Ion Mobility-Mass Spectrometry Reveals Conformational Changes in Charge Reduced Multiprotein Complexes

Characterizing intact multiprotein complexes in terms of both their mass and size by ion mobility-mass spectrometry is becoming an increasingly important tool for structural biology. Furthermore, the charge states of intact protein complexes can dramatically influence the information content of gas-phase measurements performed. Specifically, protein complex charge state has a demonstrated influence upon the conformation, mass resolution, ion mobility resolution, and dissociation properties of protein assemblies upon collisional activation. Here we present the first comparison of charge-reduced multiprotein complexes generated by solution additives and gas-phase ion-neutral reaction chemistry. While the charge reduction mechanism for both methods is undoubtedly similar, significant gas-phase activation of the complex is required to reduce the charge of the assemblies generated using the solution additive strategy employed here. This activation step can act to unfold intact protein complexes, making the data difficult to correlate with solution-phase structures and topologies. We use ion mobility-mass spectrometry to chart such conformational effects for a range of multi-protein complexes, and demonstrate that approaches to reduce charge based on ion-neutral reaction chemistry in the gas-phase consistently produce protein assemblies having compact, ‘native-like’ geometries while the same molecules added in solution generate significantly unfolded gas-phase complexes having identical charge states.     

Effects of Select Anions from the Hofmeister Series on the Gas-Phase Conformations of Protein Ions Measured with Traveling-Wave Ion Mobility Spectrometry/Mass Spectrometry

The gas-phase conformations of ubiquitin, cytochrome c, lysozyme, and α-lactalbumin ions, formed by electrospray ionization (ESI) from aqueous solutions containing 5 mM ammonium perchlorate, ammonium iodide, ammonium sulfate, ammonium chloride, ammonium thiocyanate, or guanidinium chloride, are examined using traveling-wave ion mobility spectrometry (TWIMS) coupled to time-of-flight (TOF) mass spectrometry (MS). For ubiquitin, cytochrome c, and α-lactalbumin, adduction of multiple acid molecules results in no significant conformational changes to the highest and lowest charge states formed from aqueous solutions, whereas the intermediate charge states become more compact. The transition to more compact conformers for the intermediate charge states occurs with fewer bound H₂SO₄ molecules than HClO₄ or HI molecules, suggesting ion-ion or salt-bridge interactions are stabilizing more compact forms of the gaseous protein. However, the drift time distributions for protein ions of the same net charge with the highest levels of adduction of each acid are comparable, indicating that these protein ions all adopt similarly compact conformations or families of conformers. No significant change in conformation is observed upon the adduction of multiple acid molecules to charge states of lysozyme. These results show that the attachment of HClO₄, HI, or H₂SO₄ to multiply protonated proteins can induce compact conformations in the resulting gas-phase protein ions. In contrast, differing Hofmeister effects are observed for the corresponding anions in solution at higher concentrations.     

Impact of limited oxidation on protein ion mobility and structure of importance to footprinting by radical probe mass spectrometry

The effect of hydroxyl radical induced oxidation on the collision cross-sections of hen egg lysozyme and bovine ubiquitin was investigated by travelling wave ion mobility mass spectrometry for the first time. The oxidized ions of lysozyme and ubiquitin share common collision cross-sections with their unoxidized counterparts suggesting that they share common structures that were unaffected by limited oxidation. In the case of bovine ubiquitin, two distinct conformers were detected for the protein in its unoxidized and oxidized states though no change in the levels of each was observed upon oxidation. This supports the validity of Radical Probe Mass Spectrometry (RP-MS) using an electrical discharge source for protein footprinting experiments. Travelling wave ion mobility mass spectrometry has been used for the first time to confirm that limited oxidation does not have an impact on the global structure of proteins.     

