High-resolution ion mobility separations coupled to mass spectrometry: What's next?

Herein, I provide a personal perspective on high-resolution multipass ion mobility spectrometry-mass spectrometry (IMS-MS), with a specific emphasis on cyclic (cIMS) and structures for lossless ion manipulations (SLIM IMS)-based separations. My overarching goal for this perspective was to detail what I believe will be the key important areas in which IMS-MS will help shape the bioanalytical community and especially omics-based research.

Isolating the Contributions from Moments of Inertia in Isotopic Shifts Measured by High-Resolution Cyclic Ion Mobility Separations.

The unexpected finding that isotopomers (i.e., isotopic isomers) can be separated with high-resolution ion mobility spectrometry-mass spectrometry (IMS-MS) has raised new structural considerations affecting an ion's mobility, namely its center of mass (CoM) and moments of inertia (MoI). Unfortunately, thus far, no studies have attempted to experimentally isolate either CoM or MoI, as they are intrinsically linked by their definitions, where MoI is calculated in relation to CoM. In this study, we designed and synthesized four isotopically labeled tetrapropylammonium (TAA3) ions, each with a unique mass distribution. Three of the synthesized TAA3 ions were labeled symmetrically, thus having identical CoM but differing MoI, which we verified using density functional theory (DFT) calculations. Consequently, we were able to isolate the effect of MoI changes in high-resolution IMS-MS separations. Cyclic ion mobility spectrometry-mass spectrometry (cIMS-MS) separations of the isotopically labeled TAA3 variants revealed isotopic mobility shifts attributable solely to changes in MoI. A 60-m cIMS-MS separation demonstrated that two nominally isobaric TAA3 pseudoisotopomers could be partially resolved, showcasing potential feasibility for isotopomer separations on commercially available IMS-MS platforms. With our previously established collision cross section (CCS) calibration protocol, we also quantified the relationship between MoI and CCS. Our results represent the first demonstration of IMS-MS separations based solely on MoI differences. We believe these findings will contribute important evidence to the growing body of literature on the physical nature of isotopic shifts in IMS-MS separations and work toward more accurate CCS predictions.

Multiplatform High-Definition Ion Mobility Separations of the Largest Epimeric Peptides.

Ion mobility spectrometry (IMS) coupled to mass spectrometry (MS) has become a versatile tool to fractionate complex mixtures, distinguish structural isomers, and elucidate molecular geometries. Along with the whole MS field, IMS/MS advances to ever larger species. A topical proteomic problem is the discovery and characterization of d-amino acid-containing peptides (DAACPs) that are critical to neurotransmission and toxicology. Both linear IMS and FAIMS previously disentangled d/l epimers with up to ∼30 residues. In the first study using all three most powerful IMS methodologies─trapped IMS, cyclic IMS, and FAIMS─we demonstrate baseline resolution of the largest known d/l peptides (CHH from Homarus americanus with 72 residues) with a dynamic range up to 100. This expands FAIMS analyses of isomeric modified peptides, especially using hydrogen-rich buffers, to the ∼50-100 residue range of small proteins. The spectra for d and l are unprecedentedly strikingly similar except for a uniform shift of the separation parameter, indicating the conserved epimer-specific structural elements across multiple charge states and conformers. As the interepimer resolution tracks the average for smaller DAACPs, the IMS approaches could help search for yet larger DAACPs. The a priori method to calibrate cyclic (including multipass) IMS developed here may be broadly useful.

Nonthreaded Isomers of Sungsanpin and Ulleungdin Lasso Peptides Inhibit H1299 Cancer Cell Migration.

