Correlation between the shape of the ion mobility signals and the stepwise folding process of polylactide ions

In the field of polymer characterization, the use of ion mobility mass spectrometry (IMMS) remains mainly devoted to the temporal separation of cationized oligomers according to their charge states, molecular masses and macromolecular architectures in order to probe the presence of different structures. When analyzing multiply charged polymer ions by IMMS, the most striking feature is the observation of breaking points in the evolution of the average collision cross sections with the number of monomer units. Those breaking points are associated to the folding of the polymer chain around the cationizing agents. Here, we scrutinize the shape of the arrival time distribution (ATD) of polylactide ions and associate the broadening as well as the loss of symmetry of the ATD signals to the coexistence of different populations of ions attributed to the transition from opened to folded stable structures. The observation of distinct distributions reveals the absence of folded/extended structure interconversion on the ion mobility time scale (1–10 ms) and then on the lifetime of ions within the mass spectrometer at room temperature. In order to obtain information on the possible interconversion between the different observed populations upon ion activation, we performed IM–IM–MS experiments (tandem ion mobility measurements). To do so, mobility‐selected ions were activated by collisions before a second mobility measurement. Interestingly, the conversion by collisional activation from a globular structure into a (partially) extended structure, i.e. the gas phase unfolding of the ions, was not observed in the energetic regime available with the used experimental setup. The absence of folded/extended interconversion, even upon collisional activation, points to the fact that the polylactide ions are ‘frozen’ in their specific 3D structure during the desolvation/ionization electrospray processes. Copyright © 2017 John Wiley & Sons, Ltd.     

Conformer-dependent proton-transfer reactions of ubiquitin ions

The conformations of ubiquitin ions before and after being exposed to proton transfer reagents have been studied by using ion mobility/mass spectrometry techniques. Ions were produced by electrospray ionization and exposed to acetone, acetophenone, n-butylamine, and 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene. Under the conditions employed, the +4 to +13 charge states were formed and a variety of conformations, which we have characterized as compact, partially folded, and elongated, have been observed. The low charge state ions have cross sections that are similar to those calculated for the crystal conformation. High charge states favor unfolded conformations. The ion mobility distributions recorded after ions have been exposed to each base show that the lowest charge state that is formed during proton-transfer reactions favors a compact conformation. More open conformations are observed for the higher charge states that remain after reaction. The results show that for a given charge state, the apparent gas-phase acidities of the different conformations are ordered as compact < partially folded < elongated.     

Transfer of structural elements from compact to extended states in unsolvated ubiquitin

Multidimensional ion mobility spectrometry techniques (IMS-IMS and IMS-IMS-IMS) combined with mass spectrometry are used to study structural transitions of ubiquitin ions in the gas phase. It is possible to select and activate narrow distributions of compact and partially folded conformation types and examine new distributions of structures that are formed. Different compact conformations unfold, producing a range of new partially folded states and three resolvable peaks associated with elongated conformers. Under gentle activation conditions, the final populations of the three elongated forms depend on the initial structures of the selected ions. This requires that some memory of the compact state (most likely secondary structure) is preserved along the unfolding pathway. Activation of selected, partially folded intermediates (formed from specific compact states) leads to elongated state populations that are consistent with the initial selected compact form-evidence that intermediates not only retain elements of initial structure but also are capable of transmitting structure to final states.     

Evidence for unfolding and refolding of gas-phase cytochrome <Emphasis Type="Italic">c</Emphasis> ions in a Paul trap

The folding pathways of gas-phase cytochrome c ions produced by electrospray ionization have been studied by an ion trapping/ion mobility technique that allows conformations to be examined over extended timescales (10 ms to 10 s). The results show that the +9 charge state emerges from solution as a compact structure and then rapidly unfolds into several substantially more open structures, a transition that requires 30-60 ms; over substantially longer timescales (250 ms to 10 s) elongated states appear to refold into an array of folded structures. The new folded states are less compact than those that are apparent during the initial unfolding. Apparently, unfolding to highly open conformations is a key step that must occur before +9 ions can sample more compact states that are stable at longer times.     