Ion mobility-mass spectrometry

This review article compares and contrasts various types of ion mobility-mass spectrometers available today and describes their advantages for application to a wide range of analytes. Ion mobility spectrometry (IMS), when coupled with mass spectrometry, offers value-added data not possible from mass spectra alone. Separation of isomers, isobars, and conformers; reduction of chemical noise; and measurement of ion size are possible with the addition of ion mobility cells to mass spectrometers. In addition, structurally similar ions and ions of the same charge state can be separated into families of ions which appear along a unique mass-mobility correlation line. This review describes the four methods of ion mobility separation currently used with mass spectrometry. They are (1) drift-time ion mobility spectrometry (DTIMS), (2) aspiration ion mobility spectrometry (AIMS), (3) differential-mobility spectrometry (DMS) which is also called field-asymmetric waveform ion mobility spectrometry (FAIMS) and (4) traveling-wave ion mobility spectrometry (TWIMS). DTIMS provides the highest IMS resolving power and is the only IMS method which can directly measure collision cross-sections. AIMS is a low resolution mobility separation method but can monitor ions in a continuous manner. DMS and FAIMS offer continuous-ion monitoring capability as well as orthogonal ion mobility separation in which high-separation selectivity can be achieved. TWIMS is a novel method of IMS with a low resolving power but has good sensitivity and is well intergrated into a commercial mass spectrometer. One hundred and sixty references on ion mobility-mass spectrometry (IMMS) are provided.     

Formation of multimeric steroid metal adducts and implications for isomer mixture separation by traveling wave ion mobility spectrometry

Steroid analysis is essential to the fields of medicine and forensics, but such analyses can present some complex analytical challenges. While chromatographic methods require long acquisition times and often provide incomplete separation, ion mobility spectrometry (IMS) as coupled to mass spectrometry (MS) has demonstrated significant promise for the separation of steroids, particularly in concert with metal adduction and multimerization. In this study, traveling wave ion mobility spectrometry (TWIMS) was employed to separate multimer steroid metal adducts of isomers in mixtures. The results show the ability to separate steroid isomers with a decrease in resolution compared with single component standards because of the formation of heteromultimers. Additionally, ion‐neutral collision cross sections (CCS) of the species studied were measured in the mixtures and compared with CCSs obtained in single component standards. Good agreement between these values suggests that the CCS may aid in identification of unknowns. Furthermore, a complex mixture composed of five sets of steroid isomers were analyzed, and distinct features for each steroid component were identified. This study further demonstrated the potential of TWIMS‐MS methods for the rapid and isomer‐specific study of steroids in biological samples for use either in tandem with or without chromatographic separation.     

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.     

Enhancements in travelling wave ion mobility resolution

The use of ion mobility separation to determine the collision cross-section of a gas-phase ion can provide valuable structural information. The introduction of travelling-wave ion mobility within a quadrupole/time-of-flight mass spectrometer has afforded routine collision cross-section measurements to be performed on a range of ionic species differing in gas-phase size/structure and molecular weight at physiologically relevant concentrations. Herein we discuss the technical advances in the second-generation travelling-wave ion mobility separator, which result in up to a four-fold increase in mobility resolution. This improvement is demonstrated using two reverse peptides (mw 490 Da), small ruthenium-containing anticancer drugs (mw 427 Da), a cisplatin-modified protein (mw 8776 Da) and the noncovalent tetradecameric chaperone complex GroEL (mw 802 kDa). What is also shown are that the collision cross-sections determined using the second-generation mobility separator correlate well with the previous generation and theoretically derived values.     

A Computational Model for Protein Ionization by Electrospray Based on Gas-Phase Basicity

Identifying the key factor(s) governing the overall protein charge is crucial for the interpretation of electrospray-ionization mass spectrometry data. Current hypotheses invoke different principles for folded and unfolded proteins. Here, first we investigate the gas-phase structure and energetics of several proteins of variable size and different folds. The conformer and protomer space of these proteins ions is explored exhaustively by hybrid Monte-Carlo/molecular dynamics calculations, allowing for zwitterionic states. From these calculations, the apparent gas-phase basicity of desolvated protein ions turns out to be the unifying trait dictating protein ionization by electrospray. Next, we develop a simple, general, adjustable-parameter-free model for the potential energy function of proteins. The model is capable to predict with remarkable accuracy the experimental charge of folded proteins and its well-known correlation with the square root of protein mass.     

