Protein-Protein Binding Affinities in Solution Determined by Electrospray Mass Spectrometry
Protein-Protein Binding Affinities in Solution Determined by Electrospray Mass Spectrometry
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DOI：
10.1007/s13361-010-0052-1
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发表时间：
2011-03-01
影响因子：
3.2
通讯作者：
Konermann, Lars
中科院分区：
文献类型：
--
作者：
Liu, Jiangjiang;Konermann, Lars
电喷雾电离（ESI）允许多蛋白复合物转移到气相，从而提供了一种通过质谱（MS）监测这些非共价组件的化学计量学的简单方法。然而，目前尚不清楚的是，所测量的自由和结合物种的离子丰度比是否适用于确定溶液相结合亲和力（K (d)值）。许多类型的质谱仪使用rf-only四极作为离子引导。这项工作表明，用于这些设备的设置是确保均匀传输行为的关键因素，这是有意义的亲和测量的先决条件。使用牛β -乳球蛋白和血红蛋白作为模型系统，证明了在精心调整的条件下，“直接”ESI-MS方法能够提供与先前发表的溶液相数据非常一致的K (d)值。在测试的几个离子源中，常规ESI发射器以1 μ L min（-1）的压力驱动流量工作，获得了最理想的结果。这些实验中的潜在问题包括构象诱导的电离效率差异、无意碰撞诱导的解离和esi诱导的聚类伪影。可以进行一些简单的测试来评估这些因素在所使用的条件下是否普遍存在。此外，该方法的保真度可以通过在广泛的浓度范围内进行测量来仔细检查。总的来说，这项工作支持了直接ESI-MS方法测定溶液中蛋白质-蛋白质复合物结合亲和力的可行性。
Electrospray ionization (ESI) allows the transfer of multi-protein complexes into the gas phase, thereby providing a simple approach for monitoring the stoichiometry of these noncovalent assemblies by mass spectrometry (MS). It remains unclear, however, whether the measured ion abundance ratios of free and bound species are suitable for determining solution-phase binding affinities (K (d) values). Many types of mass spectrometers employ rf-only quadrupoles as ion guides. This work demonstrates that the settings used for these devices are a key factor for ensuring uniform transmission behavior, which is a prerequisite for meaningful affinity measurements. Using bovine beta-lactoglobulin and hemoglobin as model systems, it is demonstrated that under carefully adjusted conditions the "direct" ESI-MS approach is capable of providing K (d) values that are in good agreement with previously published solution-phase data. Of the several ion sources tested, a regular ESI emitter operated with pressure-driven flow at 1 mu L min(-1) provided the most favorable results. Potential problems in these experiments include conformationally-induced differences in ionization efficiencies, inadvertent collision-induced dissociation, and ESI-induced clustering artifacts. A number of simple tests can be conducted to assess whether or not these factors are prevalent under the conditions used. In addition, the fidelity of the method can be scrutinized by performing measurements over a wide concentration range. Overall, this work supports the viability of the direct ESI-MS approach for determining binding affinities of protein-protein complexes in solution.