Large anhydrous polyalanine ions: evidence for extended helices and onset of a more compact state.

Ion mobility measurements and molecular modeling calculations have been used to examine the conformations of large multiply charged polyalanine peptides. Two series of [Ala(n)+3H](3+) conformations which do not interconvert during the 10 to 30 ms experimental timescales are observed: a family of elongated structures for n = 18 to 39 and a series of more compact conformations for n = 24 to 41. The more compact state becomes the dominant conformer type for n > 32. Molecular modeling studies and comparisons of calculated collision cross sections with experiment indicate that the elongated ions have extended helical conformations. We suggest that the more compact state corresponds to a new conformer type: a folded hinged helix-coil state in which helical and coil regions have similar physical dimensions. The competition between extended and compact states is rationalized by considering differences in charge stabilization and entropy.     

Charge-state dependent compaction and dissociation of protein complexes: insights from ion mobility and molecular dynamics

Collapse to compact states in the gas phase, with smaller collision cross sections than calculated for their native-like structure, has been reported previously for some protein complexes although not rationalized. Here we combine experimental and theoretical studies to investigate the gas-phase structures of four multimeric protein complexes during collisional activation. Importantly, using ion mobility-mass spectrometry (IM-MS), we find that all four macromolecular complexes retain their native-like topologies at low energy. Upon increasing the collision energy, two of the four complexes adopt a more compact state. This collapse was most noticeable for pentameric serum amyloid P (SAP) which contains a large central cavity. The extent of collapse was found to be highly correlated with charge state, with the surprising observation that the lowest charge states were those which experience the greatest degree of compaction. We compared these experimental results with in vacuo molecular dynamics (MD) simulations of SAP, during which the temperature was increased. Simulations showed that low charge states of SAP exhibited compact states, corresponding to collapse of the ring, while intermediate and high charge states unfolded to more extended structures, maintaining their ring-like topology, as observed experimentally. To simulate the collision-induced dissociation (CID) of different charge states of SAP, we used MS to measure the charge state of the ejected monomer and assigned this charge to one subunit, distributing the residual charges evenly among the remaining four subunits. Under these conditions, MD simulations captured the unfolding and ejection of a single subunit for intermediate charge states of SAP. The highest charge states recapitulated the ejection of compact monomers and dimers, which we observed in CID experiments of high charge states of SAP, accessed by supercharging. This strong correlation between theory and experiment has implications for further studies as well as for understanding the process of CID and for applications to gas-phase structural biology more generally.     

Conformational studies of Zn-Ligand-Hexose diastereomers using ion mobility measurements and density functional theory calculations

Ion mobility studies and density functional theory calculations were used to study the structures of [Zn/diethylenetriamine/Hexose/Cl]+ complexes in an effort to probe differences in the three-dimensional conformations. This information allows us to gain insight into the structure of these complexes before collisional activation, which is the first step in understanding the stereoselective dissociations observed under collisionally activated conditions. The collision cross sections obtained from the ion mobility measurements showed that the mannose structure is more compact than the galactose and glucose complexes, respectively. Using density functional theory, candidate structures for each of the experimentally observed complexes were generated. Two criteria were used to determine the most likely structures of these complexes before activation: (1) The allowed relative energies of the molecules (between 0-90 kJ/mol) and (2) collision cross section agreement (within 2%) between the theoretically determined structures and the experimentally determined cross section. It was found that the identity of the monosaccharide made a difference in the overall conformation of the metal-ligand-monosaccharide complex. For glucose and galactose, metal coordination to O(6) was found to be favorable, with the monosaccharide occupying the 4C1 chair conformation, while for mannose, O(2) metal coordination was found with the monosaccharide in a B3,0 conformation. Coordination numbers varied between four and six for the Zn(II) metal centers. Given these results, it appears that the stereochemistry of the monosaccharide influences the conformation and metal coordination sites of the Zn(II)/monosaccharide/dien complex. These differences may influence the dissociation products observed under collisionally activated conditions.     

