Recommendations for reporting ion mobility mass spectrometry measurements

© 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc. Here we present a guide to ion mobility mass spectrometry experiments, which covers both linear and nonlinear methods: what is measured, how the measurements are done, and how to report the results, including the uncertainties of mobility and collision cross section values. The guide aims to clarify some possibly confusing concepts, and the reporting recommendations should help researchers, authors and reviewers to contribute comprehensive reports, so that the ion mobility data can be reused more confidently. Starting from the concept of the definition of the measurand, we emphasize that (i) mobility values (K0) depend intrinsically on ion structure, the nature of the bath gas, temperature, and E/N; (ii) ion mobility does not measure molecular surfaces directly, but collision cross section (CCS) values are derived from mobility values using a physical model; (iii) methods relying on calibration are empirical (and thus may provide method-dependent results) only if the gas nature, temperature or E/N cannot match those of the primary method. Our analysis highlights the urgency of a community effort toward establishing primary standards and reference materials for ion mobility, and provides recommendations to do so. © 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc

Interpreting the Collision Cross Sections of Native-like Protein Ions: Insights from Cation-to-Anion Proton-Transfer Reactions

The effects of charge state on structures of native-like cations of serum albumin, streptavidin, avidin, and alcohol dehydrogenase were probed using cation-to-anion proton-transfer reactions (CAPTR), ion mobility, mass spectrometry, and complementary energy-dependent experiments. The CAPTR products all have collision cross-section (Ω) values that are within 5.5% of the original precursor cations. The first CAPTR event for each precursor yields products that have smaller Ω values and frequently exhibit the greatest magnitude of change in Ω resulting from a single CAPTR event. To investigate how the structures of the precursors affect the structures of the products, ions were activated as a function of energy prior to CAPTR. In each case, the Ω values of the activated precursors increase with increasing energy, but the Ω values of the CAPTR products are smaller than the activated precursors. To investigate the stabilities of the CAPTR products, the products were activated immediately prior to ion mobility. These results show that additional structures with smaller or larger Ω values can be populated and that the structures and stabilities of these ions depend most strongly on the identity of the protein and the charge state of the product, rather than the charge state of the precursor or the number of CAPTR events. Together, these results indicate that the excess charges initially present on native-like ions have a modest, but sometimes statistically significant, effect on their Ω values. Therefore, potential contributions from charge state should be considered when using experimental Ω values to elucidate structures in solution

Effects of Drift Gas Selection on the Ambient-Temperature, Ion Mobility Mass Spectrometry Analysis of Amino Acids

Ion mobility (IM) separates ions based on their response to an electric field in the presence of a drift gas. Because of its speed and sensitivity, the integration of IM and mass spectrometry (MS) offers many potential advantages for the analysis of small molecules. To determine the effects that drift gas selection has on the information content of IM separations, absolute collision cross sections (Ω) with He, N₂, Ar, CO₂, and N₂O were measured for the 20 common amino acids using low-pressure, ambient-temperature ion mobility experiments performed in a radio frequency-confining drift cell. The drift gases were selected to span a range of masses, geometries, and polarizabilities. The information content of each separation was quantified using its peak capacity, which depended on factors contributing to widths of peaks as well as the range of Ω relative to the average Ω for the analytes. The selectivity of each separation was quantified by calculating the peak-to-peak resolution for each pairwise combination of amino acid ions. The number of pairs that were resolved depended strongly on the peak capacity, but the identities of the pairs resolved also depended on the drift gas. Therefore, results using different drift gases are partially orthogonal and provide complementary chemical information. The temperatures and pressures used for these experiments are similar to those used in many IM-MS instruments, therefore, the outcomes of this research are applicable to optimizing the information content of a wide range of contemporary and future IM-MS experiments

Effects of Charge State, Charge Distribution, and Structure on the Ion Mobility of Protein Ions in Helium Gas: Results from Trajectory Method Calculations

