Ion Mobility Mass Spectrometry Research Articles

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.

Cyclable Variable Path Length Multilevel Structures for Lossless Ion Manipulations (SLIM) Platform for Enhanced Ion Mobility Separations.

Ion mobility-mass spectrometry (IMS-MS) is used to analyze complex samples and provide structural information on unknown compounds. As the complexity of samples increases, there is a need to improve the resolution of IMS-MS instruments to increase the rate of molecular identification. This work evaluated a cyclable and variable path length (and hence resolving power) multilevel Structures for Lossless Ion Manipulations (SLIM) platform to achieve a higher resolving power than what was previously possible. This new multilevel SLIM platform has eight separation levels connected by ion escalators, yielding a total path length of ∼88 m (∼11 m per level). Our new multilevel SLIM can also be operated in an "ion cycling" mode by utilizing a set of return ion escalators that transport ions from the eighth level back to the first, allowing even extendable path lengths (and higher IMS resolution). The platform has been improved to enhance ion transmission and IMS separation quality by reducing the spacing between SLIM boards. The board thickness was reduced to minimize the ions' escalator residence time. Compared to the previous generation, the new multilevel SLIM demonstrated better transmission for a set of phosphazene ions, especially for the low-mobility ions. For example, the transmission of m/z 2834 ions was improved by a factor of ∼3 in the new multilevel SLIM. The new multilevel SLIM achieved 49% better resolving powers for GRGDS1+ ions in 4 levels than our previous 4-level SLIM. The collision cross-section-based resolving power of the SLIM platform was tested using a pair of reverse sequence peptides (SDGRG1+, GRGDS1+). We achieved 1100 resolving power using 88 m of path length (i.e., 8 levels) and 1400 following an additional pass through the eight levels. Further evaluation of the multilevel SLIM demonstrated enhanced separation for positively and negatively charged brain total lipid extract samples. The new multilevel SLIM enables a tunable high resolving power for a wide range of ion mobilities and improved transmission for low-mobility ions.

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.

High-Throughput Extracellular Matrix Proteomics of Human Lungs Enabled by Photocleavable Surfactant and diaPASEF.

The extracellular matrix (ECM) is a complex assembly of proteins that provide interstitial scaffolding and elastic recoil for human lungs. The pulmonary extracellular matrix is increasingly recognized as an independent bioactive entity, by creating biochemical and mechanical signals that influence disease pathogenesis, making it an attractive therapeutic target. However, the pulmonary ECM proteome ("matrisome") remains challenging to analyze by mass spectrometry due to its inherent biophysical properties and relatively low abundance. Here, we introduce a strategy designed for rapid and efficient characterization of the human pulmonary ECM using the photocleavable surfactant Azo. We coupled this approach with trapped ion mobility MS with diaPASEF to maximize the depth of matrisome coverage. Using this strategy, we identify nearly 400 unique matrisome proteins with excellent reproducibility that are known to be important in lung biology, including key core matrisome proteins.

AutonoMS: Automated Ion Mobility Metabolomic Fingerprinting.

Automation is dramatically changing the nature of laboratory life science. Robotic lab hardware that can perform manual operations with greater speed, endurance, and reproducibility opens an avenue for faster scientific discovery with less time spent on laborious repetitive tasks. A major bottleneck remains in integrating cutting-edge laboratory equipment into automated workflows, notably specialized analytical equipment, which is designed for human usage. Here we present AutonoMS, a platform for automatically running, processing, and analyzing high-throughput mass spectrometry experiments. AutonoMS is currently written around an ion mobility mass spectrometry (IM-MS) platform and can be adapted to additional analytical instruments and data processing flows. AutonoMS enables automated software agent-controlled end-to-end measurement and analysis runs from experimental specification files that can be produced by human users or upstream software processes. We demonstrate the use and abilities of AutonoMS in a high-throughput flow-injection ion mobility configuration with 5 s sample analysis time, processing robotically prepared chemical standards and cultured yeast samples in targeted and untargeted metabolomics applications. The platform exhibited consistency, reliability, and ease of use while eliminating the need for human intervention in the process of sample injection, data processing, and analysis. The platform paves the way toward a more fully automated mass spectrometry analysis and ultimately closed-loop laboratory workflows involving automated experimentation and analysis coupled to AI-driven experimentation utilizing cutting-edge analytical instrumentation. AutonoMS documentation is available at https://autonoms.readthedocs.io.

