Ion-neutral Collision Cross Sections Research Articles

General Method to Obtain Collision Cross-Section Values in Multipass High-Resolution Cyclic Ion Mobility Separations.

In recent years, ion mobility spectrometry-mass spectrometry (IMS-MS) has advanced the field of omics-based research, especially with the development of high-resolution platforms; however, these separations have generally been qualitative in nature. The rotationally averaged ion neutral collision cross section (CCS) is one of the only quantitative metrics available for aiding in characterizing biomolecules in IMS-MS. However, determining the CCS of an ion for multipass IMS systems, such as in cyclic ion mobility-mass spectrometry (cIMS-MS) and structures for lossless ion manipulations, has been challenging due to the lack of methods available for calculating CCS when more than a single pass is required for separation as well as the laborious nature of requiring calibrants and unknown compounds to be subjected to identical number of passes, which may not be possible in certain instances because of peak splitting, high levels of diffusion, etc. Herein, we present a general method that uses average ion velocities for calculating CCS values in cIMS-MS-based separations. Initially, we developed calibration curves using common CCS calibrants [i.e., tetra-alkylammonium salts, polyalanine, and hexakis(fluoroalkoxy)phosphazines] at different traveling wave (TW) conditions and the calculated cIMS CCS values were within ∼1% error or less compared to previously established drift tube IMS CCS measurements. Since it has been established that glycans can split into their α/β anomers, we utilized this method for two glycan species, 2α-mannobiose and melibiose. Both glycans were analyzed at the same TW conditions as the calibrants, and we observed anomer splitting at pathlengths of 20 m for 2α-mannobiose and 40 m for melibiose and thus assigned two unique CCS values for each glycan, which is the first time this has ever been done. We have demonstrated that the use of average ion velocities is a robust approach for obtaining CCS values with good agreement to CCS measurements from the previous literature and anticipate that this methodology can be applied to any IMS-MS platform that utilizes multipass separations. Our future work aims to incorporate this methodology for the development of a high-resolution CCS database to aid in the characterization of human milk oligosaccharides.

Ion-Neutral Collision Cross Section as a Function of the Static Dipole Polarizability and the Ionization Energy of the Ion.

Ion mobility spectrometry is becoming more and more popular as a fast, efficient, and sensitive tool for the separation and identification of ionized molecules in the gas phase. An ion traveling through a drift tube at atmospheric pressure under the influence of an electric field collides with the buffer gas molecules. The mobility of the ion depends inversely on the ion-neutral collision cross section. In the simplest hard-sphere approximation, the collision cross section is the area of the conventional geometric cross section. However, deviations are expected because of the physical interactions between the colliding species. More than a century ago, Langevin described a model for the interaction between a point-charge ion and a polarizable atom (molecule). Since then, the model has been modified many times to include better approximations of the interaction potential, usually preserving the point-charge nature of the ion. Although more advanced approaches allow for considering polarizable ions with dissimilar sizes and shapes, still explicit analytical dependencies on the properties of the ion remain elusive. In this work, an extended version of the Langevin model is proposed and solved using algebraic perturbation theory. A simple analytical expression of the collision cross section depending explicitly on both the static dipole polarizability and the ionization energy of the ion is found. The equation is validated using ion mobility data. Surprisingly, even low-level calculations of the polarizability tensors produce results that are consistent with the experimental observations. This fact makes the equation very attractive for helping applications in different areas, such as the deconvolution of mobilograms of protomers, ion-molecule chemical kinetics, and others.

Collision Cross-Section Measurements of Collision-Induced Dissociation Precursor and Product Ions in an FTICR-MS and an IM-MS: A Comparative Study.

