Ion Mobility Mass Spectrometry Research Articles

Cyclic Ion Mobility-Mass Spectrometry and Tandem Collision Induced Unfolding for Quantification of Elusive Protein Biomarkers.

Sensitive analytical techniques that are capable of detecting and quantifying disease-associated biomolecules are indispensable in our efforts to understand disease mechanisms and guide therapeutic intervention through early detection, accurate diagnosis, and effective monitoring of disease. Parkinson's Disease (PD), for example, is one of the most prominent neurodegenerative disorders in the world, but the diagnosis of PD has primarily been based on the observation of clinical symptoms. The protein α-synuclein (α-syn) has emerged as a promising biomarker candidate for PD, but a lack of analytical methods to measure complex disease-associated variants of α-syn has prevented its widespread use as a biomarker. Antibody-based methods such as immunoassays and mass spectrometry-based approaches have been used to measure a limited number of α-syn forms; however, these methods fail to differentiate variants of α-syn that display subtle differences in only the sequence and structure. In this work, we developed a cyclic ion mobility-mass spectrometry method that combines multiple stages of activation and timed ion selection to quantify α-syn variants using both mass- and structure-based measurements. This method can allow for the quantification of several α-syn variants present at physiological levels in biological fluid. Taken together, this approach can be used to galvanize future efforts aimed at understanding the underlying mechanisms of PD and serves as a starting point for the development of future protein-structure-based diagnostics and therapeutic interventions.

Ion mobility-tandem mass spectrometry of mucin-type O-glycans

The dense O-glycosylation of mucins plays an important role in the defensive properties of the mucus hydrogel. Aberrant glycosylation is often correlated with inflammation and pathology such as COPD, cancer, and Crohn’s disease. The inherent complexity of glycans and the diversity in the O-core structure constitute fundamental challenges for the analysis of mucin-type O-glycans. Due to coexistence of multiple isomers, multidimensional workflows such as LC-MS are required. To separate the highly polar carbohydrates, porous graphitized carbon is often used as a stationary phase. However, LC-MS workflows are time-consuming and lack reproducibility. Here we present a rapid alternative for separating and identifying O-glycans released from mucins based on trapped ion mobility mass spectrometry. Compared to established LC-MS, the acquisition time is reduced from an hour to two minutes. To test the validity, the developed workflow was applied to sputum samples from cystic fibrosis patients to map O-glycosylation features associated with disease.

Exploring the Aggregation Propensity of PHF6 Peptide Segments of the Tau Protein Using Ion Mobility Mass Spectrometry Techniques.

Peptide and protein aggregation involves the formation of oligomeric species, but the complex interplay between oligomers of different conformations and sizes complicates their structural elucidation. Using ion mobility mass spectrometry (IM-MS), we aim to reveal these early steps of aggregation for the Ac-PHF6-NH2 peptide segment from tau protein, thereby distinguishing between different oligomeric species and gaining an understanding of the aggregation pathway. An important factor that is often neglected, but which can alter the aggregation propensity of peptides, is the terminal capping groups. Here, we demonstrate the use of IM-MS to probe the early stages of aggregate formation of Ac-PHF6-NH2, Ac-PHF6, PHF6-NH2, and uncapped PHF6 peptide segments. The aggregation propensity of the four PHF6 segments is confirmed using thioflavin T fluorescence assays and transmission electron microscopy. A novel approach based on post-IM fragmentation and quadrupole selection on the TIMS-Qq-ToF (trapped ion mobility) spectrometer was developed to enhance oligomer assignment, especially for the higher-order aggregates. This approach pushes the limits of IM identification of isobaric species, whose signatures appear closer to each other with increasing oligomer size, and provides new insights into the interpretation of IM-MS data. In addition, TIMS collision cross section values are compared with traveling wave ion mobility (TWIMS) data to evaluate potential instrumental bias in the trapped ion mobility results. The two IM-MS instrumental platforms are based on different ion mobility principles and have different configurations, thereby providing us with valuable insight into the preservation of weakly bound biomolecular complexes such as peptide aggregates.