Collision cross section (Ω) values of gas-phase ions of proteins and protein complexes are used to probe the structures of the corresponding species in solution. Ions of many proteins exhibit increasing Ω-values with increasing charge state but most Ω-values calculated for protein ions have used simple collision models that do not explicitly account for charge. Here we use a combination of ion mobility mass spectrometry experiments with helium gas and trajectory method calculations to characterize the extents to which increases in experimental Ω-values with increasing charge state may be attributed to increased momentum transfer concomitant with enhanced long-range interactions between the protein ion and helium atoms. Ubiquitin and C-to-N terminally linked diubiquitin ions generated from different solution conditions exhibit more than a 2-fold increase in Ω with increasing charge state. For native and energy-relaxed models of the proteins and most methods for distributing charge, Ω-values calculated using the trajectory method increase by less than 1% over the range of charge states observed from typical solution conditions used for native mass spectrometry. However, the calculated Ω-values increase by 10% to 15% over the full range of charge states observed from all solution conditions. Therefore, contributions from enhanced ion-induced dipole interactions with increasing charge state are significant but without additional structural changes can account for only a fraction of the increase in Ω observed experimentally. On the basis of these results, we suggest guidelines for calculating Ω-values in the context of applications in biophysics and structural biology

Ion Mobility of Proteins in Nitrogen Gas: Effects of Charge State, Charge Distribution, and Structure

Ion mobility is emerging as a rapid and sensitive tool for structural characterization. Collision cross-section (Ω) values determined using ion mobility are often compared to values calculated for candidate structures generated through molecular modeling. Several methods exist for calculating Ω values, but the trajectory method explicitly includes contributions from long-range, ion–neutral interactions. Recent implementations of the trajectory method have significantly reduced its expense and have made applications to proteins far more tractable. Here, we use ion mobility experiments and trajectory method calculations to characterize the effects of charge state, charge distribution, and structure on the ion mobility of proteins in nitrogen gas. These results show that ion-induced dipole interactions contribute significantly to the Ω values of these ions with nitrogen gas, even for the modestly charged ions commonly observed in native mass spectrometry experiments. Therefore, these interactions contribute significantly to the values measured in most structural biology and biophysics applications of ion mobility using nitrogen gas. Comparisons between the reciprocal mobilities of protein ions in helium gas and in nitrogen gas show that there are significant, noncorrelated differences between these values. As a consequence, it is challenging to estimate the errors associated with interconverting between helium- and nitrogen-based mobilities without extensive characterization in both gases, even for ions of proteins with similar sequences. Therefore, we recommend reporting Ω and mobility values that are based on the predominant gas present in the separation and applying additional caution when comparing results from mobility experiments performed using different gases

Structural Dynamics of Native-Like Ions in the Gas Phase: Results from Tandem Ion Mobility of Cytochrome <i>c</i>

Ion mobility (IM) is a gas-phase separation technique that is used to determine the collision cross sections of native-like ions of proteins and protein complexes, which are in turn used as restraints for modeling the structures of those analytes in solution. Here, we evaluate the stability of native-like ions using tandem IM experiments implemented using structures for lossless ion manipulations (SLIM). In this implementation of tandem IM, ions undergo a first dimension of IM up to a switch that is used to selectively transmit ions of a desired mobility. Selected ions are accumulated in a trap and then released after a delay to initiate the second dimension of IM. For delays ranging from 16 to 33 231 ms, the collision cross sections of native-like, 7+ cytochrome <i>c</i> ions increase monotonically from 15.1 to 17.1 nm². The largest products formed in these experiments at near-ambient temperature are still far smaller than those formed in energy-dependent experiments (∼21 nm²). However, the collision cross section increases by ∼2% between delay times of 16 and 211 ms, which may have implications for other IM experiments on these time scales. Finally, two subpopulations from the full population were each mobility selected and analyzed as a function of delay time, showing that the three populations can be differentiated for at least 1 s. Together, these results suggest that elements of native-like structure can have long lifetimes at near-ambient temperature in the gas phase but that gas-phase dynamics should be considered when interpreting results from IM

Effects of Polarity on the Structures and Charge States of Native-Like Proteins and Protein Complexes in the Gas Phase

