Soft-Landing of Size-Separated Macromolecules for Structural Analysis by Cryo-TEM

Abstract Large numbers of clinically relevant protein structures remain inaccessible to cryogenic electron microscopy (Cryo-EM) due to challenges in sample preparation. Various "soft-landing” approaches currently in development aim to overcome this issue by first size-separating target proteins by native mass spectrometry (MS) and then directing their deposition onto an EM sample grid. These techniques have enormous potential to advance structural biology and drive drug discovery but face inherent obstacles for preserving protein structure during MS- based separation. Following electrospray ionization, rapid desiccation of proteins under high-vacuum required for mass analysis and high energy ballistic trajectories of analyte ions pose fundamental challenges for the advancement of MS-based soft-landing methods. NanoEngineering Corporation (NEC) is therefore partnering with the University of Michigan to circumvent these limitations by integrating soft-landing with an alternative low- cost separation technology known as Electrospray Differential Mobility Analysis (ES-DMA). Our high resolution ES-DMA instruments have recently demonstrated angstrom-scale sizing of protein complexes in the gas phase and preliminary data shows our ability to resolve and collect higher-order oligomeric assemblies that are currently intractable to chromatographic separation. By operating at atmospheric pressures and reduced charge states, ES-DMA offers several unique advantages over existing soft-landing approaches, including at least 50-fold less energetic deposition and the ability to maintain protein hydration. In Phase I we will exploit these properties to design, build, and test an ES-DMA soft landing system capable of achieving greater structural preservation and higher resolution cryo-EM data through improved control over target protein dehydration, deposition, and orientation. As proof of principle we aim to achieve separation and deposition of clinically relevant protein targets, such as higher-order oligomeric states of DNAJB6, a protein chaperone implicated in Alzheimer’s and Parkinson’s disease, and specific subsets of polymorphic dimer populations detected in therapeutic antibody preparations. Phase II efforts will focus on realizing the potential of this technology to generate high resolution cryo-EM data of these and similar targets for the first time. By avoiding the need for custom modification of expensive MS instrumentation this innovation will greatly improve access to soft-landing for structural biologists while at the same time overcoming limitations associated with MS-based separation and expanding the scope of potential protein targets. Commercialization will target over 100 cryo-EM labs in academia and industry as potential customers. We also anticipate that this sizing/deposition technology will open additional markets in bio- nanomaterials sciences for applications in engineering and diagnostics.