Abstract 7090: A scaled proteomic discovery study for prostate cancer diagnostic signatures using Proteograph workflow with trapped ion mobility mass spectrometry

Abstract Background and Aims: The low cancer specificity of serum Prostate-Specific Antigen (PSA), the principal biomarker for prostate cancer detection, leads to a high frequency of unwarranted prostate biopsies. To alleviate this deficit, we executed a proteomic discovery study seeking PSA reflex signatures using the Proteograph™ Product Suite, a multi-nanoparticle-based deep plasma/serum proteomics workflow (Seer, Inc.), with timsTOF Pro mass spectrometry (Bruker) to interrogate over 900 serum specimens from patients that underwent prostate biopsy due to elevated PSA and/or abnormal digital rectal exam. Methods: Our study followed rigorous design principles including uniform collection, randomization and blinding of specimens, all of which were processed with the Proteograph Product Suite. Liquid chromatography-mass spectrometry (LC-MS) analyses leveraged the Bruker timsTOF Pro MS platform utilizing 30-minute reversed-phase chromatography and a label-free dia-PASEF (data independent acquisition - parallel accumulation serial fragmentation) data acquisition method. Peptides deriving from a chosen subset of specimens designed to maximize peptide diversity were pooled, fractionated and analyzed using DDA (data-dependent acquisition)-PASEF to build a spectral reference library. Results: The DIA-NN algorithm was employed through the cloud-based Proteograph Analysis Suite (PAS) to search LC-MS data. Leveraging our study-specific spectral library proteomic depth achieved was approximately 3600 protein groups identified (median) per patient serum specimen and more than 4400 in at least 25% of samples across the study. Customized machine learning workflows and the SeerML pipeline were used to identify and assess diagnostic power of new proteomic signatures. Conclusions: Our data demonstrate the remarkable proteomic depth achievable in a scaled patient serum specimen discovery study using a combination of Proteograph and timsTOF platforms. This significant effort revealed serum proteomic signatures with increased diagnostic performance over that provided by PSA blood measurement and traditional patient-associated metadata alone, providing a foundation for future clinical tests aimed to reduce the current high frequency of unnecessary prostate biopsy. Citation Format: Mark Flory, Matthew Chang, Jessie May Cartier, Jane Lange, Travis Moore, James McGann, Ryan Kopp, Michael Liss, Robin Leach, Brenna Albracht, James Dai, Michael Krawitzky, Eltaher Elgierari, Jessica Chu, Paul Pease, Max Mahoney, Shadi Ferdosi, Ryan Benz, Khatereh Motamedchaboki, Asim Siddiqui, Mahdi Zamanighomi, Amir Alavi, Harendra Guturu, Daniel Hornburg, Serafim Batzoglou. A scaled proteomic discovery study for prostate cancer diagnostic signatures using Proteograph workflow with trapped ion mobility mass spectrometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7090.

Rapid Characterization of Phospholipids from Biological Matrix Enabled by Indium Tin Oxide (ITO) Coated Slide Assisted Enrichment MALDI Mass Spectrometry

AbstractLipidomic analysis of human serum is essential to monitor the individual's health status. Herein, we develop a facile strategy for rapid characterization of phospholipids in human serum via indium tin oxide (ITO) coated glass slide solid phase extraction MALDI mass spectrometry (ITO‐SPE‐MALDI‐MS). Phospholipid species are retained on ITO slide via solid phase extraction owing to the unique property of the ITO material; the measurement of phospholipid species from 1 μl human serum within 2 min is achievable. A comparison of ITO‐SPE strategy with conventional extraction methods was further carried out using liquid chromatography‐mass spectrometry (LC‐MS) and ion‐mobility mass spectrometry (IM‐MS), resulting in a comparable enrichment performance for the phospholipid analysis. Furthermore, rapid lipidomic profiling of serum samples from human colorectal cancer patients and cell lines was demonstrated. Our results indicate that ITO‐SPE‐MALDI‐MS provides a higher throughput strategy for the analysis of phospholipid species in complex biological mixtures, showcasing its potential for applications in the analysis of clinical biofluids.

