Brian Clowers - US grants

With support from the Chemical Measurement and Imaging Program in the Division of Chemistry, Professor Davis at Azusa Pacific University APU) and Professor Clowers at Washington State University (WSU) are pursuing a new approach for detecting and characterizing chemicals at trace levels using a technique termed Radiative Ion-Ion Neutralization (RIIN). Under the guidance of Drs. Davis and Clowers, a combined team of undergraduate and graduate researchers advance an innovative approach to chemical detection that has the potential to directly benefit a number of existing technologies routinely used for environmental monitoring, medical diagnostics, and threat detection in both the civilian and military domains. Current methods for detecting trace chemicals in the field often rely upon technology that not only ignores diagnostically-useful information, but restricts the chemical detection limits that may be reached. Successful characterization of the Radiative Ion-Ion Neutralization (RIIN) mechanism enhances the levels of information provided from rapid chemical measurements and impact fields from preliminary security screening to biochemistry. Through the implementation of this project, students from APU have the opportunity to travel to WSU during the summer academic months to work with Dr. Clowers and his students in a graduate-level research environment while students from WSU have the opportunity to travel to APU during the academic year to gain experience leading research and mentoring undergraduate research assistants.

Existing approaches for ion detection at high pressure are inherently limited by the signal-to-noise performance characteristics of analog circuitry. At present, no compatible ion amplification technology is capable of single ion-counting under atmospheric conditions. This has constrained the growth and development of ion mobility spectrometry as a trace analytical tool. Radiative Ion-Ion Neutralization (RIIN) provides a new means of recording gas-phase ion signals that also integrates optical spectroscopy with traditional ion mobility measurements. The development and characterization of the RIIN detection platform is pursued across three objectives: 1) Development of RIIN as a quantitative gas-phase ion-transduction mechanism; 2) Determination of the RIIN photon release mechanism; and 3) Leveraging wavelength resolved RIIN signals for chemical identification. The unique signal transduction provided by the RIIN system directly benefits platforms focused on characterizing atmospheric pressure gas-phase ions and adds a highly informative second dimension of information that can more precisely assess the chemical content of complex mixtures. An equally important goal of this work is the integration of RIIN-informed gas-phase spectroscopy into the ion mobility experiment. The resulting multidimensional platform represents both a new analytical methodology and means of directly probing the chemical functional groups for gas-phase ions.

With support from the Chemical Measurement and Imaging Program in the Division of Chemistry, the research groups of Professor Brian H. Clowers (Washington State University) and Professor Christopher J. Hogan (University of Minnesota) are working to improve the performance and capabilities of ion mobility spectrometry (IMS), a chemical analysis method with applications ranging from rapid screening for explosives in airports to detailed insight into the structure and function of biomolecules associated with healthy and diseased organisms. The team is devising means of using selective interactions with solvent molecules and other gaseous additives within the IMS instrument in order to enhance the ability to separate and identify molecules in complex mixtures. By combining these methods with mass spectrometry (a powerful complementary technique) and new computational tools, the research aims to provide an innovative workflow that is broadly applicable to chemical analysis. Students engaged in this research obtain unique interdisciplinary training enhanced by opportunities to work ?out-of-discipline? as part of their studies. Research opportunities are also made available to undergraduate and high school students.

