Protein and peptide isomerization arising from sequence variation, epimerization, or post-translational modification directly influences biological structure and function, necessitating rapid and reliable methods for isomer delineation. Ion mobility spectrometry-mass spectrometry (IMS-MS) provides fast, gas-phase separations of peptide isomers, yet conventional mobility measurements offer limited information beyond bulk structural changes (i.e., compaction or elongation). Comparatively, isotopic shifts, or the measurement of differences in arrival time between light and heavy isotopologues, have been shown to be orthogonal to conventional IMS-MS separations as well as able to probe ion fine structure related to changes in center of mass and moments of inertia. In this work, we probed the use of isotopic dimethylation to introduce isomer-specific isotopic shifts as measured with high-resolution cyclic ion mobility separations. We observed that shifts in arrival time were diagnostic for a range of peptide isomers, including lysine positional isomers and β-amyloid aspartic acid isomers. The magnitude and direction of these shifts varied systematically with peptide sequence and, in several cases, defied predictions based solely on reduced mass theory. Molecular modeling revealed that these effects arise from isomer-dependent changes in mass distribution, specifically alterations in center of mass and moments of inertia after isotopic labeling. We also observed direct orthogonality of our isotopic shifts to absolute arrival times, highlighting how our strategy can provide an additional dimension of information to traditional IMS-MS measurements. Overall, isotopic dimethylation-based isotopic shifts offer a broadly applicable and structurally sensitive complement to the existing IMS-MS toolbox for peptide isomer characterization.

Enantiomers play important roles in biology and the pharmaceutical industry but remain notoriously difficult to separate and characterize. Gold-standard chromatography and nuclear magnetic resonance-based methods are slow, laborious, and expensive, motivating the development of rapid methodologies for enantiomer characterization. While ion mobility spectrometry-mass spectrometry (IMS-MS) has emerged as a rapid technique for chiral analysis, current approaches often rely on derivatization, complicated chiral complexes, or large host molecules that can produce complex spectra. Herein, we investigate small carbohydrates as chiral adducts for the ion mobility separation of amino acid and drug enantiomers. Six commercially available nonreducing or reduced carbohydrates were evaluated as chiral adduct molecules using cyclic IMS-MS. All carbohydrate adducts readily formed simple 1:1 complexes in the MS dimension that produced two discrete IMS peaks for each enantiomeric pair and remained sufficiently stable for extended path length separations. The carbohydrate panel enabled chiral separation of all 15 amino acid and drug enantiomer pairs at pathlengths as low as 1 m. Relative arrival time measurements further enabled unbiased comparison of separation performance across analyte-adduct pairs. The developed carbohydrate-based method was used to quantify enantiomeric excess down to a 99:1 molar ratio with estimated limits of detection down to 5 nM. These results demonstrate that small carbohydrates are effective chiral adducts for IMS-MS-based enantiomer separations and that simple analyte-adduct complexes can provide rapid and broadly applicable enantiomer differentiation without derivatization or the use of multimolecule complexes.

Ion mobility spectrometry-mass spectrometry (IMS-MS) has emerged as a mainstream analytical technique for rapidly separating challenging molecular species as well as being complementary to existing omics-based workflows. With new technological advancements and commercial instrumentation, the ability to perform high-resolution measurements has become more available to users. This has opened the door for tackling previously intractable isomeric species as well as newer avenues of research focusing on fundamental ion structure. In this young investigator perspective, I will highlight the area of research known as isotopic shifts, or rather the separations of isotopologues and isotopomers with IMS-MS, and its applications and fundamental studies.

Recently, the use of mass distribution-based isotopic shifts in high-resolution ion mobility spectrometry-mass spectrometry-based separations have enabled isomer delineation by measuring the relative arrival times of their heavy and light isotopologues. However, all previous efforts to induce such shifts have focused solely on the introduction of one type of isotopic substitution for a given molecule or isomer set. Herein, for the first time, we present a two-dimensional isotopic labeling strategy where two unique derivatizations are performed on various steroid isomer molecules to induce two distinct isotopic shifts and thus simultaneously measure them in a single ion mobility separations experiment. Derivatization strategies were chosen to target two specific functional groups in these steroids (i.e., hydroxyl and carbonyl), and heavy-labeled versions of the derivatizing reagents were used to induce isotopic shifts at each of these positions. We found that isotopic shifts were orthogonal to one another, diagnostic for certain steroid isomers, and that the simultaneous analysis of two different isotopic shifts was necessary for complete characterization of each steroid isomer set. We envision this multidimensional isotopic shift strategy as a new method for delineating amongst isomeric molecules, especially those with several different functional groups causing their isomerism.

