A Preparative Mass Spectrometer to Deposit Intact Large Native Protein Complexes.
A Preparative Mass Spectrometer to Deposit Intact Large Native Protein Complexes.
复制标题
制备质谱计沉积完整的大型天然蛋白复合物。
DOI：
10.1021/acsnano.2c04831
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发表时间：
2022-09-27
期刊：
影响因子：
17.1
通讯作者：
Rauschenbach, Stephan
中科院分区：
文献类型：
--
作者：
Fremdling, Paul;Esser, Tim K.;Saha, Bodhisattwa;Makarov, Alexander A.;Fort, Kyle L.;Reinhardt-Szyba, Maria;Gault, Joseph;Rauschenbach, Stephan
电喷雾离子束沉积 (ES-IBD) 是一种多功能工具，可用于研究从小型金属簇到大型蛋白质组装体的分子结构和反应性。它将分子温和地带入气相，在那里可以精确地操纵和纯化它们，然后受控沉积到各种基材上。与成像技术相结合，可以获得明确分子的直接结构信息，这对于测试和解释间接质谱技术的结果至关重要。迄今为止，离子束沉积实验仅限于全球范围内的少数定制仪器，并且没有商业替代品。在这里，我们展示了一个为流行的商业 MS 平台（Thermo Scientific Q Exactive UHMR 质谱仪）添加离子束沉积功能的模块。这种组合显着降低了与定制仪器相关的开销，同时受益于既定的高性能和可靠性。我们展示了当前的性能特征，包括各种分子的光束强度、着陆能量控制和沉积点尺寸。结合原子力显微镜（AFM）和透射电子显微镜（TEM），我们区分了近乎天然的蛋白质和未折叠的蛋白质，并显示了脱水和沉积后蛋白质组装体的天然形状的保留。此外，我们使用酶测定来量化非共价蛋白质复合物沉积在干燥表面上后的活性。总之，这些结果不仅表明 ES-IBD 在结构生物学应用中具有巨大潜力，而且还概述了其充分发挥潜力所需解决的挑战。
Electrospray ion-beam deposition (ES-IBD) is a versatile tool to study the 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 on 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 Q Exactive UHMR mass spectrometer). 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 the native shape of protein assemblies after dehydration and deposition. Further, we use an enzymatic assay to quantify the activity of a noncovalent protein complex after deposition on a dry surface. Together, these results not only 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.
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影响因子：
15
作者：
Anggara K;Zhu Y;Delbianco M;Rauschenbach S;Abb S;Seeberger PH;Kern K
通讯作者：
Kern K
DOI：
10.1093/pnasnexus/pgac153
发表时间：
2022-09
期刊：
PNAS NEXUS
影响因子：
--
作者：
Esser, Tim K.;Bohning, Jan;Fremdling, Paul;Agasid, Mark T.;Costin, Adam;Fort, Kyle;Konijnenberg, Albert;Gilbert, Joshua D.;Bahm, Alan;Makarov, Alexander;Robinson, Carol V.;Benesch, Justin L. P.;Baker, Lindsay;Bharat, Tanmay A. M.;Gault, Joseph;Rauschenbach, Stephan
通讯作者：
Rauschenbach, Stephan
影响因子：
3.3
作者：
Laskin, Julia;Wang, Peng;Hadjar, Omar
通讯作者：
Hadjar, Omar
DOI：
10.1016/1044-0305(92)87067-9
发表时间：
1992-05-01
影响因子：
3.2
作者：
DOUGLAS, DJ;FRENCH, JB
通讯作者：
FRENCH, JB
影响因子：
14.8
作者：
Hernandez, Helena;Robinson, Carol V.
通讯作者：
Robinson, Carol V.