Silica-EmbeddedProteins Enable Higher Yield and AtomicIon Detection in Deep-UV Laser-Assisted Atom Probe Tomography

Abstract Determining the 3D structure of proteins is a long-standing challenge in the life sciences and is of paramount importance for understanding fundamental biological processes and advancing pharmaceutical development. Experimental techniques such as X-ray diffraction, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM) have been instrumental in determining numerous protein structures. However, these methods are not universally applicable, and new complementary approaches are still needed. Atom probe tomography (APT), a form of mass spectrometry capable of reconstructing the 3D chemical composition of materials with near-atomic resolution, has been proposed as a potential tool for the structural analysis of proteins. Although recent progress has been made, no existing methodology has yet demonstrated both high throughput and reproducibility. In this work, we apply deep-UV laser-assisted APT to study green fluorescent protein (GFP) embedded within an amorphous silica matrix. This approach enables the detection of several thousand proteins within a single measurement, representing a significant advancement toward structural analysis of proteins by APT. To assess whether the embedding procedure preserves the structural integrity of proteins, we combined experimental measurements with molecular dynamics simulations, which strongly suggest that the initial stages of silica encapsulation do not disrupt the native folding of GFP. Overall, these findings demonstrate the potential of deep-UV laser APT as a promising method for structural biology.