FBXL5 Regulates IRP2 Stability in Iron Homeostasis via an Oxygen-Responsive [2Fe2S] Cluster.

Cellular iron homeostasis is dominated by FBXL5-mediated degradation of iron regulatory protein 2 (IRP2), which is dependent on both iron and oxygen. However, how the physical interaction between FBXL5 and IRP2 is regulated remains elusive. Here, we show that the C-terminal substrate-binding domain of FBXL5 harbors a [2Fe2S] cluster in the oxidized state. A cryoelectron microscopy (cryo-EM) structure of the IRP2-FBXL5-SKP1 complex reveals that the cluster organizes the FBXL5 C-terminal loop responsible for recruiting IRP2. Interestingly, IRP2 binding to FBXL5 hinges on the oxidized state of the [2Fe2S] cluster maintained by ambient oxygen, which could explain hypoxia-induced IRP2 stabilization. Steric incompatibility also allows FBXL5 to physically dislodge IRP2 from iron-responsive element RNA to facilitate its turnover. Taken together, our studies have identified an iron-sulfur cluster within FBXL5, which promotes IRP2 polyubiquitination and degradation in response to both iron and oxygen concentrations.

Recognition of the Diglycine C-End Degron by CRL2KLHDC2 Ubiquitin Ligase.

Aberrant proteins can be deleterious to cells and are cleared by the ubiquitin-proteasome system. A group of C-end degrons that are recognized by specific cullin-RING ubiquitin E3 ligases (CRLs) has recently been identified in some of these abnormal polypeptides. Here, we report three crystal structures of a CRL2 substrate receptor, KLHDC2, in complex with the diglycine-ending C-end degrons of two early-terminated selenoproteins and the N-terminal proteolytic fragment of USP1. The E3 recognizes the degron peptides in a similarly coiled conformation and cradles their C-terminal diglycine with a deep surface pocket. By hydrogen bonding with multiple backbone carbonyls of the peptides, KLHDC2 further locks in the otherwise degenerate degrons with a compact interface and unexpected high affinities. Our results reveal the structural mechanism by which KLHDC2 recognizes the simplest C-end degron and suggest a functional necessity of the E3 to tightly maintain the low abundance of its select substrates.

Disordered region encodes α-crystallin chaperone activity toward lens client γD-crystallin.

Small heat-shock proteins (sHSPs) are a widely expressed family of ATP-independent molecular chaperones that are among the first responders to cellular stress. Mechanisms by which sHSPs delay aggregation of client proteins remain undefined. sHSPs have high intrinsic disorder content of up to ~60% and assemble into large, polydisperse homo- and hetero-oligomers, making them challenging structural and biochemical targets. Two sHSPs, HSPB4 and HSPB5, are present at millimolar concentrations in eye lens, where they are responsible for maintaining lens transparency over the lifetime of an organism. Together, HSPB4 and HSPB5 compose the hetero-oligomeric chaperone known as α-crystallin. To identify the determinants of sHSP function, we compared the effectiveness of HSPB4 and HSPB5 homo-oligomers and HSPB4/HSPB5 hetero-oligomers in delaying the aggregation of the lens protein γD-crystallin. In chimeric versions of HSPB4 and HSPB5, chaperone activity tracked with the identity of the 60-residue disordered N-terminal regions (NTR). A short 10-residue stretch in the middle of the NTR ("Critical sequence") contains three residues that are responsible for high HSPB5 chaperone activity toward γD-crystallin. These residues affect structure and dynamics throughout the NTR. Abundant interactions involving the NTR Critical sequence reveal it to be a hub for a network of interactions within oligomers. We propose a model whereby the NTR critical sequence influences local structure and NTR dynamics that modulate accessibility of the NTR, which in turn modulates chaperone activity.

Effects of charge on protein ion structure: Lessons from cation-to-anion, proton-transfer reactions.