Sustained off-resonance irradiation-cross-sectional areas by Fourier transform ion cyclotron resonance mass spectrometry (SORI-CRAFTI) is an FTICR-MS strategy to collisionally activate precursor ions and then measure their ion-neutral collision cross sections, as well as those of selected products, at the same time. We benchmarked SORI-CRAFTI using protonated leucine-enkephalin, to excellent agreement (typically within 1-2%) with previous studies performed via collision-induced dissociation-ion mobility (CID-IMS). SORI-CRAFTI was then applied to alkali metal-cationized leucine-enkephalin and compared with CID-IMS via precursor/product cross-section ratios. Qualitative agreement between SORI-CRAFTI and CID-IMS was excellent (again, usually within 1-2%); however, neither SORI-CRAFTI nor CID-IMS could determine if metalated leucine-enkephalin was present in its canonical or zwitterionic form. When SORI-CRAFTI was used on [2.2.2]-cryptand+Cs+, SORI activation resulted in a 5% decrease in collision cross section, consistent with migration of the externally bound Cs+ into the cryptand's cavity and similar to the cross section observed when electrospraying from an isopropanol-rich solvent. Thus, SORI-CRAFTI is useful for studying gas-phase ion chemistry of small- to medium-sized molecules and host-guest systems.

Determining Collision Cross Sections from Differential Ion Mobility Spectrometry.

The experimental determination of ion-neutral collision cross sections (CCSs) is generally confined to ion mobility spectrometry (IMS) technologies that operate under the so-called low-field limit or those that enable empirical calibration strategies (e.g., traveling wave IMS; TWIMS). Correlation of ion trajectories to CCS in other non-linear IMS techniques that employ dynamic electric fields, such as differential mobility spectrometry (DMS), has remained a challenge since its inception. Here, we describe how an ion's CCS can be measured from DMS experiments using a machine learning (ML)-based calibration. The differential mobility of 409 molecular cations (m/z: 86-683 Da and CCS 110-236 Å2) was measured in a N2 environment to train the ML framework. Several open-source ML routines were tested and trained using DMS-MS data in the form of the parent ion's m/z and the compensation voltage required for elution at specific separation voltages between 1500 and 4000 V. The best performing ML model, random forest regression, predicted CCSs with a mean absolute percent error of 2.6 ± 0.4% for analytes excluded from the training set (i.e., out-of-the-bag external validation). This accuracy approaches the inherent statistical error of ∼2.2% for the MobCal-MPI CCS calculations employed for training purposes and the <2% threshold for matching literature CCSs with those obtained on a TWIMS platform.

Reevaluating the Role of Polarizability in Ion Mobility Spectrometry

With the expanding commercial availability of gas-phase separation systems that incorporate gas-phase mobility, there is a concurrent rise in efforts to cast the gas-phase mobility coefficient in terms of an ion-neutral collision cross-section (CCS). The motivating factors for this trend are varied, but many aim to complement experimental results with computationally generated CCS values from in silico structural approximations. Unfortunately, the current paradigm for relating experimental mobility results to computationally derived structures relies upon empirical approaches, including a myriad of variables that do not realistically bound the comparison. In this Critical Insight, we advocate for the development of a self-consistent experimental and computational framework that uses laboratory results to constrain the scope of the modeling effort. This paper aims to prompt discussion, challenge assumptions, and promote the development of more efficient, accurate computational techniques within the gas-phase ion measurement community. Specifically, we postulate whether experimental deviations from Langevin's polarization limit (Kpol) are suitable to estimate the relative contributions of hard-sphere collisions and long-range interactions within CCS values. Not surprisingly, different molecule classes exhibit different trends in the K/Kpol ratio when normalized for reduced mass, and the most common IMS calibrants (e.g., tune mix, polyalanine, tetraalkylammonium salts) follow different polarizability trends than many of the analytes probed in the literature. Succinctly, if gas-phase ion structure is largely invariant based upon the colliding neutral and newly developed experimental efforts can quantitatively capture ion polarizability, then modeling efforts describing a target analyte must be self-consistent as the collision neutral is changed in silico.

Trapped Ion Mobility Spectrometry of Native Macromolecular Assemblies.