Lasso peptides are a structurally distinct class of biologically active natural products defined by their short sequences with impressively interlocked tertiary structures. Their characteristic peptide [1]rotaxane motif confers marked proteolytic and thermal resiliency, and reports on their diverse biological functions have been credited to their exceptional sequence variability. Because of these unique properties, taken together with improved technologies for their biosynthetic production, lasso peptides are emerging as a designable scaffold for peptide-based therapeutic discovery and development. Although the defined structure of lasso peptides is recognized for its remarkable properties, the role of the motif in imparting bioactivity is less understood. For example, sungsanpin and ulleungdin are natural lasso peptides that similarly exhibit encouraging cell migration inhibitory activities in A549 lung carcinoma epithelial cells, despite sharing only one-third of the sequence homology. We hypothesized that the shape of the lasso motif is beneficial for the preorganization of the conserved residues, which might be partially retained in variants lacking the threaded structure. Herein, we describe solid-phase peptide synthesis strategies to prepare acyclic, head-to-side chain (branched), and head-to-tail (macrocyclic) cyclic variants based on the sungsanpin (Sun) and ulleungdin (Uln) sequences. Proliferation assays and time-lapse cell motility imaging studies were used to evaluate the cell inhibitory properties of natural Sun compared with the synthetic Sun and Uln isomers. These studies demonstrate that the lasso motif is not a required feature to slow cancer cell migration and more generally show that these nonthreaded isomers can retain similar activity to the natural lasso peptide despite the differences in their overall structures.

Implementation of charged microdroplet-based derivatization of bile acids on a cyclic ion mobility spectrometry-mass spectrometry platform.

Herein, we report the first implementation of charged microdroplet-based derivatization on a commercially-available cyclic ion mobility spectrometry-mass spectrometry platform. We have demonstrated the potential of our approach to improve separability of challenging isomers, but more importantly to rapidly screen derivatization reactions through droplet chemistry. Additionally, the use of cyclic ion mobility separations and tandem mass spectrometry reveals insights into product formation that would be lost with single stage mass spectrometry. Overall, we anticipate broad utility of our methodology owing to the simple design and setup for performing these droplet-based reactions and future work coupling these reactions online with liquid chromatography.

Coupling Isotopic Shifts with Collision Cross-Section Measurements for Carbohydrate Characterization in High-Resolution Ion Mobility Separations.

Herein, we introduce a two-dimensional strategy to better characterize carbohydrate isomers. In a single experiment, we can derive cyclic ion mobility-mass spectrometry (cIMS-MS)-based collision cross-section (CCS) values in conjunction with measuring isotopic shifts through the relative arrival times of light and heavy isotopologues. These isotopic shifts were introduced by permethylating carbohydrates with either light, CH3, or heavy, CD3, labels at every available hydroxyl group to generate a light/heavy pair of isotopologues for every individual species analyzed. We observed that our calculated CCS values, which were exclusively measured for the light isotopologues, were orthogonal to our measured isotopic shifts (i.e., relative arrival time values between heavy and light permethylated isotopologues). Our permethylation-induced isotopic shifts scaled well with increasing molecular weight, up to ∼m/z 1300, expanding the analysis of isotopic shifts to molecules 3-4 times as large as those previously studied. Our presented use of coupling CCS values with the measurement of isotopic shifts in a single cIMS-MS experiment is a proof-of-concept demonstration that our two-dimensional approach can improve the characterization of challenging isomeric carbohydrates. We envision that our presented 2D approach will have broad utility for varying molecular classes as well as being amenable to many forms of derivatization.

Permethylation and Metal Adduction: A Toolbox for the Improved Characterization of Glycolipids with Cyclic Ion Mobility Separations Coupled to Mass Spectrometry.

Lipids are an important class of molecules involved in various biological functions but remain difficult to characterize through mass-spectrometry-based methods because of their many possible isomers. Glycolipids, specifically, play important roles in cell signaling but display an even greater level of isomeric heterogeneity as compared to other lipid classes stemming from the introduction of a carbohydrate and its corresponding linkage position and α/ß anomericity at the headgroup. While liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) remains the gold standard technique in lipidomics, it is still unable to characterize all isomeric species, thus presenting the need for new, orthogonal, methodologies. Ion mobility spectrometry-mass spectrometry (IMS-MS) can provide an additional dimension of information that supplements LC-MS/MS workflows, but has seen little use for glycolipid analyses. Herein, we present an analytical toolbox that enables the characterization of various glycolipid isomer sets using high-resolution cyclic ion mobility separations coupled with mass spectrometry (cIMS-MS). Specifically, we utilized a combination of both permethylation and metal adduction to fully resolve isomeric sphingolipids and ceramides with our cIMS-MS platform. We also introduce a new metric that can enable comparing peak-to-peak resolution across varying cIMS-MS pathlengths. Overall, we envision that our presented methodologies are highly amenable to existing LC-MS/MS-based workflows and can also have broad utility toward other omics-based analyses.