Native mass spectrometry and ion mobility spectrometry were used to investigate the gas-phase structures of selected cations and anions of proteins and protein complexes with masses ranging from 6 to 468 kDa. Under the same solution conditions, the average charge states observed for all native-like anions were less than those for the corresponding cations. Using an rf-confining drift cell, similar collision cross sections were measured in positive and negative ion mode suggesting that anions and cations have very similar structures. This result suggests that for protein and protein complex ions within this mass range, there is no inherent benefit to selecting a specific polarity for capturing a more native-like structure. For peptides and low-mass proteins, polarity and charge-state dependent structural changes may be more significant. The charged-residue model is most often used to explain the ionization of large macromolecules based on the Rayleigh limit, which defines the upper limit of charge that a droplet can hold. Because ions of both polarities have similar structures and the Rayleigh limit does not depend on polarity, these results cannot be explained by the charged-residue model alone. Rather, the observed charge-state distributions are most consistent with charge-carrier emissions during the final stages of analyte desolvation, with lower charge-carrier emission energies for anions than the corresponding cations. These results suggest that the observed charge-state distributions in most native mass spectrometry experiments are determined by charge-carrier emission processes; although the Rayleigh limit may determine the gas-phase charge states of larger species, e.g., virus capsids

Effects of Solution Structure on the Folding of Lysozyme Ions in the Gas Phase

The fidelity between the structures of proteins in solution and protein ions in the gas phase is critical to experiments that use gas-phase measurements to infer structures in solution. Here we generate ions of lysozyme, a 129-residue protein whose native tertiary structure contains four internal disulfide bonds, from three solutions that preserve varying extents of the original native structure. We then use cation-to-anion proton-transfer reactions (CAPTR) to reduce the charge states of those ions in the gas phase and ion mobility to probe their structures. The collision cross section (Ω) distributions of each CAPTR product depends to varying extents on the original solution, the charge state of the product, and the charge state of the precursor. For example, the Ω distributions of the 6+ ions depend strongly on the original solutions conditions and to a lesser extent on the charge state of the precursor. Energy-dependent experiments suggest that very different structures are accessible to disulfide-reduced and disulfide-intact ions, but similar Ω distributions are formed at high energy for disulfide-intact ions from denaturing and from aqueous conditions. The Ω distributions of the 3+ ions are all similar but exhibit subtle differences that depend more strongly on the original solutions conditions than other factors. More generally, these results suggest that specific CAPTR products may be especially sensitive to specific elements of structure in solution

Analysis of Native-Like Ions Using Structures for Lossless Ion Manipulations

Ion mobility separation of native-like protein and protein complex ions expands the structural information available through native mass spectrometry analysis. Here, we implement Structures for Lossless Ion Manipulations (SLIM) for the analysis of native-like ions. SLIM has been shown previously to operate with near lossless transmission of ions up to 3000 Da in mass. Here for the first time, SLIM was used to separate native-like protein and protein complex ions ranging in mass from 12 to 145 kDa. The resulting arrival-time distributions were monomodal and were used to determine collision cross section values that are within 3% of those determined from radio-frequency-confining drift cell measurements. These results are consistent with the retention of native-like ion structures throughout these experiments. The apparent resolving powers of native-like ions measured using SLIM are as high as 42, which is the highest value reported directly from experimental data for the native-like ion of a protein complex. Interestingly, the apparent resolving power depends strongly on the identity of the analyte, suggesting that the arrival-time distributions of these ions may have contributions from an ensemble of structures in the gas phase that is unique to each analyte. These results suggest that the broad range of emerging SLIM technologies may all be adaptable to the analysis of native-like ions, which will enable future applications in the areas of structural biology, biophysics, and biopharmaceutical characterization

Ion Mobility Mass Spectrometry of Peptide Ions: Effects of Drift Gas and Calibration Strategies

One difficulty in using ion mobility (IM) mass spectrometry (MS) to improve the specificity of peptide ion assignments is that IM separations are performed using a range of pressures, gas compositions, temperatures, and modes of separation, which makes it challenging to rapidly extract accurate shape parameters. We report collision cross section values (Ω) in both He and N₂ gases for 113 peptide ions determined directly from drift times measured in a low-pressure, ambient temperature drift cell with radio-frequency (rf) ion confinement. These peptide ions have masses ranging from 231 to 2969 Da, Ω_He of 89–616 Ų, and Ω_N₂ of 151–801 Ų; thus, they are ideal for calibrating results from proteomics experiments. These results were used to quantify the errors associated with traveling-wave Ω measurements of peptide ions and the errors concomitant with using drift times measured in N₂ gas to estimate Ω_He. More broadly, these results enable the rapid and accurate determination of calibrated Ω for peptide ions, which could be used as an additional parameter to increase the specificity of assignments in proteomics experiments