Ion Mobility Mass Spectrometry for Large Synthetic Molecules: Expanding the Analytical Toolbox.

Understanding the composition, structure and stability of larger synthetic molecules is crucial for their design, yet currently the analytical tools commonly used do not always provide this information. In this perspective, we show how ion mobility mass spectrometry (IM-MS), in combination with tandem mass spectrometry, complementary techniques and computational methods, can be used to structurally characterize synthetic molecules, make and predict new complexes, monitor disassembly processes and determine stability. Using IM-MS, we present an experimental and computational framework for the analysis and design of complex molecular architectures such as (metallo)supramolecular cages, nanoclusters, interlocked molecules, rotaxanes, dendrimers, polymers and host-guest complexes.

Taming Conformational Heterogeneity on Ion Racetrack to Unveil Principles that Drive Membrane Permeation of Cyclosporines.

Our study reveals the underlying principles governing the passive membrane permeability in three large N-methylated macrocyclic peptides (N-MeMPs): cyclosporine A (CycA), Alisporivir (ALI), and cyclosporine H (CycH). We determine a series of conformers required for robust passive membrane diffusion and those relevant to other functions, such as binding to protein targets or intermediates, in the presence of solvent additives. We investigate the conformational interconversions and establish correlations with the membrane permeability. Nuclear magnetic resonance (NMR) and cyclic ion-mobility spectrometry-mass spectrometry (cIMS-MS) are employed to characterize conformational heterogeneity and identify cis-amides relevant for good membrane permeability. In addition, ion mobility selected cIMS-MS and infrared (IR) multiple-photon dissociation (IRMPD) spectroscopy experiments are conducted to evaluate the energy barriers between conformations. We observe that CycA and ALI, both cyclosporines with favorable membrane permeabilities, display multiple stable and well-defined conformers. In contrast, CycH, an epimer of CycA with limited permeability, exhibits fewer and fewer stable conformers. We demonstrate the essential role of the conformational shift from the aqueous cis MeVal11-MeBmt1 state (A1) to the closed conformation featuring cis MeLeu9-MeLeu10 (C1) in facilitating membrane permeation. Additionally, we highlight that the transition from A1 to the all-trans open conformation (O1) is specifically triggered by the presence of CaCl2. We also capture a set of conformers with cis Sar3-MeLeu4, MeLeu9-MeLeu10, denoted as I. Conformationally selected cIMS-MS and IRMPD data of [CycA+Ca]2+ show immediate repopulation of the original population distribution, suggesting that CaCl2 smooths out the energy barriers. Finally, our work presents an improved sampling molecular dynamics approach based on a refined force field that not only consistently and accurately captures established conformers of cyclosporines but also exhibits strong predictive capabilities for novel conformers.

Investigating a Novel Neurodegenerative Disease Toxic Mechanism Involving Lipid Binding Specificity of Amyloid Oligomers.

Exploring the mechanisms underlying the toxicity of amyloid oligomers (AOs) presents a significant opportunity for discovering cures and developing treatments for neurodegenerative diseases. Recently, using a combination of ion mobility spectrometry-mass spectrometry (IMS-MS) and X-ray crystallography (XRC), we showed that the peptide KVKVLWDVIEV, which is the G95W mutant of αB-Crystallin (90-100) and abbreviated as G6W, self-assembles up to a dodecamer that structurally resembles lipid transport proteins. The glycine to tryptophan mutation promotes not only larger oligomers and enhanced cytotoxicity in brain slices than the wild type but also a narrow hydrophobic cavity suitable for fatty acid or phospholipid binding. Here, we determine the plausibility of a novel cytotoxic mechanism where the G6W's structural motif could perturb lipid homeostasis by determining its lipid binding selectivity and specificity. We show that the G6W oligomers have a strong affinity toward unsaturated phospholipids with a preference toward phospholipids containing 16-C alkyl chains. Molecular dynamics simulations demonstrate how an unsaturated, 16-C phospholipid fits tightly inside and outside G6W's hydrophobic cavity. This binding is exclusive to the G6W peptide, as other amyloid oligomers with different atomic structures, including its wildtype αB-Crystallin (90-100) and several superoxide dismutase 1 (SOD1) peptides that are known to self-assemble into amyloid oligomers (SOD1P28K and SOD1WG-GW), do not experience the same strong binding affinity. While the existing chaperone-lipid hypothesis on amyloid toxicity suggests amyloid-lipid complexes perforate cell membranes, our work provides a new outlook, indicating that soluble amyloid oligomers disrupt lipid homeostasis via selective protein-ligand interactions. The toxic mechanisms may arise from the formation of unique amyloid oligomer structures assisted by lipid ligands or impaired lipid transports.