The structural elucidation of native macromolecular assemblies has been a subject of considerable interest in native mass spectrometry (MS), and more recently in tandem with ion mobility spectrometry (IMS-MS), for a better understanding of their biochemical and biophysical functions. In the present work, we describe a new generation trapped ion mobility spectrometer (TIMS), with extended mobility range (K0 = 0.185-1.84 cm2·V-1·s-1), capable of trapping high-molecular-weight (MW) macromolecular assemblies. This compact 4 cm long TIMS analyzer utilizes a convex electrode, quadrupolar geometry with increased pseudopotential penetration in the radial dimension, extending the mobility trapping to high-MW species under native state (i.e., lower charge states). The TIMS capabilities to perform variable scan rate (Sr) mobility measurements over short time (100-500 ms), high-mobility resolution, and ion-neutral collision cross-section (CCSN2) measurements are presented. The trapping capabilities of the convex electrode TIMS geometry and ease of operation over a wide gas flow, rf range, and electric field trapping range are illustrated for the first time using a comprehensive list of standards varying from CsI clusters (n = 6-73), Tuning Mix oligomers (n = 1-5), common proteins (e.g., ubiquitin, cytochrome C, lysozyme, concanavalin (n = 1-4), carbonic anhydrase, β clamp (n = 1-4), topoisomerase IB, bovine serum albumin (n = 1-3), topoisomerase IA, alcohol dehydrogenase), IgG antibody (e.g., avastin), protein-DNA complexes, and macromolecular assemblies (e.g., GroEL and RNA polymerase (n = 1-2)) covering a wide mass (up to m/z 19 000) and CCS range (up to 22 000 Å2 with <0.6% relative standard deviation (RSD)).

Ion Mobility Shift of Isotopologues in a High Kinetic Energy Ion Mobility Spectrometer (HiKE-IMS) at Elevated Effective Temperatures.

Ion mobility spectrometers (IMS) separate ions mainly by ion-neutral collision cross section and to a lesser extent by ion mass and effective temperature. When investigating isotopologues, the difference in collision cross section can be assumed negligible. Since the mobility shift of isotopologues is thus mainly caused by their difference in mass and effective temperature, the investigation of isotopologues can provide important insights into the theory of ion mobility. However, in classical IMS operated at ambient pressure, cluster formation with neutral molecules occurs, which significantly influences the mobility shift of isotopologues and thus makes a sound investigation of the effect of ion mass and effective temperature on the ion mobility difficult. In this work, the relative ion mobility of several organic compounds and their 13C-labeled isotopologues is studied in a High Kinetic Energy Ion Mobility Spectrometer (HiKE-IMS) at high reduced electric fields up to 120 Td, which allows the investigation of nonclustered ion species and thus enables a sound investigation of the mobility shift of isotopologues. The results show that the measured relative ion mobilities of isotopologues having the same effective temperature and, thus, their ion mass dominating the relative ion mobility agree well with theoretical relative ion mobilities predicted by the theory of ion mobility.

Evaluation of Trapped Ion Mobility Spectrometry Source Conditions Using Benzylammonium Thermometer Ions.

A key aspect of reduced pressure ion mobility spectrometry (IMS) experiments is to identify experimental conditions that minimize the role of collisional energy transfer that allows for assessing effective ion-neutral collision cross sections of metabolites, peptides, and proteins in "native-like" or compact states. Across two separate experimental campaigns using a prototype trapped ion mobility spectrometer (TIMS) coupled to a time-of-flight mass spectrometer, we present independent findings that support the results recently published by Morsa et al. using a different set of thermometer ions (Morsa et al. Anal. Chem. 2020, 92 (6), 4573-4582). First, using five para-substituted benzylammonium ions, we conducted survival yield experiments to assess ion internal energy across different experimental settings. Results from the present set of experiments illustrate that greater ion heating occurs at lower pressures and higher voltage settings applied to the TIMS. At the "softest" settings where the benzylammonium thermometer ions have an effective average energy of 1.73 eV, we observe the majority of bradykinin in the compact state. Under more extreme operating conditions where the energy of the benzylammonium ions varies from 1.83 to 1.86 eV, the bradykinin transitions from the compact to the elongated state. In addition to independently confirming the findings of Morsa et al., we also report the mobilities for the benzylammonium parent and fragment ions using the tandem drift-tube-TIMS calibration procedure described by Naylor et al. ( J. Am. Soc. Mass Spectrom. 2019, 30 (10), 2152-2162).