Resolution and structural transitions of elongated states of ubiquitin

Electrospray ionization, combined with two-dimensional ion mobility spectrometry and mass spectrometry, is used to produce, select, and activate distributions of elongated ions, [M + 11H]11+ to [M + 13H]13+, of ubiquitin. The analysis makes it possible to examine state-to-state transitions for structural types, and transition diagrams associated with the efficiencies of structural changes are presented. The +11 and +12 charge states can form four resolvable states while only one state is formed for [M + 13H]13+. Some conformations, which appear to belong to the same family based on mobility analysis of different charge states, undergo similar transitions, others do not. Activation of ions that exist in low-abundance conformations, having mobilities that fall in between sharp peaks associated with higher abundances species, shows that the low-abundance forms undergo efficient (approximately 90 to 100%) conversion into states associated with well-defined peaks. This efficiency is significantly higher than the approximately 10 to 60% efficiency of transitions of structures associated with well-defined peaks. The formation of sharp features from a range of low-intensity species with different cross sections indicates that large regions of conformation space must be unfavorable or inaccessible in the gas phase. These results are compared with several previous IMS measurements of this system as well as information about gas-phase structure provided by other techniques.     

How Hot are Your Ions in TWAVE Ion Mobility Spectrometry?

Effective temperatures of ions during traveling wave ion mobility spectrometry (TWIMS) analysis were measured using singly protonated leucine enkephalin dimer as a chemical thermometer by monitoring dissociation of the dimer into monomer, as well as the subsequent dissociation of monomer into a-, b-, and y-ions, as a function of instrumental parameters. At fixed helium cell and TWIMS cell gas flow rates, the extent of dissociation does not vary significantly with either the wave velocity or wave height, except at low (<500 m/s) wave velocities that are not commonly used. Increasing the flow rate of nitrogen gas into the TWIMS cell and decreasing the flow rate of helium gas into the helium cell resulted in greater dissociation. However, the mobility distributions of the fragment ions formed by dissociation of the dimer upon injection into the TWIMS cell are nearly indistinguishable from those of fragment ions formed in the collision cell prior to TWIMS analysis for all TWIMS experiments. These results indicate that heating and dissociation occur when ions are injected into the TWIMS cell, and that the effective temperature subsequently decreases to a point at which no further dissociation is observed during the TWIMS analysis. An upper limit to the effective ion temperature of 449 K during TWIMS analysis is obtained at a helium flow rate of 180 mL/min, TWIMS flow rate of 80 mL/min, and traveling wave height of 40 V, which is well below previously reported values. Effects of ion heating in TWIMS on gas-phase protein conformation are presented.     

Size dependence of the folding of multiply charged sodium cationized polylactides revealed by ion mobility mass spectrometry and molecular modelling

Ion mobility spectrometry coupled with mass spectrometry was used to experimentally determine the three-dimensional structure of multiply charged sodium cationized polylactides (PLA). In particular, the experiments were conducted to evaluate the influence of the charge state and the size on the gas-phase conformation of cationized PLA. The measured collision cross sections were then compared to calculated values obtained by computational chemistry methods. The most striking feature was the experimental and theoretical observation of a breaking point in the quasilinear relationship between the average collision cross sections and the number of monomer units for the triply charged cations. This breaking point was theoretically demonstrated, for the doubly and triply charged cations, to be associated with a significant folding of the polymer chains around the cationizing agents. The occurrence of such breaking points could be exploited to correlate the charge state of the most intense ion series observed upon electrospray ionization with the number-average molecular mass of a polymer.     

Design, synthesis, and traveling wave ion mobility mass spectrometry characterization of iron(II)- and ruthenium(II)-terpyridine metallomacrocycles

