论文标题
准备完整的大型天然蛋白质复合物的制备质谱仪
A preparative mass spectrometer to deposit intact large native protein complexes
论文作者
论文摘要
电喷雾离子梁沉积(ES-IBD)是研究从小金属簇到大蛋白质组件的分子结构和反应性的多功能工具。它将分子轻轻地进入气相,可以准确地操纵和纯化它们,然后将控制沉积在各种底物上。结合成像技术,可以获得明确定义分子的直接结构信息,这对于通过间接质谱技术的测试和解释结果至关重要。迄今为止,离子光束沉积实验仅限于全球少数定制仪器,并且没有商业选择。在这里,我们提出了一个模块,该模块将离子光束沉积功能添加到流行的商业MS平台(Thermo Scientific $^{\ MATHRM {tm}}} $ Q QUARTIVE $^{\ MATHRM {TM}} $ UHMR)。这种组合大大减少了与自定义工具相关的间接费用,同时受益于既定的高性能和可靠性。我们介绍了当前的性能特征,包括梁强度,着陆 - 能量控制和广泛分子的沉积点大小。与原子力显微镜(AFM)和透射电子显微镜(TEM)结合使用,我们将近元素与展开的蛋白质区分开,并在脱水和沉积后显示出蛋白质组件天然形状的保留。此外,我们使用酶法测定法来量化沉积A干燥表面后非共价蛋白质复合物的活性。总之,这些结果表明ES-IBD在结构生物学中的应用中具有很大的潜力,但也概述了为其全部发挥其全部潜力所需解决的挑战。
Electrospray ion-beam deposition (ES-IBD) is a versatile tool to study structure and reactivity of molecules from small metal clusters to large protein assemblies. It brings molecules gently into the gas phase where they can be accurately manipulated and purified, followed by controlled deposition onto various substrates. In combination with imaging techniques, direct structural information of well-defined molecules can be obtained, which is essential to test and interpret results from indirect mass spectrometry techniques. To date, ion-beam deposition experiments are limited to a small number of custom instruments worldwide, and there are no commercial alternatives. Here we present a module that adds ion-beam deposition capabilities to a popular commercial MS platform (Thermo Scientific$^{\mathrm{TM}}$ Q Exactive$^{\mathrm{TM}}$ UHMR). This combination significantly reduces the overhead associated with custom instruments, while benefiting from established high performance and reliability. We present current performance characteristics including beam intensity, landing-energy control, and deposition spot size for a broad range of molecules. In combination with atomic force microscopy (AFM) and transmission electron microscopy (TEM), we distinguish near-native from unfolded proteins and show retention of native shape of protein assemblies after dehydration and deposition. Further, we use an enzymatic assay to quantify activity of an non-covalent protein complex after deposition an a dry surface. Together, these results indicate a great potential of ES-IBD for applications in structural biology, but also outline the challenges that need to be solved for it to reach its full potential.