Enabling resolution of isomeric peptides using tri-state ion gating and Fourier-transform ion mobility spectrometry

Using Fourier-Transform ion gate modulation technique, we compare the ability of the tri-state ion shutter (3S-IS) to the two-state ion shutter (2S-IS) in separating three pairs of isomeric peptide including 1.Gly-Arg-Gly-Asp-Ser (GRGDS) / Ser-Asp-Gly-Arg-Gly (SDGRG); 2.Sar-Arg-Gly-Asp-Ser-Pro (SRGDSP) / Gly-Arg-Gly-Asp-Thr-Pro (GRGTP); 3.Kemptide / (Val6, Ala7)-Kemptide using electrospray ionization and ion mobility spectrometry. Mobility separation was evaluated for peptide individually and as simple mixtures. Baseline resolution of both singly and doubly charged ions of the isomeric pentapeptide mixture of GRGDS / SDGRG was attainable with the described IMS system using the 3S-IS configuration, illustrating the capacity of the present instrument to resolve isomeric compounds with differences in ion neutral collision cross section (CCS) of less than 1% for the singly charged ions. However, with the 2S-IS, both singly and doubly charged ions of the same peptide mixture were unresolved in the mobility domain. To our knowledge, this is the first-time baseline separation has been reported for the singly charged ions of the isomeric reversed sequence pentapeptide mixture using Fourier transformed drift tube IMS with nitrogen as the drift gas. For all the peptide mixtures, the ion counts for the ion mixture recorded with the 3S-IS were substantially higher (> 50%) in comparison to the 2S-IS. The resolving power of the instrument ranged between 82 to 128 for the target analyte ions analyzed in a mixture using the 3S-IS. Whereas, the resolving power of the 2S-IS ranged between 60 and 100 for the target analytes. Overall, a 20% increase in resolving power was obtained with the 3S-IS in comparison to the 2S-IS. Separation of the different isomeric peptide ion mixture depicted in this present study clearly shows the unique size-to-charge separation ability of IMS that complements the mass-to-charge ratio measurement capacity of mass spectrometry.

Breaking Down Structural Diversity for Comprehensive Prediction of Ion-Neutral Collision Cross Sections.

Identification of unknowns is a bottleneck for large-scale untargeted analyses like metabolomics or drug metabolite identification. Ion mobility-mass spectrometry (IM-MS) provides rapid two-dimensional separation of ions based on their mobility through a neutral buffer gas. The mobility of an ion is related to its collision cross section (CCS) with the buffer gas, a physical property that is determined by the size and shape of the ion. This structural dependency makes CCS a promising characteristic for compound identification, but this utility is limited by the availability of high-quality reference CCS values. CCS prediction using machine learning (ML) has recently shown promise in the field, but accurate and broadly applicable models are still lacking. Here we present a novel ML approach that employs a comprehensive collection of CCS values covering a wide range of chemical space. Using this diverse database, we identified the structural characteristics, represented by molecular quantum numbers (MQNs), that contribute to variance in CCS and assessed the performance of a variety of ML algorithms in predicting CCS. We found that by breaking down the chemical structural diversity using unsupervised clustering based on the MQNs, specific and accurate prediction models for each cluster can be trained, which showed superior performance than a single model trained with all data. Using this approach, we have robustly trained and characterized a CCS prediction model with high accuracy on diverse chemical structures. An all-in-one web interface (https://CCSbase.net) was built for querying the CCS database and accessing the predictive model to support unknown compound identifications.

Assessing the Impact of Drift Gas Polarizability in Polyatomic Ion Mobility Experiments.