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/ß 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.

Hydrogen-Deuterium-Exchange-Based Mass Distribution Shifts in High-Resolution Cyclic Ion Mobility Separations.

The mass distribution of ions influences separations in ion mobility spectrometry-mass spectrometry (IMS-MS). Herein, we introduce a method to induce mass distribution shifts for various analytes using hydrogen-deuterium exchange (HDX) immediately prior to ionization using a dual syringe approach. By replacing labile hydrogens on analytes with deuteriums, we were able to differentiate isomers using separations of isotopologues. For each analyte studied, every possible level of deuteration (from undeuterated to fully deuterated) was generated and then separated using cyclic ion mobility spectrometry-mass spectrometry (cIMS-MS). The information gained from such separations (relative arrival times; tRel. values) was found to be orthogonal to conventional IMS-MS separations. Additionally, the observed shifts were linearly additive with increasing deuteration, suggesting that this methodology could be extended to analytes with a larger number of labile hydrogens. For one isomer pair, as few as two deuteriums were able to produce a large enough mass distribution shift to differentiate isomers. In another experiment, we found that the mass distribution shift was large enough to overcome the reduced mass contribution, resulting in a "flipped" arrival time where the heavier deuterated isotopologue arrived before the lighter one. In this work, we present a proof-of-concept demonstration that mass-distribution-based shifts, tRel. values, could potentially act as an added dimension to characterize molecules in IMS-MS. We anticipate, along with future work in this area, that mass-distribution-based shifts could enable the identification of unknown molecules through a database-driven approach in an analogous fashion to collision cross section (CCS) measurements.

Assessing the Use of Host-Guest Chemistry in Conjunction with Cyclic Ion Mobility Separations for the Linkage-Specific Characterization of Human Milk Oligosaccharides.

Human milk oligosaccharides (HMOs) are a class of glycans that are highly abundant in human milk and contribute to the healthy growth of an infant's immune system. While new advancements in analytical methodologies have been made in glycomics, the high degree of isomeric heterogeneity and lack of authentic standards have made the high-resolution separation and accurate characterization of linkage positioning of all HMO species very challenging. Herein, we present an evaluation of the use of host-guest chemistry in conjunction with cyclic ion mobility spectrometry-mass spectrometry (cIMS-MS)-based separations for the identification of linkage positioning in three pairs of di-, tetra-, and hexasaccharide HMO isomers that only differ in the positioning of one glycosidic linkage (ß1,3 versus ß1,4). Suitable hosts, such as α/ß cyclodextrins, cucurbit[n]urils (n = 5, 7), crown ethers, cyclic peptides, and an ionophore, were used to assess host-guest inclusion complex formation as well as linkage-specific cIMS-MS trends. Our results indicated a linkage-specific trend for the [M + 2α + 2H]2+ cyclodextrin-based host-guest inclusion complexes where the ß1,3 linkage-containing isomers were always higher mobility than the ß1,4 linkage-containing ones as well one for the [M + α + ß + 2H]2+ complexes where the ß1,4 linkage-containing isomers were always higher mobility than the ß1,3 linkage-containing ones. We also observed diagnostic mobility fingerprints for the cucurbituril-based complexes. We anticipate that linkage-specific and mobility fingerprint trends can potentially aid in identifying linkage positioning for other HMO isomers as well as in complex human milk samples.

Isomer and Conformer-Specific Mass Distribution-Based Isotopic Shifts in High-Resolution Cyclic Ion Mobility Separations.

Herein, we present the use of mass distribution-based isotopic shifts in high-resolution cyclic ion mobility spectrometry-mass spectrometry (cIMS-MS)-based separations to characterize various isomeric species as well as conformers. Specifically, by using the observed relative arrival time values for the isotopologues found in the isotopic envelope after long pathlength cIMS-MS separations, we were able to distinguish dibromoaniline, dichloroaniline, and quaternary ammonium salt isomers, as well as a pair of 25-hydroxyvitamin D3 conformers based on their respective mass distribution-based shifts. Our observed shifts were highly reproducible and broadly applied to the isotopologues of various atoms (i.e., Cl, Br, and C). Additionally, through a control experiment, we determined that such shifts are indeed pathlength-independent, thus demonstrating that our presented methodology could be readily extended to other high-resolution IMS-MS platforms. These results are the first characterization of conformers using mass distribution-based IMS-MS shifts, as well as the first use of a commercial cIMS-MS platform to characterize isomers via their mass distribution-based shifts. We anticipate that our methodology will have broad applicability for biological analytes and that mass distribution-based shifts could potentially act as an added dimension of analysis in existing IMS-MS workflows in omics-based research. Specifically, we envision that the development of a database of these mass distribution-based shifts could, for example, enable the identification of unknown metabolites in complex matrices.