Salt-Bridged Peptide Anion Gaseous Structures Enable Efficient Negative Ion Electron Capture Dissociation.

We previously discovered that electron attachment to gaseous peptide anions can occur within a relatively narrow electron energy range. The resulting charge-increased radical ions undergo dissociation analogous to conventional cation electron capture/transfer dissociation (ECD/ETD), thus enabling a novel tandem mass spectrometry (MS/MS) technique that we termed negative ion electron capture dissociation (niECD). We proposed that gaseous zwitterionic structures are required for niECD with electron capture either occurring at or being directed by a positively charged site. Here, we further evaluate this zwitterion mechanism by performing niECD of peptides derivatized to alter their ability to form zwitterionic gaseous structures. Introduction of a fixed positive charge tag, a highly basic guanidino group, or a highly acidic sulfonate group to promote zwitterionic structures in singly charged anions, rescued the niECD ability of a peptide refractory to niECD in its unmodified form. We also performed a systematic study of five sets of synthetic peptides with decreasing zwitterion propensity and found that niECD efficiency decreased accordingly, further supporting the zwitterion mechanism. However, traveling-wave ion mobility-mass spectrometry experiments, performed to gain further insight into the gas-phase structures of peptides showing high niECD efficiency, exhibited an inverse correlation between the orientationally averaged collision cross sections and niECD efficiency. These results indicate that compact salt-bridged structures are also a requirement for effective niECD.

Exploring the potential of using ion mobility-mass spectrometry to separate matrix interferences from analytes in food control

During residue analysis in complex matrices for food safety purposes, interfering signals can sometimes overlap with those of the analyte of interest. Access to an additional separation dimension besides chromatographic and mass separation, such as ion mobility, can aid in removing interfering signals, allowing for correct analyte identification in these cases. In our laboratory, during routine LC−MS/MS analysis of liver samples for growth promoter residues, an interfering signal was found that matches the retention time and m/z values for stanozolol, a synthetic anabolic steroid. In the present work, the performance of a liquid chromatography coupled to ion mobility mass spectrometry (LC−IM−MS) method has been evaluated to study whether this LC−MS/MS false positive in liver samples could be eliminated by LC−IM−MS analysis. A cyclic ion mobility system already allowed the separation of stanozolol from the interfering peak after only one pass, showing a significant improvement compared to the conventional LC−MS/MS method. Additionally, collisional cross section (CCS) values were calculated and successfully compared with those from literature for identification purposes, eventually allowing both the identification and quantification of stanozolol in this complex matrix.

Potential of ion mobility mass spectrometry in cellulose ether analysis: substitution pattern of hydroxyethyl celluloses