New metallomacrocycles composed of 2,2':6',2″-terpyridine (tpy) ligands and Ru(II) or Fe(II) transition metal ions were prepared by stepwise directed assembly and characterized by 2D diffusion NMR spectroscopy (DOSY), electrospray ionization traveling wave ion mobility mass spectrometry (ESI TWIM MS), and molecular modeling. The supramolecular polymers synthesized include a homonuclear all-Ru hexamer as well as heteronuclear hexamer and nonamer with alternating Ru/Ru/Fe metal centers. ESI MS yields several charge states from each supramacromolecule. If ESI is interfaced with TWIM MS, overlapping charge states and the isomeric components of an individual charge state are separated based on their unique drift times through the TWIM region. From experimentally measured drift times, collision cross-sections can be deduced. The collision cross-sections obtained for the synthesized supramacromolecules are in good agreement with those predicted by molecular modeling for macrocyclic structures. Similarly, the hydrodynamic radii of the synthesized complexes derived from 2D DOSY NMR experiments agree excellently with the radii calculated for macrocyclic architectures, confirming the ESI TWIM MS finding. ESI TWIM MS and 2D DOSY NMR spectroscopy provide an alternative approach for the structural analysis of supramolecules that are difficult or impossible to crystallize, such as the large macrocyclic assemblies investigated. ESI TWIM MS will be particularly valuable for the characterization of supramolecular assemblies not available in the quantity or purity required for NMR studies.     

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.     

Structural analysis of prion proteins by means of drift cell and traveling wave ion mobility mass spectrometry

The prion protein (PrP) is implicitly involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs). The conversion of normal cellular PrP (PrP^C), a protein that is predominantly α-helical, to a β-sheet-rich isoform (PrP^Sc), which has a propensity to aggregate, is the key molecular event in prion diseases. During its short life span, PrP can experience two different pH environments; a mildly acidic environment, whilst cycling within the cell, and a neutral pH when it is glycosyl phosphatidylinositol (GPI)-anchored to the cell membrane. Ion mobility (IM) combined with mass spectrometry has been employed to differentiate between two conformational isoforms of recombinant Syrian hamster prion protein (SHaPrP). The recombinant proteins studied were α-helical SHaPrP(90-231) and β-sheet-rich SHaPrP(90-231) at pH 5.5 and pH 7.0. The recombinant proteins have the same nominal mass-to-charge ratio (m/z) but differ in their secondary and tertiary structures. A comparison of traveling-wave (T-Wave) ion mobility and drift cell ion mobility (DCIM) mass spectrometry estimated and absolute cross-sections showed an excellent agreement between the two techniques. The use of T-Wave ion mobility as a shape-selective separation technique enabled differentiation between the estimated cross-sections and arrival time distributions (ATDs) of α-helical SHaPrP(90-231) and β-sheet-rich SHaPrP(90-231) at pH 5.5. No differences in cross-section or ATD profiles were observed between the protein isoforms at pH 7.0. The findings have potential implications for a new ante-mortem screening assay, in bodily fluids, for prion misfolding diseases such as TSEs.     

Decharging of globular proteins and protein complexes in electrospray

Electrospray ionization mass spectrometry (ESI-MS) is a valuable tool in structural biology for investigating globular proteins and their biomolecular interactions. During the electrospray ionization process, proteins become desolvated and multiply charged, which may influence their structure. Reducing the net charge obtained during the electrospray process may be relevant for studying globular proteins. In this report we demonstrate the effect of a series of inorganic and organic gas-phase bases on the number of charges that proteins and protein complexes attain. Solution additives with very strong gas-phase basicities (GB) were identified among the so-called "proton sponges". The gas-phase proton affinities (PA) of the compounds that were added to the aqueous protein solutions ranged from 700 to 1050 kJ mol(-1). Circular dichroism studies showed that in these solutions the proteins retain their globular structures. The size of the proteins investigated ranged from the 14.3 kDa lysozyme up to the 800 kDa tetradecameric chaperone complex GroEL. Decharging of the proteins in the electrospray process by up to 60 % could be achieved by adding the most basic compounds rather than the more commonly used ammonium acetate additive. This decharging process probably results from proton competition events between the multiply protonated protein ions and the basic additives just prior to the final desolvation. We hypothesize that such globular protein species, which attain relatively few charges during the ionization event, obtain a gas-phase structure that more closely resembles their solution-phase structure. Thus, these basic additives can be useful in the study of the biologically relevant properties of globular proteins by using mass spectrometry.     

