Surface-induced Dissociation Mass Spectrometry as a Structural Biology Tool.

Native mass spectrometry (nMS) is evolving into a workhorse for structural biology. The plethora of online and offline preparation, separation, and purification methods as well as numerous ionization techniques combined with powerful new hybrid ion mobility and mass spectrometry systems has illustrated the great potential of nMS for structural biology. Fundamental to the progression of nMS has been the development of novel activation methods for dissociating proteins and protein complexes to deduce primary, secondary, tertiary, and quaternary structure through the combined use of multiple MS/MS technologies. This review highlights the key features and advantages of surface collisions (surface-induced dissociation, SID) for probing the connectivity of subunits within protein and nucleoprotein complexes and, in particular, for solving protein structure in conjunction with complementary techniques such as cryo-EM and computational modeling. Several case studies highlight the significant role SID, and more generally nMS, will play in structural elucidation of biological assemblies in the future as the technology becomes more widely adopted. Cases are presented where SID agrees with solved crystal or cryoEM structures or provides connectivity maps that are otherwise inaccessible by "gold standard" structural biology techniques.

Native Mass Spectrometry and Surface Induced Dissociation Provide Insight into the Post-Translational Modifications of Tetrameric AQP0 Isolated from Bovine Eye Lens.

Aquaporin-0 (AQP0) is a tetrameric membrane protein and the most abundant membrane protein in the eye lens. Interestingly, there is little to no cellular turnover once mature lens fiber cells are formed, and hence, age-related modifications accumulate with time. While bottom-up mass spectrometry-based approaches can provide identification of post-translational modifications, they cannot provide information on how these modifications coexist in a single chain or complex. Native mass spectrometry, however, enables the transfer of the intact complex into the gas-phase allowing modifications to be identified at the tetramer level. Here, we present the use of native mass spectrometry and surface-induced dissociation to study the post-translational modifications of AQP0 isolated and purified from bovine eye lens, existing as multiple forms due to the different modification states naturally present.

Simulation of Energy-Resolved Mass Spectrometry Distributions from Surface-Induced Dissociation.

Understanding the relationship between protein structure and experimental data is crucial for utilizing experiments to solve biochemical problems and optimizing the use of sparse experimental data for structural interpretation. Tandem mass spectrometry (MS/MS) can be used with a variety of methods to collect structural data for proteins. One example is surface-induced dissociation (SID), which is used to break apart protein complexes (via a surface collision) into intact subcomplexes and can be performed at multiple laboratory frame SID collision energies. These energy-resolved MS/MS experiments have shown that the profile of the breakages depends on the acceleration energy of the collision. It is possible to extract an appearance energy (AE) from energy-resolved mass spectrometry (ERMS) data, which shows the relative intensity of each type of subcomplex as a function of SID acceleration energy. We previously determined that these AE values for specific interfaces correlated with structural features related to interface strength. In this study, we further examined the structural relationships by developing a method to predict the full ERMS plot from the structure, rather than extracting a single value. First, we noted that for proteins with multiple interface types, we could reproduce the correct shapes of breakdown curves, further confirming previous structural hypotheses. Next, we demonstrated that interface size and energy density (measured using Rosetta) correlated with data derived from the ERMS plot (R2 = 0.71). Furthermore, based on this trend, we used native crystal structures to predict ERMS. The majority of predictions resulted in good agreement, and the average root-mean-square error was 0.20 for the 20 complexes in our data set. We also show that if additional information on cleavage as a function of collision energy could be obtained, the accuracy of predictions improved further. Finally, we demonstrated that ERMS prediction results were better for the native than for inaccurate models in 17/20 cases. An application to run this simulation has been developed in Rosetta, which is freely available for use.

Relative interfacial cleavage energetics of protein complexes revealed by surface collisions.

To fulfill their biological functions, proteins must interact with their specific binding partners and often function as large assemblies composed of multiple proteins or proteins plus other biomolecules. Structural characterization of these complexes, including identification of all binding partners, their relative binding affinities, and complex topology, is integral for understanding function. Understanding how proteins assemble and how subunits in a complex interact is a cornerstone of structural biology. Here we report a native mass spectrometry (MS)-based method to characterize subunit interactions in globular protein complexes. We demonstrate that dissociation of protein complexes by surface collisions, at the lower end of the typical surface-induced dissociation (SID) collision energy range, consistently cleaves the weakest protein:protein interfaces, producing products that are reflective of the known structure. We present here combined results for multiple complexes as a training set, two validation cases, and four computational models. We show that SID appearance energies can be predicted from structures via a computationally derived expression containing three terms (number of residues in a given interface, unsatisfied hydrogen bonds, and a rigidity factor).