Ion mobility mass spectrometry (ESI-tims-ToF–MS, syringe pump infusion) has been applied to glucose and oligosaccharide ethers derived from hydroxyethyl-methyl celluloses (HEMC) and hydroxyethyl celluloses (HEC) after permethylation and partial depolymerization: by hydrolysis without or with subsequent reductive amination with m-amino benzoic acid (mABA) or by reductive cleavage. As model compounds without tandem substitution methoxyethylated methylcellulose was used. Regioisomeric glucose ethers were separated according to their ion mobility, and positions of substitution could be assigned. Glucose ethers including isomers with tandem substitution showed additional signals with a smaller collision cross-section (CCS) than core-substituted isomers. Positional isomers of cellobiose ethers were only partly resolved due to too high complexity but showed a characteristic fingerprint that might allow classifying samples. Relative intensities of signals of glucose ether isomers could only be quantified in case of ABA derivatives with its fixed charge, while sodium adducts of methoxyethyl ethers showed an influence of the MeOEt position on ion yield. Results were in very good agreement with reference analysis. [M + Na]+ adducts of α- and β-anomers of glucose derivatives were separated in IM, complicating position assignment. This could be overcome by reductive cleavage of the permethylated HE(M)C yielding 1,5-anhydroglucitol-terminated oligosaccharides, showing the best resolved fingerprints of the cellobiose ethers of a particular cellulose ether. With this first application of ion mobility MS to the analysis of complex cellulose ethers, the promising potential of this additional separation dimension in mass spectrometry is demonstrated and discussed.Graphical abstract

Computational tools and algorithms for ion mobility spectrometry-mass spectrometry.

Ion mobility spectrometry-mass spectrometry (IMS-MS or IM-MS) is a powerful analytical technique that combines the gas-phase separation capabilities of IM with the identification and quantification capabilities of MS. IM-MS can differentiate molecules with indistinguishable masses but different structures (e.g., isomers, isobars, molecular classes, and contaminant ions). The importance of this analytical technique is reflected by a staged increase in the number of applications for molecular characterization across a variety of fields, from different MS-based omics (proteomics, metabolomics, lipidomics, etc.) to the structural characterization of glycans, organic matter, proteins, and macromolecular complexes. With the increasing application of IM-MS there is a pressing need for effective and accessible computational tools. This article presents an overview of the most recent free and open-source software tools specifically tailored for the analysis and interpretation of data derived from IM-MS instrumentation. This review enumerates these tools and outlines their main algorithmic approaches, while highlighting representative applications across different fields. Finally, a discussion of current limitations and expectable improvements is presented.

Exploring the Effects of Cyclosporin A to Isocyclosporin A Rearrangement on Ion Mobility Separation.

Cyclosporin A (CycA) is a peptide secondary metabolite derived from fungi that plays a crucial role in transplantation surgery. Cyclic traveling wave ion mobility mass spectrometry (IM-MS) revealed an N → O peptidyl shift in singly protonated CycA to isocyclosporin A (isoA), whereas no such isomerization was observed for doubly protonated and sodiated molecules. CycA and isoA were able to be separated by considering doubly protonated precursors using a specific ion fragment. In parallel, sodium ion stabilization facilitated the simultaneous separation and quantitation of singly charged cyclosporin isomers with the limit of detection and coefficient of determination of 1.3% and 0.9908 for CycA in isoA and 1.0% and 0.9830 for isoA in CycA, respectively. Finally, 1H-13C gHSQC NMR experiments permitted parallel recording of up to 11 cyclosporin conformers. The ratios were determined by integrating the volume of cross-peaks of the upfield resonating hydrogen in the diastereotopic methylene group of sarcosine-3.

Impact of Source Conditions on Collision Cross Section Determination by Trapped Ion Mobility Spectrometry.

Collision cross section (CCS) values determined in ion mobility-mass spectrometry (IM-MS) are increasingly employed as additional descriptors in metabolomics studies. CCS values must therefore be reproducible and the causes of deviations must be carefully known and controlled. Here, we analyzed lipid standards by trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) to evaluate the effects of solvent and flow rate in flow injection analysis (FIA), as well as electrospray source parameters including nebulizer gas pressure, drying gas flow rate, and temperature, on the ion mobility and CCS values. The stability of ion mobility experiments was studied over 10 h, which established the need for a delay-time of 20 min to stabilize source parameters (mostly pressure and temperature). Modifications of electrospray source parameters induced shifts of ion mobility peaks and even the occurrence of an additional peak in the ion mobility spectra. This behavior could be essentially explained by ion-solvent cluster formation. Changes in source parameters were also found to impact CCS value measurements, resulting in deviations up to 0.8%. However, internal calibration with the Tune Mix calibrant reduced the CCS deviations to 0.1%. Thus, optimization of source parameters is essential to achieve a good desolvation of lipid ions and avoid misinterpretation of peaks in ion mobility spectra due to solvent effects. This work highlights the importance of internal calibration to ensure interoperable CCS values, usable in metabolomics annotation.