Experimental Measurements of Relative Mobility Shifts Resulting from Isotopic Substitutions with High-Resolution Cyclic Ion Mobility Separations.

Herein, we report on the experimental measurements for estimated relative mobility shifts caused by changes in mass distribution from isotopic substitutions in isotopologues and isotopomers with high-resolution cyclic ion mobility separations. By utilizing unlabeled and fully labeled isotopologues with the same isotopic substitutions (i.e., 2H or 13C), we created a highly precise mobility scale for each set analyzed to determine the magnitude of such mass distribution shifts and thus calculate estimated deviations from expected, theoretical reduced mass contributions. We observed relative mobility shifts in various isotopologues (e.g., hexadecyltrimethylammonium, sucrose, and palmitic acid species) that deviated from reduced mass theory, according to the Mason-Schamp relationship, ranging in estimated magnitude from ∼0.007% up to ∼0.1% in relative mobility. More interestingly, it was found that two deuterated palmitic acid isotopomers also differed by ∼0.03% from one another in their respective relative mobility shifts. Our results are the first report of isotopologue and isotopomer separations on a commercially available cyclic ion mobility spectrometry-mass spectrometry platform. We envision that our presented mobility scale methodology will have broad applicability in studying the effect of mass distribution changes from isotopic substitutions in other biomolecules and help pave the way for the improvement of ion mobility theory and collision cross section calculators.

Implementation of Ion Mobility Spectrometry-Based Separations in Structures for Lossless Ion Manipulations (SLIM).

Structures for Lossless Ion Manipulations (SLIM) is a powerful variant of traveling wave ion mobility spectrometry (TW-IMS) that uses a serpentine pattern of microelectrodes deposited onto printed circuit boards to achieve ultralong ion path lengths (13.5 m). Ions are propelled through SLIM platforms via arrays of TW electrodes while RF and DC electrodes provide radial confinement, establishing near lossless transmission. The recent ability to cycle ions multiple times through a SLIM has allowed ion path lengths to exceed 1000 m, providing unprecedented separation power and the ability to observe ion structural conformations unobtainable with other IMS technologies. The combination of high separation power, high signal intensity, and the ability to couple with mass spectrometry places SLIM in the unique position of being able to address longstanding proteomics and metabolomics challenges by allowing the characterization of isomeric mixtures containing low abundance analytes.

Evaluating the Utility of Temporal Compression in High-Resolution Traveling Wave-Based Cyclic Ion Mobility Separations.

Ion mobility spectrometry coupled to mass spectrometry (IMS-MS) is slowly becoming a more integral part in omics-based workflows. With the recent technological advancements in IMS-MS instrumentation, particularly those involving traveling wave-based separations, ultralong pathlengths have become readily available in commercial platforms (e.g., Select Series Cyclic IMS from Waters Corporation and MOBIE from MOBILion). However, a tradeoff exists in such ultralong pathlength separations: increasing peak-to-peak resolution at the cost of lower signal intensities and thus poorer sensitivity of measurements. Herein, we explore the utility of temporal compression, where ions are compressed in the time domain, following high-resolution cyclic ion mobility spectrometry-mass spectrometry-based separations on a commercially available, unmodified platform. We assessed temporal compression in the context of various separations including those of reverse sequence peptide isomers, chiral noncovalent complexes, and isotopologues. From our results, we demonstrated that temporal compression improves IMS peak intensities by up to a factor of 4 while only losing ∼5 to 10% of peak-to-peak resolution. Additionally, the improvement in peak quality and signal-to-noise ratio was evident when comparing IMS-MS separations with and without a temporal compression step performed. Temporal compression can readily be implemented in existing traveling wave-based IMS-MS platforms, and our initial proof-of-concept demonstration shows its promise as a tool for improving peak shapes and peak intensities without sacrificing losses in resolution.

