Ion mobility-mass spectrometry for structural proteomics

Abstract

Ion mobility coupled to mass spectrometry has been an important tool in the fields of chemical physics and analytical chemistry for decades, but its potential for interrogating the structure of proteins and multiprotein complexes has only recently begun to be realized. Today, ion mobility-mass spectrometry is often applied to the structural elucidation of protein assemblies that have failed high-throughput crystallization or NMR spectroscopy screens. Here, we highlight the technology, approaches and data that have led to this dramatic shift in use, including emerging trends such as the integration of ion mobility-mass spectrometry data with more classical (e.g., 'bottom-up') proteomics approaches for the rapid structural characterization of protein networks.

Figure 1. The challenge of structural genomics: converting protein interaction networks into protein structures

High-throughput structural genomics efforts currently underway rely heavily on technologies that can convert an isolated multiprotein complex (A) directly into a structural model of atomic resolution (dashed arrow). The stringent sample requirements involved result in relatively high failure rates for such experiments. An alternative approach (solid arrows) uses mass spectrometry data of intact complexes to generate a contact map (B), integrates ion mobility data and other constraints to build a 3D topology model (C), and utilizes homology modeling or other forms of local constraint to generate the final atomic model (D) for multiprotein complexes. This ion mobility–mass spectrometry approach is projected to be a more universal, more sensitive and higher throughput alternative to contemporary structural biology technologies.

Figure 2. Ion mobility–mass spectrometry data acquisition and basic principles

Ions are generated at the ion source (lower left) and are allowed to drift in an ion guide filled with neutral gas molecules under the influence of an electric field. The ions migrate through this region according to their size-to-charge ratio. They are then injected into a ToF mass analyzer under vacuum for m/z analysis. The resulting data are 3D, containing ion intensity, size and mass information. The various dimensions of the data can be shown as a contour plot (middle, bottom), or 2D selections in drift time or m/z (lower right). A key for the diagram is shown (upper right). IM: Ion mobility; IM–MS: Ion mobility–mass spectrometry; MS: Mass spectrometry; m/z: Mass-to-charge ratio.

Figure 3. A high-throughput screening process to discover the optimal solution conditions for protein complex topology mapping by ion mobility–mass spectrometry

(A) A 2D screen is developed by varying the composition of solutions in a stepwise fashion over several important variables (e.g., organic content). Ions produced from each solution state are then measured against basic figures of merit: MS resolving power and mass accuracy (pink), IM resolution and collision cross-section (blue), total ion intensity (green), and the percentage of current that carries signal for subcomplexes or monomeric proteins (purple). (B) Optimal solutions for each of these classes of information are identified and recorded. In many cases, the optimal solution conditions for each figure of merit are mutually exclusive. The three dots at the bottom of part (A) and part (B) indicate that additional 2D solution screens can be implemented as needed. CCS: Collision cross-section; IM: Ion mobility; m/z: Mass-to-charge ratio.

Figure 4. Flow diagram for an integrated ion mobility–mass spectrometry structural proteomics workflow

After protien complex isolation via either standard affinity purification strategies, or following overexpression and reconstitution of the complex in vitro (purple box, top), unknown protein samples are split into three channels. One portion of the sample is subjected to denaturation and enzymatic digestion for a combination of ‘top-down’ and ‘bottom-up’-type protein identification experiments. These steps are critical for forming an accurate component list for protein contact map generation. A second sample fraction is submitted to MS for intact analysis, where the assembly is dissociated using a combination of solution and gas-phase approaches to deduce protein connectivity. Information from both of the above sample streams (turquoise) is combined to assemble a protein contact map. A third sample fraction, using optimized solution conditions, is submitted for ion-mobility analysis and measurement of protein size. Ideally, this step can be performed in parallel with MS analysis of the intact protein complex and subcomplexes created by dissociation. Size information on monomers is used to refine structures of the subunits within the complex, and various subcomplexes in a stepwise fashion. A cartoon showing how ion mobility data can refine structures for both a multidomain monomer (purple) and a protein dimer (yellow/blue) are shown, and this information is combined with MS-derived contact map information to provide a complete 3D protein topology (red). This information is then further combined with other sources of information or homology modeling to provide a complete atomic model of the complex of interest. IM-MS: Ion mobility-mass spectrometry; MS: Mass spectrometry: m/z: Mass-to-charge ratio.