Cerebrosides, a class of biologically important lipids, are comprised of a monosaccharide head group along with their ceramide tail. However, their accurate characterization is challenging because of the isomerism in both the tail, from potential double bond positioning, or in the head from monosaccharide composition and αβ anomericity. In this work, we focused on tackling the identification of the β-monosaccharide head group, as either glucose or galactose, in various cerebroside isomers as well as demonstrating how our methodology could be applied to unknowns found in a porcine extract. To achieve this, we performed collision-induced dissociation prior to cyclic ion mobility separations to generate monosaccharide fragment ions from the starting cerebroside precursor ions. With this pre-cIMS CID approach, we observed that the cIMS separations of the fragment ions were diagnostic of the β-monosaccharide head group composition (i.e., glucose versus galactose), regardless of the ceramide tail length. From there, we demonstrated an example of how this methodology could also be applied to cerebrosides found in a porcine extract and a framework for how this approach could be added to existing workflows in developing collision cross section databases. Overall, we envision that our developed pre-cIMS CID-based approach will be a complementary and orthogonal tool to existing ones in glycolipidomics workflows.

Complex carbohydrates, such as N-linked glycans, are highly important biomolecules with roles ranging from signaling to recognition and immune response. Ion mobility spectrometry-mass spectrometry (IMS-MS) has emerged as a rapid and orthogonal analytical technique to condensed-phase separations for studying carbohydrates, but many challenges exist in their analyses with IMS-MS due to their isomeric and conformational heterogeneity. Specifically, glycan IMS-MS separations often display more peaks than what can be predicted based on structure and/or much broader than expected peaks presumably from their metal-adducted conformers. This has precluded IMS-MS from being routinely used to analyze complex glycans largely because of the reduction in overall peak capacity and thus difficulty in deconvolving mixtures. In this work, we present a traveling wave-based ion heating strategy that uses activating traveling wave conditions. We demonstrated that this ion heating approach can improve peak capacity for individual glycan species as well as for those in mixtures. Importantly, we did not observe any significant loss in sensitivity and comparable resolution to glycans analyzed at gentle traveling wave conditions. Additionally, we demonstrated that isomeric glycans could be repeatedly cycled resulting in scalable resolution without significant ion losses. Overall, our approach can be broadly implemented on any traveling wave-based IMS-MS platform, and we envision utility toward other molecular classes desiring improved IMS-MS peak capacities.

Human milk oligosaccharides (HMOs) are a biologically important class of carbohydrates responsible for promoting the healthy development of infants. However, to better understand their specific biological roles, analytical techniques are needed to unambiguously characterize them. While liquid chromatography-tandem mass spectrometry (LC-MS/MS) remains the gold standard for HMO analysis, new orthogonal techniques are desired for improving their isomer analysis. Ion mobility spectrometry-mass spectrometry (IMS-MS) has emerged as a complementary technique to LC-MS/MS but has seen little use toward HMO sequencing analysis beyond the construction of collision cross section (CCS) databases. In this work, we describe the use of collision-induced dissociation performed prior to high-resolution cyclic ion mobility separations (i.e., pre-cIMS CID) in conjunction with CCS measurements to characterize the linkage positioning in various HMOs irrespective of the starting precursor ion. We then demonstrated how our developed approach could be used to sequence an unknown HMO present in a purified extract. Lastly, we applied our workflow to sequence an isomeric mixture in the same extract using cIMS/cIMS instead of pre-cIMS CID. Overall, our developed approach is a first step toward standard-free de novo HMO sequencing as well as being a complementary and orthogonal method to existing LC-MS/MS-based workflows.

Recent advancements in high-resolution ion mobility separations have enabled separations of isotopologues and isotopomers, broadly termed as isotopic shifts, based on changes in their mass distributions (i.e., changes in their center of mass and moments of inertia). However, all previous experimental measurements of these isotopic shifts include contributions from both the reduced mass term as well as the mass distribution-based one. Thus, it is of great interest to develop calibration strategies to decouple the two effects and estimate their overall contributions to experimental isotopic shifts. Herein, we have developed the first such procedure to estimate the absolute contributions from reduced mass and mass distribution in isotopic shifts by generating a reduced mass-only ion mobility-based calibration curve. Using this strategy, we were able to determine that for isotopically permethylated carbohydrates that their mass distribution terms were much larger in magnitude than their reduced mass-based ones. Overall, we envision this reduced mass-only calibration methodology can be broadly applied to study any measurable isotopic shift and will enable improved understanding of ion structure in high-resolution ion mobility separations.

