Separation of triacylglycerol (TAG) isomers by cyclic ion mobility mass spectrometry.

Triacylglycerols (TAGs), a major lipid class in foods and the human body, consist of three fatty acids esterified to a glycerol backbone. They can occur in various isomeric forms, including sn-positional, cis/trans configurational, acyl chain length, double bond positional, and mixed type isomers. Separating isomeric mixtures is of great interest as different isomers can have distinct influence on mechanisms, such as digestibility, oxidative stability, or lipid metabolism. However, TAG isomer separation remains challenging with established analytical methodologies such as liquid-chromatography coupled to mass spectrometry (LC-MS). In this study, we developed a method with cyclic ion mobility mass spectrometry (cIMS-MS) for the separation and identification of all types of TAG isomers. First, the influence of different adducts (Li+, NH4+, Na+, and K+) on the separation was studied. Overall, it was concluded that the sodium adduct is the best choice to efficiently separate all types of TAG isomers. In addition, trends were found in the influence of specific structural features on the drift time order. An order of relative influence (from high to low) was established; (1) degree of unsaturation of the fatty acid(s) on an exterior position (if the total degree of unsaturation(s) is equal in both TAGs), (2) acyl chain length on the exterior positions, (3) cis/trans configuration, and (4) double bond (DB)-position. Finally, various cIMS-MS strategies were developed for the separation of mixtures containing four, five, and six isomers. To conclude, the developed methods can be used for separation of complex mixtures of TAG isomers and have great potential to be expanded to isomers of similar types of lipids such as di- and monoacylglycerols. This study also shows the potential of cIMS-MS to be used for the application on real TAG samples.

Analysis of the Mammalian Lipidome by DMS Shotgun Lipidomics.

Shotgun Lipidomics is a robust methodology for the characterization of the lipidome of complex biological samples. This assay is among the most quantitative lipidomics methods and is capable of surveying a wide breadth of lipid subclasses, both neutral and polar. The shortfalls of the technique include limitations in lipid species characterization and computationally demanding data analysis requiring isotopic and isobaric overlap correction. Differential Mobility Spectrometry (DMS) has demonstrated its utility in enabling acyl tail characterization within a Shotgun Lipidomics experiment. Here, we present a workflow for DMS Shotgun Lipidomics that measures 1400 possible lipid species. It utilizes the Shotgun Lipidomics Assistant (SLA) application, an open-source application that supervises the data analysis for an expansive Shotgun Lipidomics experiment.

A study on the determination of the metabolites of 2,4,6-Trinitrotoluene using a dual-drift tube ion mobility spectrometer.

2,4,6-Trinitrotoluene (TNT) and its four metabolites, namely 2-ADNT, 4-ADNT, 2,4-DANT, and 2,6-DANT, are highly toxic substances. These metabolites also serve as biomarkers for assessing the health of individuals exposed to TNT. In this study, a homemade DDT-IMS apparatus was utilized to detect these metabolites. Under negative detection mode, the drift times of 2-ADNT and 4-ADNT showed subtle shifts within a drift tube temperature range of 100 °C-120 °C, aiding in their differentiation. In positive detection mode for 2,4-DANT and 2,6-DANT, significant variations were observed in both the number and drift time of their positive product ions across a drift tube temperature range of 80 °C-120 °C. Consequently, optimal analytical performance for these metabolites was achieved at approximately 100 °C. Evaluation of the instrumental response during the measurement of the four metabolites in both positive and negative modes revealed that negative detection mode offered greater advantages of detecting these compounds. The working ranges for measuring the four metabolites spanned two orders of magnitude, with detection limits for each metabolite nearly below 1 ng. Notably, clear identification of the signals for these metabolites was achieved even when samples were mixed in urine, highlighting the ability of the DDT-IMS in detecting TNT metabolites. The developed DDT-IMS detection method has significant potential for enhancing environmental risk assessment and biological hazard evaluation, particularly in relation to human exposure to TNT.

Conformational Characterization of Peptides and Proteins by 193 nm Ultraviolet Photodissociation in the Collision Cell of a Trapped Ion Mobility Spectrometry-Time-of-Flight Mass Spectrometer.