Solution Dependence of the Collisional Activation of Ubiquitin [M + 7H]7+ Ions

The solution dependence of gas-phase unfolding for ubiquitin [M + 7H]⁷⁺ ions has been studied by ion mobility spectrometry-mass spectrometry (IMS-MS). Different acidic water:methanol solutions are used to favor the native (N), more helical (A), or unfolded (U) solution states of ubiquitin. Unfolding of gas-phase ubiquitin ions is achieved by collisional heating and newly formed structures are examined by IMS. With an activation voltage of 100 V, a selected distribution of compact structures unfolds, forming three resolvable elongated states (E1-E3). The relative populations of these elongated structures depend strongly on the solution composition. Activation of compact ions from aqueous solutions known to favor N-state ubiquitin produces mostly the E1 type elongated state, whereas activation of compact ions from methanol containing solutions that populate A-state ubiquitin favors the E3 elongated state. Presumably, this difference arises because of differences in precursor ion structures emerging from solution. Thus, it appears that information about solution populations can be retained after ionization, selection, and activation to produce the elongated states. These data as well as others are discussed. Figure

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Applications of collision dynamics in quadrupole mass spectrometry

Some applications of collision dynamics in the field of quadrupole mass spectrometry are presented. Previous data on the collision induced dissociation of ions in triple quadrupole mass spectrometers is reviewed. A new method to calculate the internal energy distribution of activated ions directly from the increase in the cross section for dissociation with center of mass energy is presented. This method, although approximate, demonstrates explicitly the high efficiency of transfer of translational to internal energy of organic ions. It is argued that at eV center of mass energies, collisions between protein ions and neutrals such as Ar are expected to be highly inelastic. The discovery and application of collisional cooling in radio frequency quadrupoles is reviewed. Some previously unpresented data on fragment ion energies in triple quadrupole tandem mass spectrometry are shown that demonstrate directly the loss of kinetic energy of fragment ions in the cooling process. The development of the energy loss method to measure collision cross sections of protein ions in triple quadrupole instruments is reviewed along with a new discussion of the effects of inelastic collisions in these experiments and related ion mobility experiments.     

Duplex formation and the onset of helicity in poly d(CG)n oligonucleotides in a solvent-free environment

The gas-phase conformations of a series of cytosine/guanine DNA duplexes were examined by ion mobility and molecular dynamics methods. Deprotonated duplex ions were formed by electrospray ionization, and their collision cross sections measured in helium were compared to calculated cross sections of theoretical models generated by molecular dynamics. The 4-mer (dCGCG) and 6-mer (dCGCGCG) duplexes were found to have globular conformations. Globular and helical structures were observed for the 8-mer (dCGCGCGCG) duplex, with the globular form being the more favored conformer. For the 10-mer (dCGCGCGCGCG), 14-mer (dCGCGCGCGCGCGCG), and 18-mer (dCGCGCGCGCGCGCGCGCG) duplexes, only helical structures were observed in the ion mobility measurements. Theory predicts that the helical structures are less stable than the globular forms in the gas phase and should collapse into the globular form given enough time. However, molecular dynamics simulations at 300 K indicate the helical structures are stable in aqueous solution and will retain their conformations for a limited time in the gas phase. The presence of helical structures in the ion mobility experiments indicates that the duplexes retain "solution structures" in the gas phase on the millisecond time scale.     

Mass spectrometric study of gas‐phase ions of acid β‐glucosidase (Cerezyme) and iminosugar pharmacological chaperones