Collision cross-section values, which can be determined using ion mobility experiments, are sensitive to the structures of protein ions and useful for applications to structural biology and biophysics. Protein ions with different charge states can exhibit very different collision cross-section values, but a comprehensive understanding of this relationship remains elusive. Here, we review cation-to-anion, proton-transfer reactions (CAPTR), a method for generating a series of charge-reduced protein cations by reacting quadrupole-selected cations with even-electron monoanions. The resulting CAPTR products are analyzed using a combination of ion mobility, mass spectrometry, and collisional activation. We compare CAPTR to other charge-manipulation strategies and review the results of various CAPTR-based experiments, exploring their contribution to a deeper understanding of the relationship between protein ion structure and charge state.

High-Performance Workflow for Identifying Site-Specific Crosslinks Originating from a Genetically Incorporated, Photoreactive Amino Acid.

In conventional crosslinking mass spectrometry, proteins are crosslinked using a highly selective, bifunctional chemical reagent, which limits crosslinks to residues that are accessible and reactive to the reagent. Genetically incorporating a photoreactive amino acid offers two key advantages: any site can be targeted, including those that are inaccessible to conventional crosslinking reagents, and photoreactive amino acids can potentially react with a broad range of interaction partners. However, broad reactivity imposes additional challenges for crosslink identification. In this study, we incorporate benzoylphenylalanine (BPA), a photoreactive amino acid, at selected sites in an intrinsically disordered region of the human protein HSPB5. We report and characterize a workflow for identifying and visualizing residue-level interactions originating from BPA. We routinely identify 30 to 300 crosslinked peptide spectral matches with this workflow, which is up to ten times more than existing tools for residue-level BPA crosslink identification. Most identified crosslinks are assigned to a precision of one or two residues, which is supported by a high degree of overlap between replicate analyses. Based on these results, we anticipate that this workflow will support the more general use of genetically incorporated, photoreactive amino acids for characterizing the structures of proteins that have resisted high-resolution characterization.

Drugs Form Ternary Complexes with Human Liver Fatty Acid Binding Protein 1 (FABP1) and FABP1 Binding Alters Drug Metabolism.

Liver fatty acid binding protein 1 (FABP1) binds diverse endogenous lipids and is highly expressed in the human liver. Binding to FABP1 alters the metabolism and homeostasis of endogenous lipids in the liver. Drugs have also been shown to bind to rat FABP1, but limited data are available for human FABP1 (hFABP1). FABP1 has a large binding pocket, and up to two fatty acids can bind to FABP1 simultaneously. We hypothesized that drug binding to hFABP1 results in formation of ternary complexes and that FABP1 binding alters drug metabolism. To test these hypotheses, native protein mass spectrometry (MS) and fluorescent 11-(dansylamino)undecanoic acid (DAUDA) displacement assays were used to characterize drug binding to hFABP1, and diclofenac oxidation by cytochrome P450 2C9 (CYP2C9) was studied in the presence and absence of hFABP1. DAUDA binding to hFABP1 involved high (Kd,1 = 0.2 µM) and low (Kd,2 > 10 µM) affinity binding sites. Nine drugs bound to hFABP1 with equilibrium dissociation constant (Kd) values ranging from 1 to 20 µM. None of the tested drugs completely displaced DAUDA from hFABP1, and fluorescence spectra showed evidence of ternary complex formation. Formation of DAUDA-hFABP1-diclofenac ternary complex was verified with native MS. Docking predicted diclofenac binding in the portal region of FABP1 with DAUDA in the binding cavity. The catalytic rate constant of diclofenac hydroxylation by CYP2C9 was decreased by ∼50% (P < 0.01) in the presence of FABP1. Together, these results suggest that drugs form ternary complexes with hFABP1 and that hFABP1 binding in the liver will alter drug metabolism and clearance. SIGNIFICANCE STATEMENT: Many commonly prescribed drugs bind fatty acid binding protein 1 (FABP1), forming ternary complexes with FABP1 and the fluorescent fatty acid 11-(dansylamino)undecanoic acid. These findings suggest that drugs will bind to apo-FABP1 and fatty acid-bound FABP1 in the human liver. The high expression of FABP1 in the liver, together with drug binding to FABP1, may alter drug disposition processes in vivo.

Metastability of Protein Solution Structures in the Absence of a Solvent: Rugged Energy Landscape and Glass-like Behavior.