Ultraviolet photodissociation (UVPD) has been shown to be a versatile ion activation strategy for the characterization of peptides and intact proteins among other classes of biological molecules. Combining the high-performance mass spectrometry (MS/MS) capabilities of UVPD with the high-resolution separation of trapped ion mobility spectrometry (TIMS) presents an opportunity for enhanced structural elucidation of biological molecules. In the present work, we integrate a 193 nm excimer laser in a TIMS-time-of-flight (TIMS-TOF) mass spectrometer for UVPD in the collision cell and use it for the analysis of several mass-mobility-selected species of ubiquitin and myoglobin. The resultant data displayed differences in fragmentation that could be correlated with changes in protein conformation. Additionally, this mobility-resolved UVPD strategy was applied to collision-induced unfolded ions of ubiquitin to follow changes in fragmentation patterns relating to the extent of protein unfolding. This platform and methodology offer new opportunities for exploring how conformational variations are manifested in the fragmentation patterns of gas-phase ions.

Analysis and discrimination of adhesive species using ATR-FTIR combined with Raman, and HS-GC-IMS together with multivariate statistical analysis.

Identifying the species and origin of adhesives in criminal investigations aids in narrowing inquiry scope and supporting case detection. This study introduces two advanced combined analytical techniques for distinguishing adhesive species, including attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) combined with Raman spectroscopy, and headspace gas chromatography-ion mobility spectrometry (HS-GC-IMS) together with multivariate statistical analysis. ATR-FTIR categorized seven adhesives into three groups based on the base materials, with further differentiation achieved via Raman spectra. Analysis of volatile components identified 79 volatile organic compounds (VOCs), with esters being the most concentrated. The fingerprint profile clearly illustrated the characteristic fingerprint sequence and unique marker compounds of each adhesive, effectively enabling their differentiation. Multivariate statistical analysis methods, including principal component analysis (PCA), orthogonal partial least squares-discriminant analysis (OPLS-DA), heatmap, and hierarchical cluster analysis (HCA), were utilized to visually interpret the classification of adhesives. This integrated analytical approach provides a comprehensive analysis of adhesive compositions, facilitating the diversification and precision of adhesive species identification, and broadening the scope for detecting and analyzing trace evidence in forensic science.

Simultaneous Determination of the Position and Cis-Trans Configuration of Lipid C═C Bonds via Asymmetric Derivatization and Ion Mobility-Mass Spectrometry.

The position and cis-trans configuration of C═C bonds in unsaturated lipids significantly affect their biological activities. Simultaneous identification of the position and cis-trans configuration of C═C bonds in unsaturated lipids is important; nonetheless, it still remains a challenging task. Herein, a stereoselective asymmetric reaction was used to recognize cis-trans isomers of the C═C bonds, and the derivatized precursor ions and product ions were subjected to tandem ion mobility-mass spectrometry (IM-MS) analysis. The theoretical calculation revealed that the formation of intramolecular hydrogen bonds after the cyclization reaction amplified the structural difference between diastereomers and increased the separation efficiency in IM. Consequently, a simple, sensitive, and highly selective platform for simultaneous determination of the position and cis-trans configuration of various C═C bonds in unsaturated lipids was established. It was then successfully applied to pinpoint the cis-trans geometry conversion of the located C═C bonds in lipids of the bacterial membrane under environmental stress and track the heterogeneous distribution of unsaturated lipids in rats after spinal cord injury. The present study also offers new insights into the application of IM-MS technology in resolving molecular structures and demonstrates the potential as a platform for a broad range of applications.

Quantitative Determination of Volatile N-Nitrosamines in Meat Products Using Gas Chromatography Coupled to an Ion Mobility Spectrometer with an Ammonia Dopant.

Herein, we develop a novel method using gas chromatography-ion mobility spectrometry (IMS) with an ammonia dopant for the determination of volatile N-nitrosamines in meat products. The IMS system was implemented with Fourier deconvolution multiplexing to simultaneously improve the resolving power and sensitivity. The ammonia dopant mitigated the formation of N-nitrosamine dimer ions, suppressing the ionization of interferents and shifting the reactant peak from hydrated protons to hydrated ammonium ions. The ammonium adduct product ions of N-nitrosamines were confirmed using time-of-flight mass spectrometry. The instrument limits of detection and quantitation for nitrosamines were 0.78-1.79 and 2.60-5.82 ng/mL, respectively, with recoveries between 71.39% and 110.59% using a simple water distillation method. The developed method showed satisfactory performance when applied to the detection of nitrosamines in various meat products.

