Three-Dimensional Chemical Mapping of a Single Protein in the Hydrated State with Atom Probe Tomography

Abstract

Unravelling the three-dimensional structures and compositions of biological macromolecules sheds light on their functions and also contributes to the design of future biochemical compounds and processes. Atom probe tomography (APT) is demonstrated in this research as a new and effective approach to explore the structure and chemical composition of a single protein in the hydrated state. By introducing graphene encapsulation, proteins in solution can be immobilized on a metal specimen tip, with an end radius in the range of 50 nm to allow field ionization and evaporation. Using a ferritin particle as an example, analysis of the mass spectrum and reconstructed 3D chemical maps at near-atomic resolution acquired from APT reveals the core consisting of iron and iron oxides, the peptide shell containing amino acids, and the interior interface between the iron core and the peptide shell. The quantitative distribution and proportion of iron isotopes from a single ferritin core have been determined for the first time, as well as identification of the possible sites of amino acids inside the protein shell. The complete experimental protocol is straightforward and lays a foundation for future exploration of various macromolecules in a controlled environment.

Enabling near-atomic–scale analysis of frozen water

“…The difference in our experiment is that our entire specimen was made of ice, leading to the conclusion that an electrostatic field must be concentrated at the ice-vacuum interface throughout the experiment. The expected electrostatic field intensity is also much lower in our case, compared to other recent reports where the field is sufficiently high to lead, typically, to the fragmentation of the water molecules (15,55).…”

“…The usefulness of APT in studying wet chemical systems has been limited by the challenges inherent to the analysis of liquid media. There are ongoing efforts to push the development of "cryo-APT"-although the term is slightly inappropriate as all APT experiments are performed at cryogenic temperature, typically at 20 to 80 K. The development of cryo-approaches for specimen preparation and specimen transfer (9)(10)(11) has enabled the analysis of limited volumes of frozen water (12)(13)(14) sandwiched between a needle-shaped metallic specimen and graphene sheets (15), akin to liquid cells for TEM (2). To pave the way toward studying large volumes of frozen water, strategies for specimen preparation and systematic studies are necessary to enable a precise analysis and, hence, an assessment of the perform-ance limits of cryo-APT.…”

Graphene encapsulation enables vitreous ice sample for APT and near-atomic reconstruction of nanoparticle-liquid interface

“…The authors acknowledge funding from the Australian Research Council (DP180103955) and also the contributions from coauthors in [1][2][3]. This work was performed in part at the Melbourne Centre for Nanofabrication (MCN), Victorian Node of the Australian National Fabrication Facility (ANFF).…”

“…The reconstructed 3D chemical maps further reveal the original position of ions within the frozen hydrated sample [1]. This approach allows exploring domains previously unreachable, such as a single protein in solution [2].…”

3D Sub-Nanometer Analysis of Glucose in an Aqueous Solution by Cryo-Atom Probe Tomography

“…Depending on the controlled removal of individual atoms from a sharp needle-like emitter in the form of ions, this destructive method transforms the studied sample volume into a 3D computer model with individual ions characterized chemically. One of the current trends in APT is to gain a deeper understanding of soft matter in general and liquids in particular [7][8][9][10][11][12][13][14][15][16] . While thin layers of ice, created by condensed vapour on metallic tips, have been studied previously [17][18][19][20][21] , the newly available cryo-transfer systems 22,23 facilitate the preparation and transport of APT specimens from frozen liquids with large sampled volumes.…”