Accurate Prediction of Ion Mobility Collision Cross-Section Using Ion's Polarizability and Molecular Mass with Limited Data.

The rotationally averaged collision cross-section (CCS) determined by ion mobility-mass spectrometry (IM-MS) facilitates the identification of various biomolecules. Although machine learning (ML) models have recently emerged as a highly accurate approach for predicting CCS values, they rely on large data sets from various instruments, calibrants, and setups, which can introduce additional errors. In this study, we identified and validated that ion's polarizability and mass-to-charge ratio (m/z) have the most significant predictive power for traveling-wave IM CCS values in relation to other physicochemical properties of ions. Constructed solely based on these two physicochemical properties, our CCS prediction approach demonstrated high accuracy (mean relative error of <3.0%) even when trained with limited data (15 CCS values). Given its ability to excel with limited data, our approach harbors immense potential for constructing a precisely predicted CCS database tailored to each distinct experimental setup. A Python script for CCS prediction using our approach is freely available at https://github.com/MSBSiriraj/SVR_CCSPrediction under the GNU General Public License (GPL) version 3.

Improving the Sensitivity and Linear Range of Photoionization Ion Mobility Spectrometry via Confining the Ion Recombination and Space Charge Effects Assisted by Theoretical Modeling.

Photoionization (PI) is an efficient ionization source for ion mobility spectrometry (IMS) and mass spectrometry. Its hyphenation with IMS (PI-IMS) has been employed in various on-site analysis scenarios targeting a wide range of compounds. However, the signal intensity and linear dynamic range of PI-IMS at ambient pressure usually do not follow the Beer-Lambert law predictions, and the factors causing that negative deviation remain unclear. In this work, a variable pressure PI-IMS system was developed to examine the ion loss effects from factors like ion recombination and space charge by varying its working pressure from 1 to 0.1 bar. Assisted by theoretical modeling, it was found that ion recombination could contribute up to 90% of signal intensity loss for ambient pressure PI-IMS setups. Lowering the pressure and increasing the electric field in PI-IMS helped suppress the ion recombination process and thus an optimal pressure Poptimal appeared for best signal intensity, despite the decreased net ion number density and the increased space charge effect. A simplified theoretical equation taking ion recombination as the primary ion loss factor was derived to link Poptimal with analyte concentration and electric field in PI-IMS, enabling a swift optimization of the PI-IMS performance. For example, compared to ambient pressure, PI-IMS at a Poptimal of 0.4 bar provided a signal intensity increment of more than 400% for 0.716 ppmv toluene and also expanded the linear dynamic range by more than two times. Revealing factors influencing the PI-IMS response would also benefit the applications of other chemical ionization sources in IMS or mass spectrometry (MS).

Effects of Nano-Electrospray Ionization Emitter Position on Unintentional In-Source Activation of Peptide and Protein Ions.

Native ion mobility-mass spectrometry (IM-MS) typically introduces protein ions into the gas phase through nano-electrospray ionization (nESI). Many nESI setups have mobile stages for tuning the ion signal and extent of co-solute and salt adduction. However, tuning the position of the emitter capillary in nESI can have unintended downstream consequences for collision-induced unfolding or collision-induced dissociation (CIU/D) experiments. Here, we show that relatively small variations in the nESI emitter position can shift the midpoint (commonly called the "CID50" or "CIU50") potential of CID breakdown curves and CIU transitions by as much as 8 V on commercial instruments. A spatial "map" of the shift in CID50 for the loss of heme from holomyoglobin onto the emitter position on a Waters Synapt G2-Si mass spectrometer shows that emitter positions closer to the instrument inlet can result in significantly greater in-source activation, whereas different effects are found on an Agilent 6545XT instrument for the ions studied. A similar effect is observed for CID of the singly protonated leucine enkephalin peptide and Shiga toxin 1 subunit B homopentamer on the Waters Synapt G2-Si instrument. In-source activation effects on a Waters Synapt G2-Si are also investigated by examining the RMSD between CIU fingerprints acquired at different emitter positions and the shifts in CIU50 for structural transitions of bovine serum albumin and NIST monoclonal antibody.