Surface-Induced Dissociation of Anionic vs Cationic Native-Like Protein Complexes.

Characterizing protein-protein interactions, stoichiometries, and subunit connectivity is key to understanding how subunits assemble into biologically relevant, multisubunit protein complexes. Native mass spectrometry (nMS) has emerged as a powerful tool to study protein complexes due to its low sample consumption and tolerance for heterogeneity. In nMS, positive mode ionization is routinely used and charge reduction, through the addition of solution additives, is often used, as the resulting lower charge states are often considered more native-like. When fragmented by surface-induced dissociation (SID), charge reduced complexes often give increased structural information over their "normal-charged" counterparts. A disadvantage of solution phase charge reduction is that increased adduction, and hence peak broadening, is often observed. Previous studies have shown that protein complexes ionized using negative mode generally form lower charge states relative to positive mode. Here we demonstrate that the lower charged protein complex anions activated by surface collisions fragment in a manner consistent with their solved structures, hence providing substructural information. Negative mode ionization in ammonium acetate offers the advantage of charge reduction without the peak broadening associated with solution phase charge reduction additives and provides direct structural information when coupled with SID. SID of 20S human proteasome (a 28-mer comprised of four stacked heptamer rings in an αßßα formation), for example, provides information on both substructure (e.g., splitting into a 7α ring and the corresponding ßßα 21-mer, and into α dimers and trimers to provide connectivity around the 7 α ring) and proteoform information on monomers.

Prediction of Protein Complex Structure Using Surface-Induced Dissociation and Cryo-Electron Microscopy.

A variety of techniques involving the use of mass spectrometry (MS) have been developed to obtain structural information on proteins and protein complexes. One example of these techniques, surface-induced dissociation (SID), has been used to study the oligomeric state and connectivity of protein complexes. Recently, we demonstrated that appearance energies (AE) could be extracted from SID experiments and that they correlate with structural features of specific protein-protein interfaces. While SID AE provides some structural information, the AE data alone are not sufficient to determine the structures of the complexes. For this reason, we sought to supplement the data with computational modeling, through protein-protein docking. In a previous study, we demonstrated that the scoring of structures generated from protein-protein docking could be improved with the inclusion of SID data; however, this work relied on knowledge of the correct tertiary structure and only built full complexes for a few cases. Here, we performed docking using input structures that require less prior knowledge, using homology models, unbound crystal structures, and bound+perturbed crystal structures. Using flexible ensemble docking (to build primarily subcomplexes from an ensemble of backbone structures), the RMSD100 of all (15/15) predicted structures using the combined Rosetta, cryo-electron microscopy (cryo-EM), and SID score was less than 4 Å, compared to only 7/15 without SID and cryo-EM. Symmetric docking (which used symmetry to build full complexes) resulted in predicted structures with RMSD100 less than 4 Å for 14/15 cases with experimental data, compared to only 5/15 without SID and cryo-EM. Finally, we also developed a confidence metric for which all (26/26) proteins flagged as high confidence were accurately predicted.

Investigation of sliding DNA clamp dynamics by single-molecule fluorescence, mass spectrometry and structure-based modeling.

Proliferating cell nuclear antigen (PCNA) is a trimeric ring-shaped clamp protein that encircles DNA and interacts with many proteins involved in DNA replication and repair. Despite extensive structural work to characterize the monomeric, dimeric, and trimeric forms of PCNA alone and in complex with interacting proteins, no structure of PCNA in a ring-open conformation has been published. Here, we use a multidisciplinary approach, including single-molecule Förster resonance energy transfer (smFRET), native ion mobility-mass spectrometry (IM-MS), and structure-based computational modeling, to explore the conformational dynamics of a model PCNA from Sulfolobus solfataricus (Sso), an archaeon. We found that Sso PCNA samples ring-open and ring-closed conformations even in the absence of its clamp loader complex, replication factor C, and transition to the ring-open conformation is modulated by the ionic strength of the solution. The IM-MS results corroborate the smFRET findings suggesting that PCNA dynamics are maintained in the gas phase and further establishing IM-MS as a reliable strategy to investigate macromolecular motions. Our molecular dynamic simulations agree with the experimental data and reveal that ring-open PCNA often adopts an out-of-plane left-hand geometry. Collectively, these results implore future studies to define the roles of PCNA dynamics in DNA loading and other PCNA-mediated interactions.

Extended Gas-Phase Trapping Followed by Surface-Induced Dissociation of Noncovalent Protein Complexes.