Due to the core assumptions of kinetic theory and the drive toward realizing reproducible gas-phase measurements, ion mobility experiments are commonly conducted in the presence of an inert, neat buffer gas, usually nitrogen or helium. Mixing drift gases in defined, static ratios can provide useful information not only for optimizing the separation of analytes but also for defining the interaction between the ion and neutral particle. In a foundational effort, we seek to validate the role of the drift gas polarizability on the observed mobility of the ions by systematically mixing drift gases to discretely access a range of bulk gas polarizabilities not given by pure drift gases. Compared to historical efforts to probe the role of polarizability on the ion-neutral collisional cross section where a linear relationship was assumed, the data collected in the present effort clearly illustrate a quadratic dependency of the ion-neutral particle collision cross section and polarizability (R2 > 0.999). When translating these data into the mobility dimension, we illustrate that the gas-phase mobility of polyatomic ions conforms to Blanc's law. These observations combined with considerations related to Langevin's polarization limit provide an experimental mechanism to estimate to what degree an ion-neutral interaction conforms to either the hard-sphere or induced-dipole model. To support these observations, additional comparisons are made with the respective reduced masses, polarizabilities, and mobilities of ions in mixtures where different degrees of hard-sphere interactions are present.

Liquid chromatography-ion mobility spectrometry-mass spectrometry analysis of multiple classes of steroid hormone isomers in a mixture

Methods for the analysis of steroids have long been of interest due to the multiple uses for such methods in medical applications, sports monitoring, and environmental science. The analysis of steroids involves inherent analytical hurdles due to their low biological concentrations, poor ionization efficiencies, and frequent occurrence of isomerism. One analytical technique that has been recently applied to steroid analysis is ion mobility spectrometry (IMS). While previous work has focused on the use of metal adduction and multimer formation to enhance separation through IMS analysis coupled to mass spectrometry (MS), this work furthers this approach by coupling IMS-MS with liquid chromatography (LC). Three different LC methods with varying tradeoffs between chromatographic resolution and run time were developed, with one of these achieving a resolution above 1.5 for all steroid isomers. These results also indicate that the coupling of LC to IMS-MS can increase the overall resolution of steroid isomers relative to what can be achieved by either LC or IMS alone. Furthermore, the use of LC and IMS in concert can allow for a more rapid analysis of steroid isomers than can be achieved by LC-MS alone. Finally, the IMS dimension provided for measurements of ion-neutral collision cross sections (CCSs), which were found to be in good agreement with previously reported measurements. Thus, this approach provides three complementary quantitative parameters (retention time, CCS, and mass-to-charge ratio) that can contribute the identification of analytes. Overall, the work presented here demonstrates the potential of coupling LC, IMS, and MS for the analysis of isomeric steroid hormones.

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.

Composition and charge state influence on the ion-neutral collision cross sections of protonated N-linked glycopeptides: an experimental and theoretical deconstruction of coulombic repulsion vs. charge solvation effects.

Ion mobility spectrometry (IMS) is of significant interest as a platform for glycoanalysis. While much attention has been focused on the resolution of isomeric carbohydrates and glycoconjugates, another appealing aspect of IMS is the ability to sort different classes of biomolecules into distinct regions of mass vs. mobility space. This capability has potential to greatly simplify glycoproteomic analyses, as glycosylated and non-glycosylated peptides can be rapidly partitioned in the gas phase. Nevertheless, the physical and chemical characteristics of glycopeptides that dictate their mass vs. mobility loci have yet to be systematically investigated. This report presents an IMS study of model protonated glycopeptide ions with systematically varied oligosaccharide topologies, polypeptide sequences, and charge states. In all, over 110 ion-neutral collision cross sections (CCSs) were measured and analyzed in the context of the physicochemical characteristics of the analytes. Glycan size and composition emerged as a decisive factor in dictating the CCS space occupied by the glycopeptides and exerted this influence in a charge state dependent fashion. Furthermore, elongation of the glycan group was found to either increase or decrease glycopeptide CCSs depending on the ion charge state and the size of the glycan. Molecular dynamics (MD) simulations of the gas phase structures and CCSs of selected glycopeptides revealed that the experimental observations were consistent with a glycan size and charge state dependent interplay between destabilizing coulombic repulsion effects (tending to result in more extended structures) and stabilizing charge solvation effects in which the glycan plays a major role (tending to result in more compact structures). Taken together, these IMS and MD findings suggest the possibility of predicting and delineating glycopeptide-enriched regions of mass vs. mobility space for applications in glycoproteomics.

