清华大学Xiaoyu Zhou、Hong-Wei Wang和Zheng Ouyang研究组合作取得一项新突破。他们的研究发现利用电喷雾辅助制备冷冻电镜样品可减轻界面效应。2024年4月25日出版的《自然-方法学》发表了这项成果。

研究人员介绍一种基于原生质谱电喷雾离子化的标本制备方法-ESI-cryoPrep，该方法旨在缓解大分子吸附在界面上时引起的蛋白质变性或优先取向等相关问题。通过微调喷洒参数，研究人员优化了蛋白质的保存完整性，并达到了分析目标大分子所需的冰厚度。

利用ESI-cryoPrep，研究人员制备了五种蛋白质的高质量冷冻样本，并实现了接近原子分辨率的三维重建。该研究结果表明，ESI-cryoPrep能有效地将大分子限制在无定形冰薄层的中间，有助于制备无印迹玻璃体样本。

这种保护机制的特点是不同大小的带电生物大分子在带电液滴中分布不均，可防止目标生物大分子吸附在空气-水或石墨烯-水界面上，从而避免了蛋白质颗粒的结构损坏或引入取向问题。

据了解，解决冷冻电镜标本制备过程中的界面效应仍然是一项挑战。

附：英文原文

Title: Electrospray-assisted cryo-EM sample preparation to mitigate interfacial effects

Author: Yang, Zi, Fan, Jingjin, Wang, Jia, Fan, Xiao, Ouyang, Zheng, Wang, Hong-Wei, Zhou, Xiaoyu

Issue&Volume: 2024-04-25

Abstract: Addressing interfacial effects during specimen preparation in cryogenic electron microscopy remains challenging. Here we introduce ESI-cryoPrep, a specimen preparation method based on electrospray ionization in native mass spectrometry, designed to alleviate issues associated with protein denaturation or preferred orientation induced by macromolecule adsorption at interfaces. Through fine-tuning spraying parameters, we optimized protein integrity preservation and achieved the desired ice thickness for analyzing target macromolecules. With ESI-cryoPrep, we prepared high-quality cryo-specimens of five proteins and obtained three-dimensional reconstructions at near-atomic resolution. Our findings demonstrate that ESI-cryoPrep effectively confines macromolecules within the middle of the thin layer of amorphous ice, facilitating the preparation of blotting-free vitreous samples. The protective mechanism, characterized by the uneven distribution of charged biomolecules of varying sizes within charged droplets, prevents the adsorption of target biomolecules at air–water or graphene–water interfaces, thereby avoiding structural damage to the protein particles or the introduction of dominant orientation issues.

DOI: 10.1038/s41592-024-02247-0

Source: https://www.nature.com/articles/s41592-024-02247-0