Native ion mobility/mass spectrometry is well-poised to structurally screen proteomes but characterizes protein structures in the absence of a solvent. This raises long-standing unanswered questions about the biological significance of protein structures identified through ion mobility/mass spectrometry. Using newly developed computational and experimental ion mobility/ion mobility/mass spectrometry methods, we investigate the unfolding of the protein ubiquitin in a solvent-free environment. Our data suggest that the folded, solvent-free ubiquitin observed by ion mobility/mass spectrometry exists in a largely native fold with an intact ß-grasp motif and α-helix. The ensemble of folded, solvent-free ubiquitin ions can be partitioned into kinetically stable subpopulations that appear to correspond to the structural heterogeneity of ubiquitin in solution. Time-resolved ion mobility/ion mobility/mass spectrometry measurements show that folded, solvent-free ubiquitin exhibits a strongly stretched-exponential time dependence, which simulations trace to a rugged energy landscape with kinetic traps. Unfolding rate constants are estimated to be approximately 800 to 20,000 times smaller than in the presence of water, effectively quenching the unfolding process on the time scale of typical ion mobility/mass spectrometry measurements. Our proposed unfolding pathway of solvent-free ubiquitin shares substantial characteristics with that established for the presence of solvent, including a polarized transition state with significant native content in the N-terminal ß-hairpin and α-helix. Our experimental and computational data suggest that (1) the energy landscape governing the motions of folded, solvent-free proteins is rugged in analogy to that of glassy systems; (2) large-scale protein motions may at least partially be determined by the amino acid sequence of a polypeptide chain; and (3) solvent facilitates, rather than controls, protein motions.

Quantitatively Differentiating Antibodies Using Charge-State Manipulation, Collisional Activation, and Ion Mobility-Mass Spectrometry.

Antibody-based therapeutics continue to expand both in the number of products and in their use in patients. These heterogeneous proteins challenge traditional drug characterization strategies, but ion mobility (IM) and mass spectrometry (MS) approaches have eased the challenge of higher-order structural characterization. Energy-dependent IM-MS, e.g., collision-induced unfolding (CIU), has been demonstrated to be sensitive to subtle differences in structure. In this study, we combine a charge-reduction method, cation-to-anion proton-transfer reactions (CAPTR), with energy-dependent IM-MS and varied solution conditions to probe their combined effects on the gas-phase structures of IgG1κ and IgG4κ from human myeloma. CAPTR paired with MS-only analysis improves the confidence of charge-state assignments and the resolution of the interfering protein species. Collision cross-section distributions were determined for each of the charge-reduced products. Similarity scoring was used to quantitatively compare distributions determined from matched experiments analyzing samples of the two antibodies. Relative to workflows using energy-dependent IM-MS without charge-state manipulation, combining CAPTR and energy-dependent IM-MS enhanced the differentiation of these antibodies. Combined, these results indicate that CAPTR can benefit many aspects of antibody characterization and differentiation.

Are the Gas-Phase Structures of Molecular Elephants Enduring or Ephemeral? Results from Time-Dependent, Tandem Ion Mobility.

The structural stability of biomolecules in the gas phase remains an important topic in mass spectrometry applications for structural biology. Here, we evaluate the kinetic stability of native-like protein ions using time-dependent, tandem ion mobility (IM). In these tandem IM experiments, ions of interest are mobility-selected after a first dimension of IM and trapped for up to ∼14 s. Time-dependent, collision cross section distributions are then determined from separations in a second dimension of IM. In these experiments, monomeric protein ions exhibited structural changes specific to both protein and charge state, whereas large protein complexes did not undergo resolvable structural changes on the timescales of these experiments. We also performed energy-dependent experiments, i.e., collision-induced unfolding, as a comparison for time-dependent experiments to understand the extent of unfolding. Collision cross section values observed in energy-dependent experiments using high collision energies were significantly larger than those observed in time-dependent experiments, indicating that the structures observed in time-dependent experiments remain kinetically trapped and retain some memory of their solution-phase structure. Although structural evolution should be considered for highly charged, monomeric protein ions, these experiments demonstrate that higher-mass protein ions can have remarkable kinetic stability in the gas phase.