Mass spectrometry has emerged as a useful tool in the study of proteins and protein complexes. It is of fundamental interest to explore how the structures of proteins and protein complexes are affected by the absence of solvent and how this alters with increasing time in the gas phase. Here we demonstrate that a range of protein and protein complexes can be confined within the Trap T-wave region of a modified Waters Synapt G2S instrument, including monomeric (ß-lactoglobulin), dimeric (ß-lactoglobulin and enolase), tetrameric (streptavidin, concanavalin A, and pyruvate kinase), and pentameric (C-reactive protein) complexes, ranging in size up to 237 kDa. We demonstrate that complexes can be confined within the Trap region for varying lengths of time over the range 1-60 s and with up to 86% trapping efficiency for 1 s trapping. Furthermore, using model systems, we show that these noncovalent complexes can also be fragmented by surface-induced dissociation (SID) following trapping. SID reveals similar dissociation patterns over all trapping times studied for unactivated protein complexes, suggesting that any conformational changes occurring over this time scale are insufficient to cause substantial differences in the SID spectra of these complexes. Intentional alteration of structure by cone activation produces a distinct SID spectrum, with the differences observed being conserved, in comparison to unactivated complex, after trapping. However, subtle differences in the SID spectra of the activated complex are also observed as a function of trapping time.

Illustration of SID-IM-SID (surface-induced dissociation-ion mobility-SID) mass spectrometry: homo and hetero model protein complexes.

The direct determination of the overall topology and inter-subunit contacts of protein complexes plays an integral role in understanding how different subunits assemble into biologically relevant multisubunit complexes. Mass spectrometry has emerged as a useful structural biological tool because of its sensitivity, high tolerance for heterogeneous mixtures and the fact that crystals are not required. Perturbation of subunit interfaces in solution followed by gas-phase detection using mass spectrometry is a current means of probing the disassembly and hence assembly of protein complexes. Herein, we present an alternative method that employs native mass spectrometry coupled with ion mobility and two stages of surface induced dissociation (SID) where protein complexes are dissociated into subcomplexes in the first SID stage. The subcomplexes are then separated by ion mobility and subsequently fragmented into their individual monomers in the second SID stage (SID-IM-SID), providing information on how individual subunits assemble into protein complexes with different native topologies. The results also illustrate complex dependent differences in charge redistribution onto individual monomers obtained in SID-IM-SID.

Electron capture dissociation and drift tube ion mobility-mass spectrometry coupled with site directed mutations provide insights into the conformational diversity of a metamorphic protein.

Ion mobility mass spectrometry can be combined with data from top-down sequencing to discern adopted conformations of proteins in the absence of solvent. This multi-technique approach has particular applicability for conformationally dynamic systems. Previously, we demonstrated the use of drift tube ion mobility-mass spectrometry (DT IM-MS) and electron capture dissociation (ECD) to study the metamorphic protein lymphotactin (Ltn). Ltn exists in equilibrium between distinct monomeric (Ltn10) and dimeric (Ltn40) folds, both of which can be preserved and probed in the gas-phase. Here, we further test this mass spectrometric framework, by examining two site directed mutants of Ltn, designed to stabilise either distinct fold in solution, in addition to a truncated form consisting of a minimum model of structure for Ltn10. The truncated mutant has similar collision cross sections to the wild type (WT), for low charge states, and is resistant to ECD fragmentation. The monomer mutant (CC3) presents in similar conformational families as observed previously for the WT Ltn monomer. As with the WT, the CC3 mutant is resistant to ECD fragmentation at low charge states. The dimer mutant W55D is found here to exist as both a monomer and dimer. As a monomer W55D exhibits similar behaviour to the WT, but as a dimer presents a much larger charge state and collision cross section range than the WT dimer, suggesting a smaller interaction interface. In addition, ECD on the W55D mutant yields greater fragmentation than for the WT, suggesting a less stable ß-sheet core. The results highlight the power of MS to provide insight into dynamic proteins, providing further information on each distinct fold of Ltn. In addition we observe differences in the fold stability following single or double point mutations. This approach, therefore, has potential to be a useful tool to screen for the structural effects of mutagenesis, even when sample is limited.

Expanding Native Mass Spectrometry to the Masses.

At the 33rd ASMS Sanibel Meeting, on Membrane Proteins and Their Complexes, a morning roundtable discussion was held discussing the current challenges facing the field of native mass spectrometry and approaches to expanding the field to nonexperts. This Commentary summarizes the discussion and current initiatives to address these challenges.

Evaluation of Surface-Induced Dissociation Ion Mobility-Mass Spectrometry for Lipid Structural Characterization.