CE-MS/MS and CE-timsTOF to separate and characterize intramolecular disulfide bridges of monoclonal antibody subunits and their application for the assessment of subunit reduction protocols

Characterization at the subunit level enables detailed mass spectrometric characterization of posttranslational modifications (PTMs) of monoclonal antibodies (mAbs). The implemented reduction often leaves the intramolecular disulfide bridges intact. Here, we present a capillary electrophoretic (CE) method based on a neutral-coated capillary for the separation of immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) digested and reduced mAb subunits followed by mass spectrometry (MS), MS/MS identification, and trapped ion mobility mass spectrometry (timsTOF). Our CE approach enables the separation of (i) different subunit moieties, (ii) various reduction states, and (iii) positional isomers of these partly reduced subunit moieties. The location of the remaining disulfide bridges can be determined by middle-down electron transfer higher energy collisional dissociation (EThcD) experiments. All these CE-separated variants show differences in ion mobility in the timsTOF measurements. Applying the presented CE-MS/MS method, reduction parameters such as the use of chaotropic salts were studied. For the investigated antibodies, urea improved the subunit reduction significantly, whereas guanidine hydrochloride (GuHCl) leads to multiple signals of the same subunit in the CE separation. The presented CE-MS method is a powerful tool for the disulfide-variant characterization of mAbs on the subunit level. It enables understanding disulfide bridge reduction processes in antibodies and potentially other proteins.

Three-Minute Enantioselective Amino Acid Analysis by Ultra-High-Performance Liquid Chromatography Drift Tube Ion Mobility-Mass Spectrometry Using a Chiral Core-Shell Tandem Column Approach.

Fast liquid chromatography (LC) amino acid enantiomer separation of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatives using a chiral core-shell particle tandem column with weak anion exchange and zwitterionic-type quinine carbamate selectors in less than 3 min was achieved. Enantiomers of all AQC-derivatized proteinogenic amino acids and some isomeric ones (24 in total plus achiral glycine) were baseline separated (Rs > 1.5 except for glutamic acid with Rs = 1.3), while peaks of distinct amino acids and structural isomers (constitutional isomers and diastereomers of leucine and threonine) of the same configuration overlapped to various degrees. For this reason, drift tube ion mobility-mass spectrometry was added (i.e., LC-IM-MS) as an additional selectivity filter without extending run time. The IM separation dimension in combination with high-resolution demultiplexing enabled confirmation of threonine isomers (threonine, allo-threonine, homoserine), while leucine, isoleucine, and allo-isoleucine have almost identical collisional cross-section (DTCCSN2) values and added no selectivity to the partial LC separation. Density functional theory (DFT) calculations show that IM separation of threonine isomers was possible due to conformational stabilization by hydrogen bond formation between the hydroxyl side chain and the urea group. Generally, the CCSN2 of protonated ions increased uniformly with addition of the AQC label, while outliers could be explained by consideration of intramolecular interactions and additional structural analysis. Preliminary validation of the enantioselective LC-IM-MS method for quantitative analysis showed compliance of accuracy and precision with common limits in bioanalytical methods, and applicability to a natural lipopeptide and a therapeutic synthetic peptide could be demonstrated.

Intramolecular Ring-Chain Equilibrium Elimination Strategy for Pinpointing C═C Positional and Geometric Isomers of N-Alkylpyridinium Unsaturated Fatty Acid Derivatives via Ion Mobility-Mass Spectrometry.

Free unsaturated fatty acids (UFA) are key intermediates of lipid metabolism and participate in many metabolic pathways with specific biological functions. Although various fragmentation-based methods for pinpointing C═C locations in UFA were developed, the current mass spectrometry methods are difficult to simultaneously differentiate geometric isomers and positional isomers in trace samples due to low ionization efficiency, low conversion, and low resolution. Herein, an intramolecular ring-chain equilibrium elimination strategy via 4-plex stable isotope labeling dual derivatization-assisted ion mobility-mass spectrometry was developed, thereby one-pot specifically labeling C═C and carboxyl groups among the trace and unstable UFA with high sensitivity, high efficiency, and good substrate generality. It achieved fast separation of both C═C positional and geometric isomers with high resolution, which benefited from eliminating the intramolecular ring-chain equilibrium by suppressing the formation of salt bridges between free carboxyl groups and pyridine cations. 4-plex stable isotope labeling reagents showed similar reactivity, enabling high-throughput quantitative analysis of omics. This method was successfully applied for accurate and rapid identification of the UFA composition in olive oil extract. These results suggest that the developed method provides new insight for rapid characterization of UFA C═C positional and geometric isomers in complex samples to explore disease biomarkers and food quality control indicators.

Nitrile Imines as Peptide and Oligonucleotide Photo-Cross-Linkers in Gas-Phase Ions.