The effect on the conformations and stability of gas‐phase ions of Cerezyme, a glycoprotein, when bound to three small‐molecule chaperones has been studied using intact ESI MS, collision cross section and MS/MS measurements. To distinguish between the peaks from apo and small‐molecule complex ions, Cerezyme is deglycosylated (dg‐Cer). ESI MS of dg‐Cer reveals that glycosylation accounts for 8.5% of the molecular weight. When excess chaperone, either covalent (2FGF) or noncovalent (A and B iminosugars), is added to solutions of dg‐Cer, mass spectra show peaks from 1:1 chaperone–enzyme complexes as well as free enzyme. On average, ions of the apoenzyme have 1.6 times higher cross sections when activated in the source region of the mass spectrometer. For a given charge state, ions of complexes of 2FGF and B have about 30% and 8.4% lower cross sections, respectively, compared to the apoenzyme. Thus, binding the chaperones causes the gas‐phase protein to adopt more compact conformations. The noncovalent complex ions dissociate by the loss of charged chaperones. In the gas phase, the relative stability of dg‐Cer with B is higher than that with the A, whereas in solution A binds enzyme more strongly than B. Nevertheless, the disagreement is explained based on the greater number of contacts between the B and dg‐Cer than the A and dg‐Cer (13 vs. 8), indicating the importance of noncovalent interactions within the protein–chaperone complex in the absence of solvent. Findings in this work suggest a hypothesis towards predicting a consistent correlation between gas‐phase properties to solution binding properties. Copyright © 2014 John Wiley & Sons, Ltd.     

High-pressure collisional activation mass spectrometry of aromatic halides

The high-pressure collisional activation mass spectra with methane as the reagent/collision gas are reported for five aromatic halides. The major decomposition of the protonated aromatic halides is hydrogen halide elimination. The energy-resolved mass spectra and the chemical reactivities of fragment ions with the methane collision gas are used to establish dissociation pathways and structures of fragment ions. The high-pressure collisional activation mass spectra are compared with conventional collisionally induced dissociation and chemical ionization mass spectra.     

Investigating direct current potentials that affect native protein conformation during trapped ion mobility spectrometry–mass spectrometry

Trapped ion mobility spectrometry–time-of-flight mass spectrometry (TIMS-TOFMS) has emerged as a tool to study protein conformational states. In TIMS, gas-phase ions are guided across the IM stages by applying direct current (DC) potentials (D1–6), which, however, might induce changes in protein structures through collisional activation. To define conditions for native protein analysis, we evaluated the influence of these DC potentials using the metalloenzyme bovine carbonic anhydrase (BCA) as primary test compound. The variation of DC potentials did not change BCA-ion charge and heme content but affected (relative) charge-state intensities and adduct retention. Constructed extracted-ion mobilograms and corresponding collisional cross-section (CCS) profiles gave useful insights in (alterations of) protein conformational state. For BCA, the D3 and D6 potential (which are applied between the deflection transfer and funnel 1 [F1] and the accumulation exit and the start of the ramp, respectively) had most profound effects, showing multimodal CCS distributions at higher potentials indicating gradual unfolding. The other DC potentials only marginally altered the CCS profiles of BCA. To allow for more general conclusions, five additional proteins of diverse molecular weight and conformational stability were analyzed, and for the main protein charge states, CCS profiles were constructed. Principal component analysis (PCA) of the obtained data showed that D1 and D3 exhibit the highest degree of correlation with the ratio of folded and unfolded protein (F/U) as extracted from the mobilograms obtained per set D potential. The correlation of D6 with F/U and protein charge were similar, and D2, D4, and D5 showed an inverse correlation with F/U but were correlated with protein charge. Although DC boundary values for induced conformational changes appeared protein dependent, a set of DC values could be determined, which assured native analysis of most proteins.     

Do Charge State Signatures Guarantee Protein Conformations?

The extent to which proteins in the gas phase retain their condensed-phase structure is a hotly debated issue. Closely related to this is the degree to which the observed charge state reflects protein conformation. Evidence from electron capture dissociation, hydrogen/deuterium exchange, ion mobility, and molecular dynamics shows clearly that there is often a strong correlation between the degree of folding and charge state, with the most compact conformations observed for the lowest charge states. In this article, we address recent controversies surrounding the relationship between charge states and folding, focussing also on the manipulation of charge in solution and its effect on conformation. 'Supercharging' reagents that have been used to effect change in charge state can promote unfolding in the electrospray droplet. However for several protein complexes, supercharging does not appear to perturb the structure in that unfolding is not detected. Consequently, a higher charge state does not necessarily imply unfolding. Whilst the effect of charge manipulation on conformation remains controversial, there is strong evidence that a folded, compact state of a protein can survive in the gas phase, at least on a millisecond timescale. The exact nature of the side-chain packing and secondary structural elements in these compact states, however, remains elusive and prompts further research.     