The complexity of the lipidome has necessitated the development of novel analytical approaches for the identification and structural analysis of morphologically diverse classes of lipids. At this time, a variety of dissociation techniques have been utilized to probe lipid decomposition pathways in search of structurally diagnostic fragment ions. Here, we investigate the application of surface-induced dissociation (SID), a fragmentation technique that imparts energy to the target molecule via collision with a coated surface, for the fragmentation of seven lipids across four major lipid subclasses. We have developed a tuning methodology for guiding the efficient operation of a previously developed custom SID device for molecules as small as ca. 300 Da with ion mobility analysis of the fragmentation products. SID fragmentation of the various lipids analyzed was found to generate fragment ions similar to those observed in CID spectra, but fragment ion lab frame onset energies were lower in SID due to the higher energy deposition via a more massive target. For the largest lipid evaluated (cardiolipin 18:1), SID produced chain fragment ions, which yielded analytically useful information regarding the composition of the acyl tails. Ion mobility provided an orthogonal dimension of separation and aided in assigning product ions to their precursors. Overall, the combination of SID and IM-MS is another potential methodology in the analytical toolkit for lipid structural analysis.

Protein complex heterogeneity and topology revealed by electron capture charge reduction and surface induced dissociation.

We illustrate the utility of native mass spectrometry (nMS) combined with a fast, tunable gas-phase charge reduction, electron capture charge reduction (ECCR), for the characterization of protein complex topology and glycoprotein heterogeneity. ECCR efficiently reduces the charge states of tetradecameric GroEL, illustrating Orbitrap m/z measurements to greater than 100,000 m/z. For pentameric C-reactive protein and tetradecameric GroEL, our novel device combining ECCR with surface induced dissociation (SID) reduces the charge states and yields more topologically informative fragmentation. This is the first demonstration that ECCR yields more native-like SID fragmentation. ECCR also significantly improved mass and glycan heterogeneity measurements of heavily glycosylated SARS-CoV-2 spike protein trimer and thyroglobulin dimer. Protein glycosylation is important for structural and functional properties and plays essential roles in many biological processes. The immense heterogeneity in glycosylation sites and glycan structure poses significant analytical challenges that hinder a mechanistic understanding of the biological role of glycosylation. Without ECCR, average mass determination of glycoprotein complexes is available only through charge detection mass spectrometry or mass photometry. With narrow m/z selection windows followed by ECCR, multiple glycoform m/z values are apparent, providing quick global glycoform profiling and providing a future path for glycan localization on individual intact glycoforms.

Evaluation of a Commercial TIMS-Q-TOF Platform for Native Mass Spectrometry.

Mass-spectrometry based assays in structural biology studies measure either intact or digested proteins. Typically, different mass spectrometers are dedicated for such measurements: those optimized for rapid analysis of peptides or those designed for high molecular weight analysis. A commercial trapped ion mobility-quadrupole-time-of-flight (TIMS-Q-TOF) platform is widely utilized for proteomics and metabolomics, with ion mobility providing a separation dimension in addition to liquid chromatography. The ability to perform high-quality native mass spectrometry of protein complexes, however, remains largely uninvestigated. Here, we evaluate a commercial TIMS-Q-TOF platform for analyzing noncovalent protein complexes by utilizing the instrument's full range of ion mobility, MS, and MS/MS (both in-source activation and collision cell CID) capabilities. The TIMS analyzer is able to be tuned gently to yield collision cross sections of native-like complexes comparable to those previously reported on various instrument platforms. In-source activation and collision cell CID were robust for both small and large complexes. TIMS-CID was performed on protein complexes streptavidin (53 kDa), avidin (68 kDa), and cholera toxin B (CTB, 58 kDa). Complexes pyruvate kinase (237 kDa) and GroEL (801 kDa) were beyond the trapping capabilities of the commercial TIMS analyzer, but TOF mass spectra could be acquired. The presented results indicate that the commercial TIMS-Q-TOF platform can be used for both omics and native mass spectrometry applications; however, modifications to the commercial RF drivers for both the TIMS analyzer and quadrupole (currently limited to m/z 3000) are necessary to mobility analyze protein complexes greater than about 60 kDa.

Small-molecule inhibition of c-MYC:MAX leucine zipper formation is revealed by ion mobility mass spectrometry.