Application of Group I Metal Adduction to the Separation of Steroids by Traveling Wave Ion Mobility Spectrometry.

Steroids represent an interesting class of small biomolecules due to their use as biomarkers and their status as scheduled drugs. Although the analysis of steroids is complicated by the potential for many isomers, ion mobility spectrometry (IMS) has previously shown promise for the rapid separation of steroid isomers. This work is aimed at the further development of IMS separation for the analysis of steroids. Here, traveling wave ion mobility spectrometry (TWIMS) was applied to the study of group I metal adducted steroids and their corresponding multimers for five sets of isomers. Each set of isomers had a minimum of one dimeric metal ion adduct that exhibited a resolution greater than one (i.e., approaching baseline resolution). Additionally, ion-neutral collision cross sections (CCSs) were measured using polyalanine as a calibrant, which may provide an additional metric contributing to analyte identification. Where possible, measured CCSs were compared to previously reported values. When measuring CCSs of steroid isomers using polyalanine as the calibrant, nitrogen CCS values were within 1.0% error for monomeric sodiated adducts and slightly higher for the dimeric sodiated adducts. Overall, TWIMS was found to successfully separate steroids as dimeric adducts of group I metals. Graphical Abstract ᅟ.

Fourier Transform-Ion Mobility-Orbitrap Mass Spectrometer: A Next-Generation Instrument for Native Mass Spectrometry.

A new instrument configuration for native ion mobility-mass spectrometry (IM-MS) is described. Macromolecule ions are generated by using a static ESI source coupled to an RF ion funnel, and these ions are then mobility and mass analyzed using a periodic focusing drift tube IM analyzer and an Orbitrap mass spectrometer. The instrument design retains the capabilities for first-principles determination of rotationally averaged ion-neutral collision cross sections and high-resolution measurements in both mobility and mass analysis modes for intact protein complexes. Operation in the IM mode utilizes FT-IMS modes (originally described by Knorr ( Knorr , F. J. Anal. Chem . 1985 , 57 ( 2 ), 402 - 406 )), which provides a means to overcome the inherent duty cycle mismatch for drift tube (DT)-IM and Orbitrap mass analysis. The performance of the native ESI-FT-DT-IM-Orbitrap MS instrument was evaluated using the protein complexes Gln K (MW 44 kDa) and streptavidin (MW 53 kDa) bound to small molecules (ADP and biotin, respectively) and transthyretin (MW 56 kDa) bound to thyroxine and zinc.

Characterization of Intramolecular Interactions of Cytochrome c Using Hydrogen-Deuterium Exchange-Trapped Ion Mobility Spectrometry-Mass Spectrometry and Molecular Dynamics.

Globular proteins, such as cytochrome c (cyt c), display an organized native conformation, maintained by a hydrogen bond interaction network. In the present work, the structural interrogation of kinetically trapped intermediates of cyt c was performed by correlating the ion-neutral collision cross section (CCS) and charge state with the starting solution conditions and time after desolvation using collision induced activation (CIA), time-resolved hydrogen/deuterium back exchange (HDX) and trapped ion mobility spectrometry-mass spectrometry (TIMS-MS). The high ion mobility resolving power of the TIMS analyzer allowed the identification of new ion mobility bands, yielding a total of 63 mobility bands over the +6 to +21 charge states and 20 mobility bands over the -5 to -10 charge states. Mobility selected HDX rates showed that for the same charge state, conformers with larger CCS present faster HDX rates in both positive and negative ion mode, suggesting that the charge sites and neighboring exchange sites on the accessible surface area define the exchange rate regardless of the charge state. Complementary molecular dynamic simulations permitted the generation of candidate structures and a mechanistic model of the folding transitions from native (N) to molten globule (MG) to kinetic intermediates (U) pathways. Our results suggest that cyt c major structural unfolding is associated with the distancing of the N- and C-terminal helices and subsequent solvent exposure of the hydrophobic, heme-containing cavity.