The detection and utilization of volatile metabolomics in Klebsiella pneumoniae by gas chromatography-ion mobility spectrometry.

This research aimed to analyze the volatile compounds emitted during the proliferation of Klebsiella pneumoniae (K. pneumoniae) in the laboratory setting using gas chromatography-ion mobility spectrometry (GC-IMS) and to investigate the potential of volatile metabolomics for detecting carbapenemase-producing strains of K. pneumoniae. The volatile metabolomics of K. pneumoniae were comprehensively analyzed using GC-IMS in tryptic soy broth (TSB) as the culture medium. Afterward, the growth stabilization period (T2) served as the primary time point for analysis, with the introduction of imipenem and carbapenemase inhibitors (avibactam sodium or EDTA) during the exponential growth phase (T0) to further investigate alterations in volatile molecules associated with K. pneumoniae. Standard strains were utilized as references, while clinical strains were employed for validation purposes. At T2, a total of 22 volatile organic compounds (VOCs) associated with K. pneumoniae were identified (3 VOCs found in both monomer and dimer forms). Significant differences in VOCs were observed between carbapenemase-negative and carbapenemase-positive strains, both standard and clinical, following the introduction of imipenem. Furthermore, the addition of avibactam sodium led to distinct changes in the VOC content of strains producing class A carbapenemase, while the addition of EDTA resulted in specific alterations in the volatile metabolic profiles of strains producing class B carbapenemase. GC-IMS demonstrated significant promise for analyzing bacterial volatile metabolomics, and its application in evaluating the volatolomics of K. pneumoniae may facilitate the timely detection of carbapenemase-producing strains.

Online, Bottom-up Characterization of Histone H4 4-17 Isomers.

The "Histone Code" is comprised of specific types and positions of post-translational modifications (PTMs) which produce biological signals for gene regulation and have potential as biomarkers for medical diagnostics. Previous work has shown that electron-based fragmentation improves the sequence coverage and confidence of labile PTM position assignment. Here, we evaluated two derivatization methods (e.g., irreversible - propionylation and reversible-citraconylation) for bottom-up analysis of histone H4 4-17 proteoforms using online liquid chromatography (LC), trapped ion mobility spectrometry (TIMS), and electron-based dissociation (ExD) in tandem with mass spectrometry. Two platforms were utilized: a custom-built LC-TIMS-q-ExD-ToF MS/MS based on a Bruker Impact and a commercial µLC-EAD-ToF MS/MS SCIEX instrument. Complementary LC-TIMS preseparation of H4 4-17 0-4ac positional isomer standards showed that they can be resolved in their endogenous form, while positional isomers cannot be fully resolved in their propionylated form; online LC-ExD-MS/MS provided high sequence coverage (>90%) for all H4 4-17 (0-4ac) proteoforms in both instrumental platforms. When applied to model cancer cells treated with a histone deacetylase inhibitor (HeLa + HDACi), both derivatization methods and platforms detected and confirmed H4 4-17 (0-4ac) proteolytic peptides based on their fragmentation pattern. Moreover, a larger number of HeLa + HDACi H4 4-17 proteoforms were observed combining LC-TIMS and LC-q-ExD-ToF MS/MS due to the positional isomer preseparation in the LC-TIMS domain of citraconylated H4 4-17 (0-4ac) peptides.

Introducing Molecular Hypernetworks for Discovery in Multidimensional Metabolomics Data.

Orthogonal separations of data from high-resolution mass spectrometry can provide insight into sample composition and address challenges of complete annotation of molecules in untargeted metabolomics. "Molecular networks" (MNs), as used in the Global Natural Products Social Molecular Networking platform, are a prominent strategy for exploring and visualizing molecular relationships and improving annotation. MNs are mathematical graphs showing the relationships between measured multidimensional data features. MNs also show promise for using network science algorithms to automatically identify targets for annotation candidates and to dereplicate features associated with a single molecular identity. This paper introduces "molecular hypernetworks" (MHNs) as more complex MN models able to natively represent multiway relationships among observations. Compared to MNs, MHNs can more parsimoniously represent the inherent complexity present among groups of observations, initially supporting improved exploratory data analysis and visualization. MHNs also promise to increase confidence in annotation propagation, for both human and analytical processing. We first illustrate MHNs with simple examples, and build them from liquid chromatography- and ion mobility spectrometry-separated MS data. We then describe a method to construct MHNs directly from existing MNs as their "clique reconstructions", demonstrating their utility by comparing examples of previously published graph-based MNs to their respective MHNs.