The leucine zipper interaction between MAX and c-MYC has been studied using mass spectrometry and drift time ion mobility mass spectrometry (DT IM-MS) in addition to circular dichroism spectroscopy. Peptides comprising the leucine zipper sequence with (c-MYC-Zip residues 402-434) and without a postulated small-molecule binding region (c-MYC-ZipΔDT residues 406-434) have been synthesized, along with the corresponding MAX leucine zipper (MAX-Zip residues 74-102). c-MYC-Zip:MAX-Zip complexes are observed both in the absence and in the presence of the reported small-molecule inhibitor 10058-F4 for both forms of c-MYC-Zip. DT IM-MS, in combination with molecular dynamics (MD), shows that the c-MYC-Zip:MAX-Zip complex [M+5H](5+) exists in two conformations, one extended with a collision cross section (CCS) of 1164 ± 9.3 Å(2) and one compact with a CCS of 982 ± 6.6 Å(2); similar values are observed for the two forms of c-MYC-ZipΔDT:MAX-Zip. Candidate geometries for the complexes have been evaluated with MD simulations. The helical leucine zipper structure previously determined from NMR measurements (Lavigne, P.; et al. J. Mol. Biol. 1998, 281, 165), altered to include the DT region and subjected to a gas-phase minimization, yields a CCS of 1247 Å(2), which agrees with the extended conformation we observe experimentally. More extensive MD simulations provide compact complexes which are found to be highly disordered, with CCSs that correspond to the compact form from experiment. In the presence of the ligand, the leucine zipper conformation is completely inhibited and only the more disordered species is observed, providing a novel method to study the effect of interactions of disordered systems and subsequent inhibition of the formation of an ordered helical complex.

Ion mobility mass spectrometry for peptide analysis.

The use of ion mobility mass spectrometry has grown rapidly over the last two decades. This powerful analytical platform now forms an attractive prospect for comprehensive analysis of many different molecular species, including chemically complex biological molecules. This paper describes the application of IM-MS to the study of peptides. We focus on three different ion mobility devices that are most frequently found in tandem with mass spectrometers. These are instruments using linear drift tubes (LDT), those using travelling wave ion guides (TWIGS) and those employing high field asymmetric ion mobility spectrometry (FAIMS). Each technique is described. Examples are given on the use of IM-MS for the determination of peptide structure, the study of peptides that form amyloid fibrils, and the study of complex peptide mixtures in proteomic investigations. We describe and comment on the methodologies used and the outlook for this developing analytical technique.

Surface-Induced Dissociation for Protein Complex Characterization.

Native mass spectrometry (nMS) enables intact non-covalent complexes to be studied in the gas phase. nMS can provide information on composition, stoichiometry, topology, and, when coupled with surface-induced dissociation (SID), subunit connectivity. Here we describe the characterization of protein complexes by nMS and SID. Substructural information obtained using this method is consistent with the solved complex structure, when a structure exists. This provides confidence that the method can also be used to obtain substructural information for unknowns, providing insight into subunit connectivity and arrangements. High-energy SID can also provide information on proteoforms present. Previously SID has been limited to a few in-house modified instruments and here we focus on SID implemented within an in-house-modified Q Exactive UHMR. However, SID is currently commercially available within the Waters Select Series Cyclic IMS instrument. Projects are underway that involve the NIH-funded native MS resource (nativems.osu.edu), instrument vendors, and third-party vendors, with the hope of bringing the technology to more platforms and labs in the near future. Currently, nMS resource staff can perform SID experiments for interested research groups.

Native Mass Spectrometry: Recent Progress and Remaining Challenges.

Native mass spectrometry (nMS) has emerged as an important tool in studying the structure and function of macromolecules and their complexes in the gas phase. In this review, we cover recent advances in nMS and related techniques including sample preparation, instrumentation, activation methods, and data analysis software. These advances have enabled nMS-based techniques to address a variety of challenging questions in structural biology. The second half of this review highlights recent applications of these technologies and surveys the classes of complexes that can be studied with nMS. Complementarity of nMS to existing structural biology techniques and current challenges in nMS are also addressed.

Protein quaternary structures in solution are a mixture of multiple forms.

Over half the proteins in the E. coli cytoplasm form homo or hetero-oligomeric structures. Experimentally determined structures are often considered in determining a protein's oligomeric state, but static structures miss the dynamic equilibrium between different quaternary forms. The problem is exacerbated in homo-oligomers, where the oligomeric states are challenging to characterize. Here, we re-evaluated the oligomeric state of 17 different bacterial proteins across a broad range of protein concentrations and solutions by native mass spectrometry (MS), mass photometry (MP), size exclusion chromatography (SEC), and small-angle X-ray scattering (SAXS), finding that most exhibit several oligomeric states. Surprisingly, some proteins did not show mass-action driven equilibrium between the oligomeric states. For approximately half the proteins, the predicted oligomeric forms described in publicly available databases underestimated the complexity of protein quaternary structures in solution. Conversely, AlphaFold multimer provided an accurate description of the potential multimeric states for most proteins, suggesting that it could help resolve uncertainties on the solution state of many proteins.