Fluorescence probe of Trp-cage protein conformation in solution and in gas phase

Measurements of protein unfolding in the absence of solvent, when combined with unfolding studies in solution, offer a unique opportunity to measure the effects of solvent on protein structure and dynamics. The experiments presented here rely on the fluorescence of an attached dye to probe the local conformational dynamics through interactions with a Trp residue and fields originating on charge sites. We present fluorescence measurements of thermal fluctuations accompanying conformational change of a miniprotein, Trp-cage, in solution and in gas phase. Molecular dynamics (MD) simulations are performed as a function of temperature, charge state, and charge location to elucidate the dye-protein conformational dynamics leading to the changes in measured fluorescence. The results indicate that the stability of the unsolvated protein is dominated by hydrogen bonds. Substituting asparagine for aspartic acid at position 9 results in a dramatic alteration of the solution unfolding curve, indicating that the salt bridge involving Lys8, Asp9, and Arg16 (+ - +) is essential for Trp-cage stability in solution. In contrast, this substitution results in minor changes in the unfolding curve of the unsolvated protein, showing that hydrogen bonds are the major contributor to the stability of Trp-cage in gas phase. Consistent with this hypothesis, the decrease in the number of hydrogen bonds with increasing temperature indicated by MD simulations agrees reasonably well with the experimentally derived enthalpies of conformational change. The simulation results display relatively compact conformations compared with NMR structures that are generally consistent with experimental results. The measured unfolding curves of unsolvated Trp-cage ions are invariant with the acetonitrile content of the solution from which they are formed, possibly as a result of conformational relaxation during or after desolvation. This work demonstrates the power of combined solution and gas-phase studies and of single-point mutations to identify specific noncovalent interactions which contribute to protein-fold stability. The combination of experiment and simulation is particularly useful because these approaches yield complementary information which can be used to deduce the details of structural changes of proteins in the gas phase.     

Traveling-wave ion mobility-mass spectrometry reveals additional mechanistic details in the stabilization of protein complex ions through tuned salt additives

Ion mobility–mass spectrometry is often applied to the structural elucidation of multiprotein assemblies in cases where X-ray crystallography or NMR experiments have proved challenging. Such applications are growing steadily as we continue to probe regions of the proteome that are less-accessible to such high-resolution structural biology tools. Since ion mobility measures protein structure in the absence of bulk solvent, strategies designed to more-broadly stabilize native-like protein structures in the gas-phase would greatly enable the application of such measurements to challenging structural targets. Recently, we have begun investigating the ability of salt-based solution additives that remain bound to protein ions in the gas-phase to stabilize native-like protein structures. These experiments, which utilize collision induced unfolding and collision induced dissociation in a tandem mass spectrometry mode to measure protein stability, seek to develop a rank-order similar to the Hofmeister series that categorizes the general ability of different anions and cations to stabilize gas-phase protein structure. Here, we study magnesium chloride as a potential stabilizing additive for protein structures in vacuo, and find that the addition of this salt to solutions prior to nano-electrospray ionization dramatically enhances multiprotein complex structural stability in the gas-phase. Based on these experiments, we also refine the physical mechanism of cation-based protein complex ion stabilization by tracking the unfolding transitions experienced by cation-bound complexes. Upon comparison with unbound proteins, we find strong evidence that stabilizing cations act to tether protein complex structure. We conclude by putting the results reported here in context, and by projecting the future applications of this method.     

Toward a Rational Design of Highly Folded Peptide Cation Conformations. 3D Gas-Phase Ion Structures and Ion Mobility Characterization

