Collidoscope: An Improved Tool for Computing Collisional Cross Sections with the Trajectory Method

Ion Mobility-Mass Spectrometry (IM-MS) can be a powerful tool for determining structural information about ions in the gas phase, from small covalent analytes to large, unfolded, and/or denatured proteins and complexes. For large biomolecular ions, which may have a wide variety of possible gas-phase conformations and multiple charge sites, quantitative, physically explicit modeling of collisional cross sections (CCSs) for comparison to IMS data can be challenging and time-consuming. We present a “trajectory method” (TM) based CCS calculator, named “Collidoscope”, which utilizes parallel processing and optimized trajectory sampling, and implements both He and N2 as collision gas options. Also included is a charge-placement algorithm for determining probable charge site configurations for protonated protein ions given an input geometry in pdb file format. Results from Collidoscope are compared to those from the current state-of-the-art CCS simulation suite, IMoS. Collidoscope CCSs are typically within 4% of IMoS values for ions with masses from ~18 Da to ~800 kDa. Collidoscope CCSs using x-ray crystal geometries are typically within a few percent of IM-MS experimental values for ions with mass up to ~3.5 kDa (melittin), and discrepancies for larger ions up to ~800 kDa (GroEL) are attributed in large part to changes in ion structure during and after the electrospray process. Due to its physically explicit modeling of scattering, computational efficiency, and accuracy, Collidoscope can be a valuable tool for IM-MS research, especially for large biomolecular ions

Gas Phase Stability and Unfolding of Proteins via Ion Mobility-Mass Spectrometry

The study of protein dynamics and unfolding is key to understanding the basis of misfolding and aggregational diseases such as type II diabetes, α1-antrypsin deficiency, Parkinsons’ disease and Alzheimers' disease. Common structural biology techniques, such as x-ray crystallography and cryo electron microscopy often require large quantities of high purity sample and can only capture major structural ensembles. Mass spectrometry coupled to ion mobility (IM-MS) is positioned as an ideal technique to study protein dynamics and unfolding as it allows the separation of ions based on mass, charge and conformation from low concentrations of heterogenous solutions. In this thesis I present new techniques, instrumentation and computational workflows relating to IM-MS and how these techniques can be applied to protein systems involved in misfolding and aggregational diseases

Eye lens β-crystallins are predicted by native ion mobility-mass spectrometry and computations to form compact higher-ordered heterooligomers

Eye lens crystallin proteins maintain the refractive properties of the lens but are not replaced after denucleation. Rolland et al. use native ion mobility-mass spectrometry, kinetics experiments, and computations to reveal that b-crystallins form heterodimers. These likely assemble into compact heterooligomers that enable the very high protein concentrations found in lens tissue

Linking Gas-Phase and Solution-Phase Protein Unfolding via Mobile Proton Simulations

Native mass spectrometry coupled to ion mobility (IM-MS) combined with collisional activation (CA) of ions in the gas phase (in vacuo) is an important method for the study of protein unfolding. It has advantages over classical biophysical and structural techniques as it can be used to analyze small volumes of low-concentration heterogeneous mixtures while maintaining solution-like behavior and does not require labeling with fluorescent or other probes. It is unclear, however, whether the unfolding observed during collision activation experiments mirrors solution-phase unfolding. To bridge the gap between in vacuo and in-solution behavior, we use unbiased molecular dynamics (MD) to create in silico models of in vacuo unfolding of a well-studied protein, the N-terminal domain of ribosomal L9 (NTL9) protein. We utilize a mobile proton algorithm (MPA) to create 100 thermally unfolded and coulombically unfolded in silico models for observed charge states of NTL9. The unfolding behavior in silico replicates the behavior in-solution and is in line with the in vacuo observations; however, the theoretical collision cross section (CCS) of the in silico models was lower compared to that of the in vacuo data, which may reflect reduced sampling

Extended Protein Ions are Formed by the Chain Ejection Model in Chemical Supercharging Electrospray Ionization

