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

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. ESI-cryoPrep is a cryo-EM specimen preparation method that employs electrospray ionization techniques to deposit charged macromolecule-containing droplets on EM grids. Demonstrated across various protein samples, this approach effectively prevents biomolecule adsorption at air-water or graphene-water interfaces, addressing challenges related to protein denaturation and preferred orientation.