Measurement and Theory of Gas-Phase Ion Mobility Shifts Resulting from Isotopomer Mass Distribution Changes.

The unanticipated discovery of recent ultra-high-resolution ion mobility spectrometry (IMS) measurements revealing that isotopomers─compounds that differ only in the isotopic substitution sites─can be separated has raised questions as to the physical basis for their separation. A study comparing IMS separations for two isotopomer sets in conjunction with theory and simulations accounting for ion rotational effects provides the first-ever prediction of rotation-mediated shifts. The simulations produce observable mobility shifts due to differences in gas-ion collision frequency and translational-to-rotational energy transfer. These differences can be attributed to distinct changes in the moment of inertia and center of mass between isotopomers. The simulations are in broad agreement with the observed experiments and consistent with relative mobility differences between isotopomers. These results provide a basis for refining IMS theory and a new foundation to obtain additional structural insights through IMS.

Investigating the Structure of α/ß Carbohydrate Linkage Isomers as a Function of Group I Metal Adduction and Degree of Polymerization as Revealed by Cyclic Ion Mobility Separations.

In high-resolution ion mobility spectrometry-mass spectrometry (IMS-MS)-based separations individual, pure, oligosaccharide species often produce multiple IMS peaks presumably from their α/ß anomers, cation attachment site conformations, and/or other energetically favorable structures. Herein, the use of high-resolution traveling wave-based cyclic IMS-MS to systematically investigate the origin of these multiple peaks by analyzing α1,4- and ß1,4-linked d-glucose homopolymers as a function of their group I metal adducts is presented. Across varying degrees of polymerization, and for certain metal adducts, at least two major IMS peaks with relative areas that matched the ∼40:60 ratio for the α/ß anomers of a reducing-end d-glucose as previously calculated by NMR were observed. To further validate that these were indeed the α/ß anomers, rather than other substructures, the reduced versions of several maltooligosaccharides were analyzed and all produced a single IMS peak. This result enabled the discovery of a mobility fingerprint trend: the ß anomer was always higher mobility than the α anomer for the cellooligosaccharides, while the α anomer was always higher mobility than the ß anomer for the maltooligosaccharides. For maltohexaose, a spurious, high mobility, fourth peak was present. This was hypothesized to potentially be from a highly compacted conformation. To investigate this, α-cyclodextrin, a cyclic oligosaccharide, produced similar arrival times as the high mobility maltohexaose peak. It is anticipated that these findings will aid in the data deconvolution of IMS-MS-based glycomics workflows and enable the improved characterization of biologically relevant carbohydrates.

Toward Sequencing the Human Milk Glycome: High-Resolution Cyclic Ion Mobility Separations of Core Human Milk Oligosaccharide Building Blocks.

Human milk oligosaccharides (HMOs) are an unconjugated class of glycans that have been implicated for their role in promoting the healthy development of the brain-gut axes of infants. Production of HMOs is ever-changing and specifically tailored for each infant in response to various biological factors (e.g., cognitive development, diseases, or allergies). While every HMO consists of up to only five monosaccharides, their structures can be composed of many possible glycosidic linkage positions and corresponding α/ß anomericities, linear or branched chains, and potential fucosylation/sialylation modifications, thus leading to a tremendous degree of isomeric heterogeneity. With limited availability of authentic standards for every putative HMO structure (estimated to be >200 total), new analytical methods are needed for their accurate characterization. Complete sequencing of the human milk glycome would enable a better understanding of their infant-specific biological roles and potentially lead to their widespread incorporation into infant formula. Herein, we explore the use of our high-resolution cyclic ion mobility spectrometry-mass spectrometry (cIMS-MS)-based platform for the separation of core disaccharide and trisaccharide isomer building blocks as a first step toward the sequencing of larger HMOs. By utilizing the flexible capabilities of the cIMS array, separation pathlengths were extended up to 40 m, thus enabling the resolution of all seven sets of sialylated, fucosylated galactosyllactose and lactosamine HMO building block isomers. Additionally, we assessed the utility of pre-/post-cIMS tandem mass spectrometry (MS/MS) and tandem cIMS (cIMS/cIMS) for the characterization of HMOs based on their diagnostic fragmentation patterns and mobility fingerprints. We anticipate that our presented cIMS-MS-based methodology will enable the better characterization of larger, unknown HMOs when incorporated into an overall workflow that also includes online liquid chromatography and enzymatic hydrolyses.