Supercharging electrospray ionization can be a powerful tool for increasing charge states in mass spectra and generating unfolded ion structures, yet key details of its mechanism remain unclear. The structures of highly extended protein ions and the mechanism of supercharging were investigated using ion mobility-mass spectrometry. Head-to-tail-linked polyubiquitins (Ubq1−11) were used to determine size and charge state scaling laws for unfolded protein ions formed by supercharging while eliminating amino acid composition as a potential confounding factor. Collisional cross section was found to scale linearly with mass for these ions and several other monomeric proteins, and the maximum observed charge state for each analyte scales with mass in agreement with an analytical charge state scaling law for protein ions with highly extended structures that is supported by experimental gas-phase basicities. These results indicate that these highly unfolded ions can be considered quasi-one-dimensional, and collisional cross sections modeled with the Trajectory Method in Collidoscope show that these ions are significantly more extended than linear α-helices but less extended than straight chains. The effect of internal disulfide bonds on the extent of supercharging was probed using bovine serum albumin, β-lactoglobulin, and lysozyme, each of which contains multiple internal disulfide bonds. Reduction of the disulfide bonds led to a marked increase in charge state upon supercharging without significantly altering folding in solution. This evidence supports a supercharging mechanism in which these proteins unfold before or during evaporation of the electrospray droplet and ionization occurs by the Chain Ejection Model

Electrospray Ionization of Polypropylene Glycol: Rayleigh-Charged Droplets, Competing Pathways, and Charge State-Dependent Conformations.

Recent molecular dynamics (MD) simulations from various laboratories have advanced the general understanding of electrospray ionization (ESI)-related processes. Unfortunately, computational cost has limited most of those previous endeavors to ESI droplets with radii of ∼3 nm or less, which represent the low end of the size distribution in the ESI plume. The current work extends this range by conducting simulations on aqueous ESI droplets with radii of 5.5 nm (∼23 000 water molecules). Considering that computational cost increases with r6, this is a significant step forward. We focused on the ESI process for polypropylene glycol (PPG) which is a common ESI-MS calibrant. Different chain lengths (PPG10, 30, and 60) were tested in droplets that were charged with excess Na+. Solvent evaporation and Na+ ejection, with occasional progeny droplet formation, kept the systems at 80-100% of the Rayleigh limit throughout their life cycle. PPG chains migrated to the droplet surface where they captured Na+ via binding to ether oxygens. Various possible pathways for PPG release into the gas phase were encountered. Some PPG10 runs showed ejection from the droplet surface, consistent with the ion evaporation model (IEM). In other instances, PPG was released after near-complete solvent evaporation, as envisioned by the charged residue model (CRM). A third avenue was the partial separation from the droplet to form double or single-tailed structures, with subsequent chain detachment from the droplet. This last pathway is consistent with the chain ejection model (CEM). Immediately after detachment many chains were electrostatically stretched, but they subsequently collapsed into compact conformers. Extended structures were retained only for the most highly charged ions. Our simulations were complemented by ESI-MS and ion mobility measurements. MD-predicted charge states and collision cross sections were in agreement with these experimental data, supporting the mechanistic insights obtained