Nitrile imines produced by photodissociation of 2,5-diaryltetrazoles undergo cross-linking reactions with amide groups in peptide-tetrazole (tet-peptide) conjugates and a tet-peptide-dinucleotide complex. Tetrazole photodissociation in gas-phase ions is efficient, achieving ca. 50% conversion with 2 laser pulses at 250 nm. The formation of cross-links was detected by CID-MS3 that showed structure-significant dissociations by loss of side-chain groups and internal peptide segments. The structure and composition of cross-linking products were established by a combination of UV-vis action spectroscopy and cyclic ion mobility mass spectrometry (c-IMS). The experimental absorption bands were found to match the bands calculated for vibronic absorption spectra of nitrile imines and cross-linked hydrazone isomers. The calculated collision cross sections (CCSth) for these ions were related to the matching experimental CCSexp from multipass c-IMS measurements. Loss of N2 from tet-peptide conjugates was calculated to be a mildly endothermic reaction with ΔH0 = 80 kJ mol-1 in the gas phase. The excess energy in the photolytically formed nitrile imine is thought to drive endothermic proton transfer, followed by exothermic cyclization to a sterically accessible peptide amide group. The exothermic nitrile imine reaction with peptide amides is promoted by proton transfer and may involve an initial [3 + 2] cycloaddition followed by cleavage of the oxadiazole intermediate. Nucleophilic groups, such as cysteine thiol, did not compete with the amide cyclization. Nitrile imine cross-linking to 2'-deoxycytidylguanosine was found to be >80% efficient and highly specific in targeting guanine. The further potential for exploring nitrile-imine cross-linking for biomolecular structure analysis is discussed.

A simple strategy for d/l-carnitine analysis in food samples using ion mobility spectrometry and theoretical calculations

This work presents a straightforward approach to the separation d/l-carnitine (d/l-Carn) using ion mobility–mass spectrometry (IM-MS) and theoretical calculations. Natamycin (Nat) was used as separation reagent to interact with the Carn, metal ions (G) were employed as ligand, the resultant ternary complexes [d/l-Carn + Nat + G]+ were observed experimentally. IM-MS results revealed that d/l-Carn could be baseline separated via complex formation using Li+, Na+, K+, Rb+, and Cs+, with a maximum peak separation resolution (Rp-p) of 2.91; Theoretical calculations were performed to determine the optimal conformations of [d/l-Carn + Nat + Li/K]+, and the predicted collisional cross section values were consistent with the experimental values. Conformational analysis was used to elucidate the enantiomeric separation of d/l-Carn at the molecular level via the formation of ternary complexes. Furthermore, quantitative analyses for the determination of the enantiomers were established with effective linearity and acceptable sensitivity. Finally, the proposed method was successfully applied in the determination of d/l-Carn in food samples.

Transition Metal Ion FRET-Based Probe to Study Cu(II)-Mediated Amyloid-β Ligand Binding.

Recent therapeutic strategies suggest that small peptides can act as aggregation inhibitors of monomeric amyloid-β (Αβ) by inducing structural rearrangements upon complexation. However, characterizing the binding events in such dynamic and transient noncovalent complexes, especially in the presence of natively occurring metal ions, remains a challenge. Here, we deploy a combined transition metal ion Förster resonance energy transfer (tmFRET) and native ion mobility-mass spectrometry (IM-MS) approach to characterize the structure of mass- and charge-selected Aβ complexes with Cu(II) ions (a quencher) and a potential aggregation inhibitor, a small neuropeptide named leucine enkephalin (LE). We show conformational changes of monomeric Αβ species upon Cu(II)-binding, indicating an uncoiled N-terminus and a close interaction between the C-terminus and the central hydrophobic region. Furthermore, we introduce LE labeled at the N-terminus with a metal-chelating agent, nitrilotriacetic acid (NTA). This allows us to employ tmFRET to probe the binding even in low-abundance and transient Aβ-inhibitor-metal ion complexes. Complementary intramolecular distance and global shape information from tmFRET and native IM-MS, respectively, confirmed Cu(II) displacement toward the N-terminus of Αβ, which discloses the binding region and the inhibitor's orientation.

Evaluation of Surface-Induced Dissociation Ion Mobility-Mass Spectrometry for Lipid Structural Characterization.

The complexity of the lipidome has necessitated the development of novel analytical approaches for the identification and structural analysis of morphologically diverse classes of lipids. At this time, a variety of dissociation techniques have been utilized to probe lipid decomposition pathways in search of structurally diagnostic fragment ions. Here, we investigate the application of surface-induced dissociation (SID), a fragmentation technique that imparts energy to the target molecule via collision with a coated surface, for the fragmentation of seven lipids across four major lipid subclasses. We have developed a tuning methodology for guiding the efficient operation of a previously developed custom SID device for molecules as small as ca. 300 Da with ion mobility analysis of the fragmentation products. SID fragmentation of the various lipids analyzed was found to generate fragment ions similar to those observed in CID spectra, but fragment ion lab frame onset energies were lower in SID due to the higher energy deposition via a more massive target. For the largest lipid evaluated (cardiolipin 18:1), SID produced chain fragment ions, which yielded analytically useful information regarding the composition of the acyl tails. Ion mobility provided an orthogonal dimension of separation and aided in assigning product ions to their precursors. Overall, the combination of SID and IM-MS is another potential methodology in the analytical toolkit for lipid structural analysis.