High-Precision, Gas-Phase Hydrogen/Deuterium-Exchange Kinetics by Mass Spectrometry Enabled by Exchange Standards.

Mass spectrometry (MS) has become a primary tool for identifying and quantifying biological molecules. In combination with other orthogonal techniques, such as gas-phase hydrogen/deuterium exchange (gHDX), MS is also capable of probing the structure of ions. However, gHDX kinetics can depend strongly on many factors, including laboratory temperature, instrumental conditions, and instrument platform selection. These effects can lead to high variability with gHDX measurements, which has hindered the broader adoption of gHDX for structural MS. Here we introduce an approach for standardizing gHDX measurements using cosampled standards. Quantifying the exchange kinetics for analytes relative to the exchange kinetics of the standards results in greater accuracy and precision than the underlying absolute measurements. The standardization was found to be effective for several types of analytes including small molecules and intact proteins. A subset of analytes showed deviations in their standardized exchange profiles that are attributed to field heating and the concomitant conformational isomerization. Inclusion of helium during the gHDX process for collisional cooling helps mitigate such variations in exchange kinetics related to ion heating. We anticipate that the outcomes of this research will enable the broader use of gHDX in MS-based workflows for molecular identification and isomer differentiation.

Recommendations for reporting ion mobility Mass Spectrometry measurements.

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.

Degronomics: Mapping the Interacting Peptidome of a Ubiquitin Ligase Using an Integrative Mass Spectrometry Strategy.

Human cells make use of hundreds of unique ubiquitin E3 ligases to ensure proteome fidelity and control cellular functions by promoting protein degradation. These processes require exquisite selectivity, but the individual roles of most E3s remain poorly characterized in part due to the challenges associated with identifying, quantifying, and validating substrates for each E3. We report an integrative mass spectrometry (MS) strategy for characterizing protein fragments that interact with KLHDC2, a human E3 that recognizes the extreme C-terminus of substrates. Using a combination of native MS, native top-down MS, MS of destabilized samples, and liquid chromatography MS, we identified and quantified a near complete fraction of the KLHDC2-binding peptidome in E. coli cells. This degronome includes peptides that originate from a variety of proteins. Although all identified protein fragments are terminated by diglycine or glycylalanine, the preceding amino acids are diverse. These results significantly expand our understanding of the sequences that can be recognized by KLHDC2, which provides insight into the potential substrates of this E3 in humans. We anticipate that this integrative MS strategy could be leveraged more broadly to characterize the degronomes of other E3 ligase substrate receptors, including those that adhere to the more common N-end rule for substrate recognition. Therefore, this work advances "degronomics," i.e., identifying, quantifying, and validating functional E3:peptide interactions in order to determine the individual roles of each E3.

SCF(FBXL3) ubiquitin ligase targets cryptochromes at their cofactor pocket.

The cryptochrome (CRY) flavoproteins act as blue-light receptors in plants and insects, but perform light-independent functions at the core of the mammalian circadian clock. To drive clock oscillations, mammalian CRYs associate with the Period proteins (PERs) and together inhibit the transcription of their own genes. The SCF(FBXL3) ubiquitin ligase complex controls this negative feedback loop by promoting CRY ubiquitination and degradation. However, the molecular mechanisms of their interactions and the functional role of flavin adenine dinucleotide (FAD) binding in CRYs remain poorly understood. Here we report crystal structures of mammalian CRY2 in its apo, FAD-bound and FBXL3-SKP1-complexed forms. Distinct from other cryptochromes of known structures, mammalian CRY2 binds FAD dynamically with an open cofactor pocket. Notably, the F-box protein FBXL3 captures CRY2 by simultaneously occupying its FAD-binding pocket with a conserved carboxy-terminal tail and burying its PER-binding interface. This novel F-box-protein-substrate bipartite interaction is susceptible to disruption by both FAD and PERs, suggesting a new avenue for pharmacological targeting of the complex and a multifaceted regulatory mechanism of CRY ubiquitination.

Structural characterization of small molecular ions by ion mobility mass spectrometry in nitrogen drift gas: improving the accuracy of trajectory method calculations.