Traveling wave-based ion mobility spectrometry-mass spectrometry (IMS-MS) has emerged as an analytical technique, particularly because of technological improvements for enabling the scalable resolution of challenging biomolecules. However, IMS-MS remains limited by peak broadening from diffusion in extended separations, thus precluding further gains in sensitivity and resolution. Previous efforts have attempted to overcome these issues through the development of peak compression strategies, such as compression ratio ion mobility programming (CRIMP) and temporal compression. Unfortunately, both previous compression strategies suffer from certain drawbacks related to resolution. Herein, we present a new reinjection-based spatial ion compression strategy implemented on a commercially available cyclic IMS-MS platform without hardware or software modifications. Our method involves slicing a portion of a mobility peak, storing it in the prestore, and reinjecting it under conditions ensuring a gentle transition back to separation conditions as well optimizing timing for the remerging (i.e., spatial compression) step. Our spatial ion compression approach enables reduction in peak widths, improved sensitivity without any ion losses during reinjection, and allows continued separation after the compression event. We highlight how this compression strategy can enable better feature finding by improving peak intensity and signal-to-noise of a low abundance species as well as achieving near baseline resolution of previously partially resolved compounds. Overall, our reinjection-based spatial ion peak compression strategy can be readily adaptable on any traveling wave-based IMS-MS platforms with ion storage regions (i.e., traps) and is an added one to the suite of existing compression strategies in IMS-MS measurements.

Gangliosides, a diverse class of glycosphingolipids, are highly abundant in neural tissue and have been implicated in numerous aging-related diseases. Their characterization with methods such as liquid chromatography-tandem mass spectrometry is often precluded by their structural complexity, isomeric heterogeneity, and lack of commercially available authentic standards. In this work, we coupled high-resolution cyclic ion mobility spectrometry with multiple collision-induced dissociation-based tandem mass spectrometry strategies to sequence the sialic acid positions in various ganglioside isomers. Initially, as a proof-of-concept demonstration, we were able to characterize the sialic acid positions in several GD1 and GT1 species. From there, we extended our approach to identify the location of N-glycolylneuraminic acid (NeuGc) residues in previously uncharacterized GD1 and GQ1 isomers. Our results highlight the potential of this presented methodology for the de novo characterization of gangliosides within complex biological matrices without the need for authentic standards.

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.

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 similar to 30 residues. In the first study using all three most powerful IMS methodologies & horbar;trapped IMS, cyclic IMS, and FAIMS & horbar;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 similar to 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.

Recently, ion mobility spectrometry-mass spectrometry (IMS-MS) has become more readily incorporated into various omics-based workflows. These growing applications are due to developments in instrumentation within the last decade that have enabled higher-resolution ion mobility separations. Two such platforms are the cyclic (cIMS) and structures for lossless ion manipulations (SLIM), both of which use traveling wave ion mobility spectrometry (TWIMS). High-resolution separations achieved with these techniques stem from the drastically increased pathlengths, on the order of 10 s of meters to >1 km, in both cIMS-MS and SLIM IMS-MS, respectively. Herein, we highlight recent developments and advances, for the period 2019-2023, in high-resolution traveling wave-based IMS-MS through instrumentation, calibration strategies, hyphenated techniques, and applications. Specifically, we will discuss applications including CCS calculations in multipass IMS-MS separations, coupling of IMS-MS with chromatography, imaging, and cryogenic infrared spectroscopy, and isomeric separations of glycans, lipids, and other small metabolites.

Human milk oligosaccharides (HMOs) are an important class of biomolecules responsible for the healthy development of the brain-gut axis of infants. Unfortunately, their accurate characterization is largely precluded due to a variety of reasons - there are over 200 possible HMO structures whereas only 10s of these are available as authentic analytical standards. Furthermore, their isomeric heterogeneity stemming from their many possible glycosidic linkage positions and corresponding α/β anomericities further complicates their analyses. While liquid chromatography coupled to tandem mass spectrometry remains the gold standard for HMO analyses, it often times cannot resolve all possible isomeric species and thus warrants the development of other orthogonal approaches. High-resolution ion mobility spectrometry coupled to mass spectrometry has emerged as a rapid alternative to condensed-phase separations but largely has remained limited to qualitative information related to the resolution of isomers. In this work, we have assessed the use of permethylation to improve both the resolution and sensitivity of HMO analyses with cyclic ion mobility separations coupled with mass spectrometry. In addition to this, we have developed the first-ever high-resolution collision cross-section database for permethylated HMOs using our previously established calibration protocol. We envision that this internal reference database generated from high-resolution cyclic ion mobility spectrometry-mass spectrometry will greatly aid in the accurate characterization of HMOs and provide a valuable, orthogonal, approach to existing liquid chromatography-tandem mass spectrometry-based methods.

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.

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.