Identification of Phospholipids Relevant to Cancer Tissue Using Differential Ion Mobility Spectrometry.

Phospholipids are the main building components of cell membranes and are also used for cell signaling and as energy storages. Cancer cells alter their lipid metabolism, which ultimately leads to an increase in phospholipids in cancer tissue. Surgical energy instruments use electrical or vibrational energy to heat tissues, which causes intra- and extracellular water to expand rapidly and degrade cell structures, bursting the cells, which causes the formation of a tissue aerosol or smoke depending on the amount of energy used. This gas phase analyte can then be analyzed via gas analysis methods. Differential mobility spectrometry (DMS) is a method that can be used to differentiate malignant tissue from benign tissues in real time via the analysis of surgical smoke produced by energy instruments. Previously, the DMS identification of cancer tissue was based on a 'black box method' by differentiating the 2D dispersion plots of samples. This study sets out to find datapoints from the DMS dispersion plots that represent relevant target molecules. We studied the ability of DMS to differentiate three subclasses of phospholipids (phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine) from a control sample using a bovine skeletal muscle matrix with a 5 mg addition of each phospholipid subclass to the sample matrix. We trained binary classifiers using linear discriminant analysis (LDA) and support vector machines (SVM) for sample classification. We were able to identify phosphatidylcholine, -inositol, and -ethanolamine with SVM binary classification accuracies of 91%, 73%, and 66% and with LDA binary classification accuracies of 82%, 74%, and 72%, respectively. Phosphatidylcholine was detected with a reliable classification accuracy, but ion separation setups should be adjusted in future studies to reliably detect other relevant phospholipids such as phosphatidylinositol and phosphatidylethanolamine and improve DMS as a microanalysis method and identify other phospholipids relevant to cancer tissue.

Separation of mycolic acid isomers by cyclic ion mobility-mass spectrometry.

RATIONALE: Mycobacterial species contain high concentrations of mycolic acids in their cell wall. Mycobacteria can pose a threat to both human health and the environment. Mass spectrometry lipidomic characterization can identify bacterial species and suggest targets for microbiological interventions. Due to the complex structures of mycolic acids and the possibility of isobaric isomers, multiple levels of separation are required for complete characterization. In this study, cyclic ion mobility (cIM) mass spectrometry (MS) was used for the analysis, separation and fragmentation of mycolic acids isomers from the bacterial species Gordonia amarae and Mycobacterium bovis. METHODS: Mycolic acid isomers were interrogated from cultured G. amarae biomass and commercially available M. bovis mycolic acid extracts. These were infused into a cIM-enabled quadrupole time-of-flight MS. Ions of interest were non-simultaneously selected with the quadrupole and passed around the cyclic ion mobility device multiple times. Fragment ion analysis was then performed for the resolved isomers of the quadrupole-selected ions. RESULTS: Repeated passes of the cIM device successfully resolved otherwise overlapping MA isomers, allowing isomer isolation and producing an ion-specific post-mobility fragmentation spectrum without isomeric interference. CONCLUSIONS: Mycolic acids (MA) isomers from G. amarae and M. bovis were resolved, resulting in a high mobility resolution and low interference fragmentation analysis. These revealed varying patterns of MA isomers in the two species: G. amarae's most abundant ion of each set of MA has 1-2 conformations, while the MA + 2 m/z the most abundant ion of each set has 3-6 conformations. These were resolved after 70 passes of the cyclic device. M. bovis' most abundant ion of each keto-MA set has 2 conformations, while the keto-MA + 2 m/z has 1-2 conformations. These were resolved after 75 passes.

An Untargeted Lipidomics Workflow Incorporating High-Resolution Demultiplexing (HRdm) Drift Tube Ion Mobility-Mass Spectrometry.

