Electron Microscopy of Selected Proteins and Protein Complexes Through Preparative Mass Spectrometry

通过制备型质谱法对选定的蛋白质和蛋白质复合物进行电子显微镜检查

• 批准号: EP/V051474/1
• 负责人: Stephan Rauschenbach
• 金额: $ 46.78万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FV051474%2F1
• 关键词: Electron; Microscopy; Selected; Proteins; Protein

In order to take a picture of a large biological molecule, such as a protein, it has to be frozen in a very thin sheet of ice where it can be imaged by an electron microscope.Many images of the same type of molecule are laid over another to improve the resolution, ultimately revealing atomic positions. This procedure requires a highly pure protein sample in solution and plunge freezing of water films hanging in very fine mesh grids, a procedure which is not compatible with all proteins. In particular proteins that reside in cell membranes, prefer to be at the water-air surface, where they are destroyed and thus cannot be imaged. Also protein, which are composed from many subunits cannot be purified sufficiently so that the averaging will fail produce a high resolution image.This proposal aims at developing an alternative sample preparation method, based on native electrospray mass spectrometry. Native electrospray ionisation can transfer a protein from solution into a gaseous particle with charge, which can be weighed (by mass spectrometry) and hence chemically identified. We will use this process to isolate the particle and instead of only detecting it, we will enrich one selected type of protein on the sample for electron microscopy. The major challenge thereby is to land the molecule so gentle, that it's characteristic native shape is not destroyed in the process. With mass-selected sample fabrication we can link chemical information to protein structure, which is information highly desirable in the development of medicine and biology.

为了给一个大的生物分子（比如蛋白质）拍照，它必须被冷冻在一个非常薄的冰层中，在那里它可以被电子显微镜成像。许多相同类型分子的图像叠加在一起以提高分辨率，最终显示出原子位置。这个程序需要一个高纯度的蛋白质样品在溶液中，并将悬挂在非常细的网状网格中的水膜急剧冷冻，这个程序并不适用于所有的蛋白质。特别是存在于细胞膜上的蛋白质，更喜欢在水-空气表面，在那里它们被破坏，因此无法成像。此外，蛋白质由许多亚基组成，不能被充分纯化，因此平均将无法产生高分辨率的图像。本提案旨在开发一种基于原生电喷雾质谱的替代样品制备方法。原生电喷雾电离可以将蛋白质从溶液中转移到带电荷的气体颗粒中，这种气体颗粒可以称重（通过质谱法），从而进行化学鉴定。我们将使用这个过程来分离粒子，而不是仅仅检测它，我们将在样品上富集一种选定的蛋白质用于电子显微镜。因此，主要的挑战是使分子如此温和地着陆，使其特有的天然形状在此过程中不会被破坏。通过大量选择样品的制作，我们可以将化学信息与蛋白质结构联系起来，这是医学和生物学发展中非常需要的信息。

项目成果

Cryo-EM samples of gas-phase purified protein assemblies using native electrospray ion-beam deposition.

• DOI: 10.1039/d2fd00065b
• 发表时间: 2022-11-08
• 期刊: FARADAY DISCUSSIONS
• 影响因子: 3.4
• 作者: Esser, Tim K.;Bohning, Jan;Fremdling, Paul;Bharat, Tanmay;Gault, Joseph;Rauschenbach, Stephan
• 通讯作者: Rauschenbach, Stephan

A Preparative Mass Spectrometer to Deposit Intact Large Native Protein Complexes.

• DOI: 10.1021/acsnano.2c04831
• 发表时间: 2022-09-27
• 期刊: ACS NANO
• 影响因子: 17.1
• 作者: Fremdling, Paul;Esser, Tim K.;Saha, Bodhisattwa;Makarov, Alexander A.;Fort, Kyle L.;Reinhardt-Szyba, Maria;Gault, Joseph;Rauschenbach, Stephan
• 通讯作者: Rauschenbach, Stephan