Separation of Isotopologues in Ultra-High-Resolution Ion Mobility Spectrometry.

Ion mobility spectrometry provides ion separation in the gas phase mainly based on differing ion-neutral collision cross sections, enabling powerful analysis of many isomers. However, the separation also has a miniscule mass dependence due to the acceleration and collision properties. In this work, we show for the first time that using a compact ultra-high-resolution ion mobility spectrometer with a resolving power of 250 and an UV ionization source enables the separation of isotopologues with ion mobility spectrometry. This is demonstrated for regular and perdeuterated acetone, benzene, and toluene as well as toluene-13C7 in nitrogen and in purified air as drift gas. The observed peak shifts in the ion mobility spectrum agree with the basic ion mobility equation when using nitrogen as drift gas and also agree with a combination of this equation with Blanc's law when using purified air as drift gas. For benzene and toluene, a reduction in the ion-neutral collision cross section of the isotopically replaced species is observed. Furthermore, a third peak formed from regular and perdeuterated acetone is observed, which can most likely be attributed to the exchange of a methyl group.

Oversampling Selective Accumulation Trapped Ion Mobility Spectrometry Coupled to FT-ICR MS: Fundamentals and Applications.

In the present paper, we describe the fundamentals and analytical advantages of Oversampling Selective Accumulation Trapped Ion Mobility Spectrometry (OSA-TIMS) when coupled to ultrahigh resolution mass analyzers (e.g., FT-ICR MS). During TIMS analysis, ion packages are spatially resolved based on their mobilities along the TIMS analyzer axis and multiple strategies can be utilized during the trapping and elution of the ion population of interest. In the case of OSA-TIMS-FT-ICR MS, the TIMS operation sequence, trapping conditions, and operations are optimized to increase the signal-to-noise and the number of points across the mobility domain, which leads to more accurate mobility and mass measurements. Experimental results show that accurate ion-neutral collision cross sections (<1%) can be measured using OSA-TIMS-FT-ICR MS with high mobility resolving powers (RIMS up to 250), high mass accuracy (<1 ppm), and ultrahigh mass resolution (RMS up to 600-1200k at m/z 400) in a single analysis. The analytical advantages of OSA-TIMS over SA-TIMS were illustrated for the analysis of structural peptide isomers (SDGRG and GRGDS [M + H](+)), conformational isomers (AT-hook peptide 3 KRGRGRPRK [M + 2H](+2)), and a complex mixture of polyaromatic hydrocarbons (PAH) from coal tar. Baseline separation of the structural peptide isomers SDGRG and GRGDS, [M + H](+), was observed, and three conformations were identified for the AT-hook peptide 3 KRGRGRPRK [M + 2H](+2) during OSA-TIMS-FT-ICR MS. A 2-fold increase in the number of molecular features and a 2-6-fold signal-to-noise increase was observed for OSA-TIMS when compared with SA-TIMS during the PAH analysis. This work provides the proof-of-principle for further application of OSA-TIMS-FT-ICR MS for the unsupervised analysis of complex mixtures based on the characterization of the conformational space and the assignment of chemical formulas in a single analysis.

Isomer Separation of Polybrominated Diphenyl Ether Metabolites using nanoESI-TIMS-MS.

In this paper, high-resolution nano-electrospray ionization-trapped ion mobility spectrometry coupled to mass spectrometry (nESI-TIMS-MS) is used for the study of hydroxylated polybrominated diphenyl ether (OH-PBDE) metabolites. In particular, experimental ion-neutral collision cross sections (CCS) were measured for five structural OH-PBDE isomers using TIMS-MS. Candidate structures were proposed for each IMS band observed in good agreement with the experimental CCS measurements (5% error). The analytical power of TIMS-MS to baseline and partially separate structural isomers of OH-BDE in binary and ternary mixtures is shown for single charge species with a mobility resolving power of RIMS ~ 400. This work provides the proof of concept for the analysis of low concentration OH-PBDE in environmental samples based on accurate collision cross section and mass measurements without the need for derivatization and pre-fractionation protocols, thus significantly reducing the cost and analysis time.