Dynamic Time-Warping Correction for Shifts in Ultrahigh Resolving Power Ion Mobility Spectrometry and Structures for Lossless Ion Manipulations.

Detection of arrival time shifts between ion mobility spectrometry (IMS) separations can limit achievable resolving power (Rp), particularly when multiple separations are summed or averaged, as commonly practiced in IMS. Such variations can be apparent in higher Rp measurements and are particularly evident in long path length traveling wave structures for lossless ion manipulations (SLIM) IMS due to their typically much longer separation times. Here, we explore data processing approaches employing single value alignment (SVA) and nonlinear dynamic time warping (DTW) to correct for variations between IMS separations, such as due to pressure fluctuations, to enable more effective spectrum summation for improving Rp and detection of low-intensity species. For multipass SLIM IMS separations, where narrow mobility range measurements have arrival times that can extend to several seconds, the SVA approach effectively corrected for such variations and significantly improved Rp for summed separations. However, SVA was much less effective for broad mobility range separations, such as obtained with multilevel SLIM IMS. Changes in ions' arrival times were observed to be correlated with small pressure changes, with approximately 0.6% relative arrival time shifts being common, sufficient to result in a loss of Rp for summed separations. Comparison of the approaches showed that DTW alignment performed similarly to SVA when used over a narrow mobility range but was significantly better (providing narrower peaks and higher signal intensities) for wide mobility range data. We found that the DTW approach increased Rp by as much as 115% for measurements in which 50 IMS separations over 2 s were summed. We conclude that DTW is superior to SVA for ultra-high-resolution broad mobility range SLIM IMS separations and leads to a large improvement in effective Rp, correcting for ion arrival time shifts regardless of the cause, as well as improving the detectability of low-abundance species. Our tool is publicly available for use with universal ion mobility format (.UIMF) and text (.txt) files.

Rapid cyclic ion mobility separations of monosaccharide building blocks as a first step toward a high-throughput reaction screening platform for carbohydrate syntheses.

Herein we present a new high-throughput screening method for carbohydrate syntheses based on cyclic ion mobility spectrometry-mass spectrometry (cIMS-MS)-based separations. We rapidly resolved the α/ß anomers for carbohydrates with varying protecting groups after only 5 m of cIMS-MS separation and also detected their respective unwanted anomeric impurities at levels lower than 2%. All experiments were performed in 1 minute of total acquisition time demonstrating our method's high-throughput nature. Our methodology was also extended to the separation of an isomeric mixtures of two protected disaccharides illustrating its utility beyond only monosaccharides. We envision our presented workflow as a first step toward the development of a high-throughput screening platform for the rapid and sensitive detection of α/ß anomeric selectivities and for trace isomeric/isobaric impurities.

Evaluation of Waveform Profiles for Traveling Wave Ion Mobility Separations in Structures for Lossless Ion Manipulations.

Structures for lossless ion manipulations (SLIM) have recently enabled a powerful implementation of traveling wave ion mobility spectrometry (TWIMS) for ultrahigh resolution separations; however, experimental parameters have not been optimized, and potential significant gains may be feasible. Most TWIMS separations have utilized square-shaped waveforms applied by time-dependent voltage stepping across repeating sets of electrodes, but alternative waveforms may provide further improvements to resolution. Here, we characterize five waveforms (including square and sine) in terms of their transmission efficiency, IMS resolution, and resolving power, and explore the effects of TW amplitude and speed on the performance of each. We found, consistent with previous work, separations were generally improved with higher TW amplitudes, moderately improved by lower speeds (limited by ion "surfing" with the waves), and found decreases in signal intensity at the extremes of operating conditions. The triangle and asymmetric "ramp forward" shaped profiles were found to provide modestly greater resolution and resolving power, an observation we tentatively attribute to their relatively uniform fields and minimal low-field regions.