The investigation of ion structures based on a combination of ion mobility mass spectrometry (IM-MS) experiments and theoretical collision cross section (CCS) calculations has become important to many fields of research. However, the accuracy of current CCS calculations for ions in nitrogen drift gas limits the information content of many experiments. In particular, few studies have evaluated and attempted to improve the theoretical tools for CCS calculation in nitrogen drift gas. In this study, based on high-quality experimental measurements and theoretical modeling, a comprehensive evaluation of various aspects of CCS calculations in nitrogen drift gas is performed. It is shown that the modification of the ion-nitrogen van der Waals (vdW) interaction potential enables accurate CCS predictions of 29 small ions with ca. 3% maximum relative error. The present method exhibits no apparent systematic bias with respect to ion CCS (size) and dipole moment, suggesting that the method adequately describes the long-range interactions between the ions and the buffer gas. However, the method shows limitations in reproducing experimental CCS at low temperatures (<150 K) and for macromolecular ions, and calculations for these cases should be complemented by CCS calculation methods in helium drift gas. This study presents an accurate and well-characterized CCS calculation method for ions in nitrogen drift gas that is expected to become an important tool for ion structural characterization and molecular identification. The experimental values reported here also provide a foundation for future studies aiming at developing more efficient computational tools.

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.

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.

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.

Structural Dynamics of Native-Like Ions in the Gas Phase: Results from Tandem Ion Mobility of Cytochrome c.

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 c 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.

Large-Scale Structural Characterization of Drug and Drug-Like Compounds by High-Throughput Ion Mobility-Mass Spectrometry.

Ion mobility-mass spectrometry (IM-MS) can provide orthogonal information, i.e., m/z and collision cross section (CCS), for the identification of drugs and drug metabolites. However, only a small number of CCS values are available for drugs, which limits the use of CCS as an identification parameter and the assessment of structure-function relationships of drugs using IM-MS. Here, we report the development of a rapid workflow for the measurement of CCS values of a large number of drug or drug-like molecules in nitrogen on the widely available traveling wave IM-MS (TWIM-MS) platform. Using a combination of small molecule and polypeptide CCS calibrants, we successfully determined the nitrogen CCS values of 1425 drug or drug-like molecules in the MicroSource Discovery Systems' Spectrum Collection using flow injection analysis of 384-well plates. Software was developed to streamline data extraction, processing, and calibration. We found that the overall drug collection covers a wide CCS range for the same mass, suggesting a large structural diversity of these drugs. However, individual drug classes appear to occupy a narrow and unique space in the CCS-mass 2D spectrum, suggesting a tight structure-function relationship for each class of drugs with a specific target. We observed bimodal distributions for several antibiotic species due to multiple protomers, including the known fluoroquinolone protomers and the new finding of cephalosporin protomers. Lastly, we demonstrated the utility of the high-throughput method and drug CCS database by quickly and confidently confirming the active component in a pharmaceutical product.

Collision cross sections and ion structures: development of a general calculation method via high-quality ion mobility measurements and theoretical modeling.

Ion mobility mass spectrometry (IM-MS) has become an important tool for the structural investigation of ions in the gas phase. Accurate theoretical evaluation of ion collision cross sections (CCSs) is essential for the effective application of IM-MS in structural studies. However, current theoretical tools have limitations in accurately describing a broad range of ions from small molecules to macromolecules. Significant difficulties in developing theoretical tools for CCS calculations are associated with obtaining high-quality experimental data and molecular models. In this study, we present a general CCS calculation method by employing two drift-tube IM-MS (DTIM-MS) instruments and thorough molecular modeling procedures. It is demonstrated that an appropriate description of the van der Waals (vdW) interactions is important for accurate CCS calculations in helium drift gas. By utilizing the vdW potentials from molecular mechanics force fields, it is shown that both the appropriate vdW potential-forms and their parameters are necessary for the highly reliable CCS predictions of small molecules. We further show that specific characteristics of the vdW interaction potential become less influential on the calculated CCS with increasing ion size, and that the calculated CCS values for the macromolecules converge to the values at the hard-sphere limit. Based on these results, a general CCS calculation method is presented that can be applied to ions of various sizes and compositions for the gas-phase structural studies.