value compared with solution data were found for ESSI, 19.4 +/- 3.6 mu M. Then, we determined the K(D)s of the two nucleotide binding sites of adenylate kinase (AK), where we obtained K(D)s of 2.2 +/- 0.8 mu M for the first and 19.5 +/- 8.0 mu M for the second binding site using ESSI. We found a weak charge state dependence of the KD for both protein-ligand systems, where for all ionization techniques the KD value decreases with increasing charge state. We demonstrate that ESSI is very gentle and insensitive to instrumental parameters, and the KD obtained is in good agreement with solution phase results from the literature. In addition, we tried to determine the K-D for the lymphocyte-specific kinase LCK binding to a kinase inhibitor using nanoESI due to the very low amount of sample available. In this case, we found KD values with a strong charge state dependence, which were in no case close to literature values for solution phase.
机译：我们比较了三种不同的基于电喷雾的电离技术，以研究非共价配合物质谱。比较了安装在混合四极杆飞行时间质谱仪上的标准电喷雾电离（ESI），基于芯片的nanoESI和电声喷雾电离（ESSI）的性能，以确定解离常数（K，）蛋清溶菌酶（HEWL）与N，N'，N''-三乙酰基壳三糖（NAG（3））结合的模型系统的结构图。发现ESSI的最佳KD值与溶液数据相比为19.4 +/- 3.6μM。然后，我们确定了腺苷酸激酶（AK）的两个核苷酸结合位点的K（D），得到了K（D使用ESSI，第一个结合位点为2.2 +/- 0.8μM，第二个结合位点为19.5 +/- 8.0μM。我们发现两种蛋白质-配体系统对KD的电荷状态依赖性均较弱，其中对于所有电离技术，KD值均随电荷状态的增加而降低。我们证明了ESSI非常温和且对仪器参数不敏感，并且所获得的KD与文献中的溶液相结果非常吻合。此外，由于可用样品的量非常少，我们试图使用nanoESI确定淋巴细胞特异性激酶LCK与激酶抑制剂结合的K-D。在这种情况下，我们发现KD值具有很强的电荷状态依赖性，在任何情况下都不接近解决方案阶段的文献值。

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