Protonation Isomers of Highly Charged Protein Ions Can Be Separated in FAIMS-MS

High-field asymmetric waveform ion mobility spectrometry-mass spectrometry (FAIMS-MS) can resolve over an order of magnitude more conformers for a given protein ion than alternative methods. Such an expansion in separation space results, in part, from protein ions with masses of \u3e29 kDa undergoing dipole alignment in the high electric field of FAIMS, and the resolution of ions that adopt pendular vs free rotor states. In this study, FAIMS-MS, collision-induced dissociation (CID), and travelling wave (TW) IMS-MS were used to investigate the pendular and free rotor states of protonated carbonic anhydrase II (CAII, 29 kDa). The electrospray ionization additive 1,2-butylene carbonate was used to increase protein charge states and ensure extended ion conformations were formed. For relatively high charge states in which dipole alignment occurs (30e38þ), FAIMS-MS can baseline resolve the isobaric pendular and free rotor ion populations. For TWIMS-MS, these same charge states resulted in monomodal arrival time distributions with collision cross sections corresponding to highly extended ion conformations. Interestingly, CID of FAIMS-selected pendular and free rotor ion populations resulted in significantly different frag-mentation patterns. For example, CID of the dipole aligned CAII 37þ resulted in cleavages C-terminal to residue 183, 192 and 196, whereas cleavage sites for the free rotor population occurred near residues 12 and 238. Given that the cleavage sites are ’directed’ by protonation sites in the CID of protein ions, and highly charged protein ions adopt extended conformations with the same or very similar collision cross sections, these results indicate that the pendular and free rotor populations separated in FAIMS can be attributed to protonation isomers. Moreover, the extent of protein ion charging in FAIMS-MS decreased substantially as the carrier gas flow rate decreased, indicating that ion charging in FAIMS-MS can be limited by proton-transfer reactions. Given that the total mass of proton charge carriers corresponds to less than 0.2% the mass of CAII, we anticipate that FAIMS-MS can be used to separate intact isobaric proteoforms with masses of at least ~29 kDa that result from alternative sites of post-translational modifications

A parallelized molecular collision cross section package with optimized accuracy and efficiency

Ion mobility-based separation prior to mass spectrometry has become an invaluable tool in the structural elucidation of gas-phase ions and in the characterization of complex mixtures. Application of ion mobility to structural studies requires an accurate methodology to bridge theoretical modelling of chemical structure with experimental determination of an ion's collision cross section (CCS). Herein, we present a refined methodology for calculating ion CCS using parallel computing architectures that makes use of atom specific parameters, which we have called MobCal-MPI. Tuning of ion-nitrogen van der Waals potentials on a diverse calibration set of 162 molecules returned a RMSE of 2.60% in CCS calculations of molecules containing the elements C, H, O, N, F, P, S, Cl, Br, and I. External validation of the ion-nitrogen potential was performed on an additional 50 compounds not present in the validation set, returning a RMSE of 2.31% for the CCSs of these compounds. Owing to the use of parameters from the MMFF94 forcefield, the calibration of the van der Waals potential can be extended to additional atoms defined in the MMFF94 forcefield (i.e., Li, Na, K, Si, Mg, Ca, Fe, Cu, Zn). We expect that the work presented here will serve as a foundation for facile determination of molecular CCSs, as MobCal-MPI boasts up to 64-fold speedups over traditional calculation packages.The authors would like to acknowledge the financial support provided by the Natural Sciences and Engineering Research Council (NSERC) of Canada

Interrogating the Quaternary Structure of Noncanonical Hemoglobin Complexes by Electrospray Mass Spectrometry and Collision-Induced Dissociation.

Various activation methods are available for the fragmentation of gaseous protein complexes produced by electrospray ionization (ESI). Such experiments can potentially yield insights into quaternary structure. Collision-induced dissociation (CID) is the most widely used fragmentation technique. Unfortunately, CID of protein complexes is dominated by the ejection of highly charged monomers, a process that does not yield any structural insights. Using hemoglobin (Hb) as a model system, this work examines under what conditions CID generates structurally informative subcomplexes. Native ESI mainly produced tetrameric Hb ions. In addition, noncanonical hexameric and octameric complexes were observed. CID of all these species [(αβ)2, (αβ)3, and (αβ)4] predominantly generated highly charged monomers. In addition, we observed hexamer → tetramer + dimer dissociation, implying that hexamers have a tetramer··dimer architecture. Similarly, the observation of octamer → two tetramer dissociation revealed that octamers have a tetramer··tetramer composition. Gas-phase candidate structures of Hb assemblies were produced by molecular dynamics (MD) simulations. Ion mobility spectrometry was used to identify the most likely candidates. Our data reveal that the capability of CID to produce structurally informative subcomplexes depends on the fate of protein-protein interfaces after transfer into the gas phase. Collapse of low affinity interfaces conjoins the corresponding subunits and favors CID via monomer ejection. Structurally informative subcomplexes are formed only if low affinity interfaces do not undergo a major collapse. However, even in these favorable cases CID is still dominated by monomer ejection, requiring careful analysis of the experimental data for the identification of structurally informative subcomplexes