Mass-Selective Soft-Landing of Protein Assemblies with Controlled Landing Energies
Mass-Selective Soft-Landing of Protein Assemblies with Controlled Landing Energies
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DOI：
10.1021/ac5018327
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发表时间：
2014-08-19
影响因子：
7.4
通讯作者：
Robinson, Carol V.
中科院分区：
文献类型：
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作者：
Mikhailov, Victor A.;Mize, Todd H.;Robinson, Carol V.
在真空中选择和软着陆生物纳米粒子是一种潜在的制备方法，可以分离非均质混合物，用于高分辨率结构研究或沉积纳米技术应用中的均质材料。然而，完整蛋白质组件的软着陆仍然具有挑战性，因为在实验过程中难以在大规模选择设备中操纵这些重物种并保持其结构。我们开发了一种串联质谱仪，它具有控制离子软着陆的能力，并且可以通过透射电子显微镜对软着陆粒子进行非原位可视化。通过对一组离子导向光学元件施加加速或减速电压来调节离子的动能，可以控制沉积条件。为了验证这一方法，我们研究了两种笼状蛋白复合物GroEL和铁蛋白，并研究了软着陆条件对该方法的吞吐量和蛋白质结构保存的影响。电喷雾电离后，基于质量电荷比的全息铁蛋白复合物和载铁蛋白复合物的分离使我们能够将分离的复合物独立地软着陆在适合下游透射电镜分析的网格上。在阴性染色后，软着陆配合物的图像显示它们的结构完整性在很大程度上是保守的，具有典型的铁铁蛋白中心空腔和全铁铁蛋白的铁核，在从液体到气体的相变、软着陆和真空脱水中幸存下来。
Selection and soft-landing of bionanoparticles in vacuum is potentially a preparative approach to separate heterogeneous mixtures for high-resolution structural study or to deposit homogeneous materials for nanotechnological applications. Soft-landing of intact protein assemblies however remains challenging, due to the difficulties of manipulating these heavy species in mass-selective devices and retaining their structure during the experiment. We have developed a tandem mass spectrometer with the capability for controlled ion soft-landing and ex situ visualization of the soft-landed particles by means of transmission electron microscopy. The deposition conditions can be controlled by adjusting the kinetic energies of the ions by applying accelerating or decelerating voltages to a set of ion-steering optics. To validate this approach, we have examined two cage-like protein complexes, GroEL and ferritin, and studied the effect of soft-landing conditions on the method's throughput and the preservation of protein structure. Separation, based on mass-to-charge ratio, of holo- and apo-ferritin complexes after electrospray ionization enabled us to soft-land independently the separated complexes on a grid suitable for downstream transmission electron microscopy analysis. Following negative staining, images of the soft-landed complexes reveal that their structural integrity is largely conserved, with the characteristic central cavity of apoferritin, and iron core of holoferritin, surviving the phase transition from liquid to gas, soft-landing, and dehydration in vacuum.