Recently, Professors Clowers and Hogan have demonstrated enhanced separation factors through selective interactions between gas-phase ions and organic vapor modifiers introduced into ion mobility systems. They now seek to gain a more detailed understanding of the underlying interactions of ion-vapor complexes in order to predict and enable expanded use of IM-MS and tandem IM experiments. Their approach uses an innovative, multidimensional ion mobility/hydrogen-deuterium exchange mass spectrometry strategy and first-principles modeling of gas-phase ion-neutral interactions and vapor uptake. Primary aims are to determine (1) if thermodynamics of vapor association depend upon chemical functionality in amino acids and simple peptide ions; (2) if the extent of binding can be extrapolated as a characterization tool for larger, multiply charged ions exhibiting known secondary structure; (3) if clustering dynamics quantitatively describe the behavior of ions in high-field and asymmetric waveform experiments; and (4) if molecular dynamics can accurately describe solvated ion transport.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Project Summary Advancements in biophysical techniques, such as X-ray and cryoEM, have undoubtedly accelerated determination of protein structure. However, it still remains challenging to capture snapshots of protein folding intermediates, including non-native states, and breathing motions that protein assemblies undergo to perform their biological function. Moreover, understanding how molecules, such as lipids, modulate protein structure and function is of paramount biological importance. Over the past two decades, mass spectrometry (MS) of intact protein complexes, often referred to as native MS, has emerged as an indispensable biophysical technique whereby non-covalent interactions and protein structure are preserved within the mass spectrometer. Native MS is a rapid and sensitive technique that has already provided invaluable information on subunit stoichiometry and topology, allostery and cooperativity for individual ligand binding events, including their binding thermodynamics. The coupling with ion mobility (IM), a separation technique based on molecule charge and shape, further enhances the capabilities of native MS where it has enabled collision cross section (CCS) measurements for large protein complexes, identification of different conformations for peptides and stabilizing ligands using collision induced unfolding, and insight in folded and denatured structure(s) of proteins. However, low- resolution commercial IM-MS instrumentation has not changed since its introduction 12 years ago. Herein, this proposal seeks to develop transformative native IM-MS technologies with high-resolution IM and MS capabilities that can address modern questions in structural biology, such as conformational dynamics, including those that may have remained ?hidden?, within membrane transporters under turnover conditions. In order to achieve these transformative goals, an interdisciplinary team of researchers whose expertise spans the fields of protein biophysics, expression and purification of proteins inclusive of membrane proteins, as well as traditional protein structure characterization, such as X-ray crystallography, has been assembled. Team members also possess decades of experience in the field of mass spectrometry inclusive of fundamental ion chemistry/physics, seminal contributions that have spawned MS proteomics, and related areas of analytical mass spectrometry and ion mobility- mass spectrometry. Collectively, the background and expertise of this research team is uniquely positioned to transform the field of IM-MS in the area of structural biology. In short, the proposed transformative research will lead to forefront IM-MS instrumentation that is poised to provide unprecedented insights into the structure and assembly of protein complexes and push the field into new frontiers of research.

Project Summary In addition to concentration, the orientation and conformation of proteins, carbohydrates, metabolites, and nucleic acids are essential characteristics differentiating healthy and diseased states. In fact, broadly available advances in mass spectrometry (MS), with unparalleled levels of selectivity, speed, and sensitivity, have armed researchers with new biological insights and prompt additional questions regarding molecular and biophysical parameters that differentiate disease states but transcend MS measurements. Ion mobility spectrometry (IMS) is a gas-phase separation technique that directly complements MS measurements and expands understanding regarding molecular shape and dynamics in biological systems. However, comparatively low sample utilization and separation efficiencies have hindered its broad adoption in the bioanalytical and clinical communities. With recent, broadly available technological advances in the field of printed circuit board (PCB) manufacturing a new class of ion mobility separation is enabled that largely alleviates the drawbacks of its predecessors. The Structures for Lossless Ion Manipulations (SLIM) framework achieves this goal by establishing a dynamic electric field capable of confining ionized molecules for expanded periods of time along with a means to efficiently fractionate the different classes prior to analysis using MS. Contemporary SLIM experiments achieve impressive levels of gas-phase ion separation, but focus only on one dimension of separation due to restrictions largely imposed by the underlying PCB electrode arrangements and control electronics. To cast the SLIM platform into multiple separation dimensions and achieve new levels of biologically relevant diagnostics, the present effort aims to develop and disseminate an economical tandem IMS platform that integrates a series of innovative, simplifying strategies. These include the integration of a low-cost electrode switch that expands the experimental versatility within the SLIM platform and a series of ion compression strategies aimed at creating high-density ion populations. Most importantly, and prior to MS analysis, we will exploit the highly compressed nature of the ion beams within the SLIM by subjecting these species to high intensity ultraviolet photons to induce molecular disruption and yield more information regarding the target biological system. Concurrent efforts using laser irradiation and a new class of UV-C light emitting diodes will be compared with the latter offering considerable cost-savings. The third, composite goal of this project is to address the duty cycle issues of existing SLIM concepts by fully multiplexing the tandem SLIM-ultraviolet photodissociation (UVPD) platform. With the added functionality of IMSn and the extended, multi-channel SLIM paths, the separation power of the system is anticipated to represent the state-of-the-art. At the conclusion of the proposed research we expect to realize a fully functioning, high-efficiency SLIM-UVPD framework capable of interfacing to all mass analyzers classes and ready to address a suite of biological problems ranging from metabolomics to structural biology.