Heptapeptide ions containing combinations of polar Lys, Arg, and Asp residues with non-polar Leu, Pro, Ala, and Gly residues were designed to study polar effects on gas-phase ion conformations. Doubly and triply charged ions were studied by ion mobility mass spectrometry and electron structure theory using correlated ab initio and density functional theory methods and found to exhibit tightly folded 3D structures in the gas phase. Manipulation of the basic residue positions in LKGPADR, LRGPADK, KLGPADR, and RLGPADK resulted in only minor changes in the ion collision cross sections in helium. Replacement of the Pro residue with Leu resulted in only marginally larger collision cross sections for the doubly and triply charged ions. Disruption of zwitterionic interactions in doubly charged ions was performed by converting the C-terminal and Asp carboxyl groups to methyl esters. This resulted in very minor changes in the collision cross sections of doubly charged ions and even slightly diminished collision cross sections in most triply charged ions. The experimental collision cross sections were related to those calculated for structures of lowest free energy ion conformers that were obtained by extensive search of the conformational space and fully optimized by density functional theory calculations. The predominant factors that affected ion structures and collision cross sections were due to attractive hydrogen bonding interactions and internal solvation of the charged groups that overcompensated their Coulomb repulsion. Structure features typically assigned to the Pro residue and zwitterionic COO-charged group interactions were only secondary in affecting the structures and collision cross sections of these gas-phase peptide ions.

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Ion mobility mass spectrometry of two tetrameric membrane protein complexes reveals compact structures and differences in stability and packing

Here we examined the gas-phase structures of two tetrameric membrane protein complexes by ion mobility mass spectrometry. The collision cross sections measured for the ion channel are in accord with a compact configuration of subunits, suggesting that the native-like structure can be preserved under the harsh activation conditions required to release it from the detergent micelle into the gas phase. We also found that the quaternary structure of the transporter, which has fewer transmembrane subunits than the ion channel, is less stable once stripped of detergents and bulk water. These results highlight the potential of ion mobility mass spectrometry for characterizing the overall topologies of membrane protein complexes and the structural changes associated with nucleotide, lipid, and drug binding.     

Delayed dissociation spectra of survivor ions from high-energy collisional activation

Collisional activation (CA) of large ions at kiloelectronvolt energies is accompanied by unexpectedly large losses of translational energy, which vary with the nature of the collision gas. Previous investigations have concentrated upon subsequent fragmentations occurring within a time window covering a few fis immediately following collision, using massanalyzed ion kinetic energy spectrometry. In the present work, survivor ions were selected for specified values of translational energy loss, and their internal energy contents assessed via their subsequent unimolecular fragmentation reactions within a later time window. Beam collimation was also applied when circumstances permitted to impose angular selection, thus minimizing cross talk between effects of collisional scattering and energy dispersion. It was shown that internal excitation of the reactant ion can account for only a small fraction of the observed loss of translational energy. The recoil energy of the target is thus the principal sink for the translational energy loss, since the latter was always chosen to be less than the lowest excitation energy of the target. This conclusion is shown to be consistent with theoretical models of the CA process. The practical implications of these conclusions for CA of large ions at kiloelectronvolt energies are discussed.     

Analyzing slowly exchanging protein conformations by ion mobility mass spectrometry: study of the dynamic equilibrium of prolyl oligopeptidase

Ion mobility mass spectrometry (IMMS) is a biophysical technique that allows the separation of isobaric species on the basis of their size and shape. The high separation capacity, sensitivity and relatively fast time scale measurements confer IMMS great potential for the study of proteins in slow (µs–ms) conformational equilibrium in solution. However, the use of this technique for examining dynamic proteins is still not generalized. One of the major limitations is the instability of protein ions in the gas phase, which raises the question as to what extent the structures detected reflect those in solution. Here, we addressed this issue by analyzing the conformational landscape of prolyl oligopeptidase (POP) – a model of a large dynamic enzyme in the µs–ms range – by native IMMS and compared the results obtained in the gas phase with those obtained in solution. In order to interpret the experimental results, we used theoretical simulations. In addition, the stability of POP gaseous ions was explored by charge reduction and collision‐induced unfolding experiments. Our experiments disclosed two species of POP in the gas phase, which correlated well with the open and closed conformations in equilibrium in solution; moreover, a gas‐phase collapsed form of POP was also detected. Therefore, our findings not only support the potential of IMMS for the study of multiple co‐existing conformations of large proteins in slow dynamic equilibrium in solution but also stress the need for careful data analysis to avoid artifacts. Copyright © 2016 John Wiley & Sons, Ltd.