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.

为了拍摄大型生物分子（例如蛋白质）的照片，必须将其冷冻在非常薄的冰层中，以便通过电子显微镜对其进行成像。同一类型分子的许多图像叠加在一起另一种是提高分辨率，最终揭示原子位置。该过程需要溶液中的高纯度蛋白质样品，并将悬挂在非常细的网格中的水膜骤冷，该过程并不与所有蛋白质兼容。特别是驻留在细胞膜中的蛋白质，更喜欢位于水-空气表面，在那里它们被破坏，因此无法成像。此外，由许多亚基组成的蛋白质无法充分纯化，因此平均将无法产生高分辨率图像。该提案旨在开发一种基于天然电喷雾质谱法的替代样品制备方法。天然电喷雾电离可以将蛋白质从溶液转移到带电荷的气态颗粒中，可以对其进行称重（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颗粒，而不只是检测它，我们将在电子显微镜样品上富集一种选定类型的蛋白质。因此，主要的挑战是使分子着陆得如此温和，以使其特有的天然形状在此过程中不会被破坏。通过大量选择的样品制造，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 of soft-landed ß -galactosidase: Gas-phase and native structures are remarkably similar

软着陆 α-半乳糖苷酶的冷冻电镜：气相结构和天然结构非常相似

• DOI: http://dx.10.1101/2023.08.17.553673
• 发表时间: 2023
• 作者: Esser T

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

使用自然电喷雾离子束沉积的气相纯化蛋白质组装体的冷冻电镜样品。

• DOI: http://dx.10.1039/d2fd00065b
• 发表时间: 2022
• 期刊: Faraday discussions
• 影响因子: 3.4
• 作者: Esser TK

Mass-selective and ice-free electron cryomicroscopy protein sample preparation via native electrospray ion-beam deposition

通过自然电喷雾离子束沉积进行质量选择性和无冰电子冷冻显微镜蛋白质样品制备

• DOI: http://dx.10.1093/pnasnexus/pgac153
• 发表时间: 2022
• 期刊: PNAS Nexus
• 作者: Esser T

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

用于沉积完整的大型天然蛋白质复合物的制备型质谱仪。

• DOI: http://dx.10.1021/acsnano.2c04831
• 发表时间: 2022
• 期刊: ACS nano
• 影响因子: 17.1
• 作者: Fremdling P