Supercharging with Trivalent Metal Ions in Native Mass Spectrometry

Addition of 1.0?mM LaCl₃ to aqueous ammonium acetate solutions containing proteins in their folded native forms can result in a significant increase in the molecular ion charging obtained with electrospray ionization as a result of cation adduction. In combination with m-nitrobenzyl alcohol, molecular ion charge states that are greater than the number of basic sites in the protein can be produced from these native solutions, even for lysozyme, which is conformationally constrained by four intramolecular disulfide bonds. Circular dichroism spectroscopy indicates that the conformation of ubiquitin is not measurably affected with up to 1.0?M LaCl₃, but ion mobility data indicate that the high charge states that are formed when 1.0?mM LaCl₃ is present are more unfolded than the low charge states formed without this reagent. These and other results indicate that the increased charging is a result of La³⁺ preferentially adducting onto compact or more native-like conformers during ESI and the gas-phase ions subsequently unfolding as a result of increased Coulomb repulsion. Electron capture dissociation of these high charge-state ions formed from these native solutions results in comparable sequence coverage to that obtained for ions formed from denaturing solutions without supercharging reagents, making this method a potentially powerful tool for obtaining structural information in native mass spectrometry.     

Solvent-induced conformational changes of polypeptides probed by electrospray-ionization mass spectrometry.

Electrospray-ionization (ESI) mass spectrometry is used to monitor higher order structural changes of polypeptides induced by alteration of the pH or organic solvent composition in the protein solution environment. A bimodal charge-state distribution is observed in the ESI mass spectrum of ubiquitin (relative molecular mass 8565) in solutions containing small amounts (less than 20%) of organic solvents. The distribution of peaks at high m/z (low-charge state) is found to represent the protein in its native, globular state; the higher-charge-state distribution is characteristic for a more extended conformation. Addition of methanol denaturant in excess of 40% v/v is needed to eliminate the low-charge-state distribution completely. Lesser amounts of acetonitrile, acetone, or isopropanol (approximately 20%) are required to denature the ubiquitin protein. Other proteins showing conformational effects in their ESI mass spectra are also illustrated. While the ESI spectra are related to solution phase structure, ESI-tandem mass spectrometry of multiply charged molecular ions of different conformation is suggested as a probe of gas-phase protein three-dimensional structure.     

Separation and characterization of oxidized isomeric lipid–peptide adducts by ion mobility mass spectrometry

Phospholipids are major components of cell membranes and lipoprotein complexes. They are prone to oxidation by endogenous and exogenous reactive oxygen species yielding a large variety of modified lipids including small aliphatic and phospholipid bound aldehydes and ketones. These carbonyls are strong electrophiles that can modify proteins and, thereby, alter their structures and functions triggering various pathophysiological conditions. The analysis of lipid–protein adducts by liquid chromatography‐MS is challenged by their mixed chemical nature (polar peptide and hydrophobic lipid), low abundance in biological samples, and formation of multiple isomers. Thus, we investigated traveling wave ion mobility mass spectrometry (TWIMS) to analyze lipid–peptide adducts generated by incubating model peptides corresponding to the amphipathic β₁ sheet sequence of apolipoprotein B‐100 with 1‐palmitoyl‐2‐(oxo‐nonanoyl)‐sn‐glycerophosphatidylcholine (PONPC). The complex mixture of peptides, lipids, and peptide–lipid adducts was separated by TWIMS, which was especially important for the identification of two mono‐PONPC‐peptide isomers containing Schiff bases at different lysine residues. Moreover, TWIMS separated structural conformers of one peptide–lipid adduct possessing most likely different orientations of the hydrophobic sn‐1 fatty acyl residue and head group of PONPC, relative to the peptide backbone. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

Retention of Native Protein Structures in the Absence of Solvent: A Coupled Ion Mobility and Spectroscopic Study

Can the structures of small to medium‐sized proteins be conserved after transfer from the solution phase to the gas phase? A large number of studies have been devoted to this topic, however the answer has not been unambiguously determined to date. A clarification of this problem is important since it would allow very sensitive native mass spectrometry techniques to be used to address problems relevant to structural biology. A combination of ion‐mobility mass spectrometry with infrared spectroscopy was used to investigate the secondary and tertiary structure of proteins carefully transferred from solution to the gas phase. The two proteins investigated are myoglobin and β‐lactoglobulin, which are prototypical examples of helical and β‐sheet proteins, respectively. The results show that for low charge states under gentle conditions, aspects of the native secondary and tertiary structure can be conserved.