Differentiating enantiomers by directional rotation of ions in a mass spectrometer

Conventional mass spectrometry does not distinguish between enantiomers, or mirror-image isomers. Here we report a technique to break the chiral symmetry and to differentiate enantiomers by inducing directional rotation of chiral gas-phase ions. Dual alternating current excitations were applied to manipulate the motions of trapped ions, including the rotation around the center of mass and macro movement around the center of the trap. Differences in collision cross section were induced, which could be measured by ion cloud profiling at high resolutions above 10,000. High-field ion mobility and tandem mass spectrometry analyses of the enantiomers were combined and implemented by using a miniature ion trap mass spectrometer. The effectiveness of the developed method was demonstrated with a variety of organic compounds including amino acids, sugars, and several drug molecules, as well as a proof-of-principle ligand optimization study for asymmetric hydrogenation.

Isomerism of Cyclodextrin Tetramer Induced by Alkali Halide Cluster Ions Observed by Ion Mobility Spectrometry-Mass Spectrometry.

Cyclodextrins (CDs) exhibit versatile self-assembly properties due to their hydrophilic and hydrophobic components, with applications such as drug delivery and selective binding. While research on CD self-assembly is extensive, limited studies have explored their aggregation behavior, particularly in interactions with small ionic guests. The present work investigates the structure of β-CD tetramers aggregated with alkali metal chloride clusters using ion mobility spectrometry-mass spectrometry (IMS-MS). The results revealed that diverse structures emerge in the tetramer depending on the alkali metal cluster size. Notably, the doubly charged tetramer exhibits distinct aggregation trends with specific numbers of MCl clusters for Na+ and K+ ions. After initially adopting a bucket-wheel structure with two internal cations, the structure transforms into a new isomer with a tetrahedral configuration upon cluster addition. The formation of the new isomer structure is closely linked to filling the cavity volume with MCl clusters and ionic interactions, which possibly compensate for the weakened hydrogen bonds between CDs. Theoretical calculations further support the structures, showing well-matched collision cross-section (CCS) values compared with the experimental CCS values. This study highlights the role of alkali metal chloride clusters as potential templates, leading to the formation of novel CD assemblies.

Collision-Induced Unfolding Reveals Disease-Associated Stability Shifts in Mitochondrial Transfer Ribonucleic Acids.

Ribonucleic acids (RNAs) remain challenging targets for structural biology, creating barriers to understanding their vast functions in cellular biology and fully realizing their applications in biotechnology. The inherent dynamism of RNAs creates numerous obstacles in capturing their biologically relevant higher-order structures (HOSs), and as a result, many RNA functions remain unknown. In this study, we describe the development of native ion mobility-mass spectrometry and collision-induced unfolding (CIU) for the structural characterization of a variety of RNAs. We evaluate the ability of these techniques to preserve native structural features in the gas phase across a wide range of functional RNAs. Finally, we apply these tools to study the elusive mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes-associated A3243G mutation. Our data demonstrate that our experimentally determined conditions preserve some solution-state memory of RNAs via the correlated complexity of CIU fingerprints and RNA HOS, the observation of predicted stability shifts in the control RNA samples, and the retention of predicted magnesium binding events in gas-phase RNA ions. Significant differences in collision cross section and stability are observed as a function of the A3243G mutation across a subset of the mitochondrial tRNA maturation pathway. We conclude by discussing the potential application of CIU for the development of RNA-based biotherapeutics and, more broadly, transcriptomic characterization.