Global discovery lipidomics can provide comprehensive chemical information toward understanding the intricacies of metabolic lipid disorders such as dyslipidemia; however, the isomeric complexity of lipid species remains an analytical challenge. Orthogonal separation strategies, such as ion mobility (IM), can be inserted into liquid chromatography-mass spectrometry (LC-MS) untargeted lipidomic workflows for additional isomer separation and high-confidence annotation, and the emergence of high-resolution ion mobility (HRIM) strategies provides marked improvements to the resolving power (Rp > 200) that can differentiate small structural differences characteristic of isomers. One such HRIM strategy, high-resolution demultiplexing (HRdm), utilizes multiplexed drift tube ion mobility spectrometry (DTIMS) with post-acquisition algorithmic deconvolution to access high IM resolutions while retaining the measurement precision inherent to the drift tube technique; however, HRdm has yet to be utilized in untargeted studies. In this manuscript, a proof-of-concept study using ATP10D dysfunctional murine models was investigated to demonstrate the utility of HRdm-incorporated untargeted lipidomic analysis pipelines. Total lipid features were found to increase by 2.5-fold with HRdm compared to demultiplexed DTIMS as a consequence of more isomeric lipids being resolved. An example lipid, PC 36:5, was found to be significantly higher in dysfunctional ATP10D mice with two resolved peaks observed by HRdm that were absent in both the functional ATP10D mice and the standard demultiplexed DTIMS acquisition mode. The benefits of utilizing HRdm for discerning isomeric lipids in untargeted workflows have the potential to enhance our analytical understanding of lipids related to disease complexity and biologically relevant studies.

Identification of Key Volatile Compounds in Tilapia during Air Frying Process by Quantitative Gas Chromatography-Ion Mobility Spectrometry.

Air frying as a new roasting technology has potential for roasted fish production. In this study, the changes in volatile compounds (VCs) during air frying of tilapia were studied by quantitative gas chromatography-ion mobility spectrometry, followed by the identification of key VCs based on their odor activity value (OAV). There were 34 verified VCs, of which 16 VCs were identified as the key VCs with OAV ≥ 1. Most of the VCs were improved by air frying and peaked at 20 min. During the air frying, the total sulfhydryl content markedly decreased, while the protein carbonyl and MDA content significantly increased, suggesting the enhancement in the oxidation of lipids and proteins. The correlation network among the chemical properties and key VCs was constructed. The change in total sulfhydryl, protein carbonyl, and MDA showed significant correlation with most of the key VCs, especially 2-methyl butanal, ethyl acetate, and propanal. The results indicated that the oxidation of lipids and proteins contributed the most to the flavor improvement in air-fried tilapia. This study provides a crucial reference for the volatile flavor improvement and pre-cooked product development of roasted tilapia.

A hyper-fast gas chromatograph coupled to an ion mobility spectrometer with high repetition rate and flow-optimized ion source to resolve the short chromatographic peaks.

By combining the high selectivity of a gas chromatograph (GC) with the high sensitivity and decent selectivity of an ion mobility spectrometer (IMS), GC-IMS have become increasingly popular in many applications. However, most GC suffer from long analysis times. In contrast, an hyper-fast GC allows for extremely fast analysis in the tens of seconds while reaching comparably high resolution. In turn, coupling such hyper-fast GC with IMS requires sufficiently high repetition rate of recording full IMS spectra to resolve the short GC peaks. Therefore, we present a drift tube IMS with 100 Hz repetition rate. Key is a small effective detector volume combined with short drift length. Therefore, the ion source of the IMS combines a small reaction region with an extended field-switching ion shutter and optimized gas flows. To resolve even the shortest GC peaks with a full width at half maximum of 100 ms, a short drift length of just 41 mm was used, achieving a measurement time of 10 ms per spectrum and hence ten data points across the shortest GC peak. To avoid condensation of the sample, the entire IMS was heated isothermally to 120 °C. Despite short drift times and high temperatures, the IMS still reaches high resolving power of Rp = 60. The hyper-fast GC-IMS reaches low detection limits in the low ppbV range. For demonstration, ketone mixes and three different hop varieties were analyzed in <30 s.

Fast and broad-coverage lipidomics enabled by ion mobility-mass spectrometry.

Aberrant lipid metabolism has been widely recognized as a hallmark of various diseases. However, the comprehensive analysis of distinct lipids is challenging due to the complexity of lipid molecular structures, wide concentration ranges, and numerous isobaric and isomeric lipids. Usually, liquid chromatography-mass spectrometry (LC-MS)-based lipidomics requires a long time for chromatographic separation to achieve optimal separation and selectivity. Ion mobility (IM) adds a new separation dimension to LC-MS, significantly enhancing the coverage, sensitivity, and resolving power. We took advantage of the rapid separation provided by ion mobility and optimized a fast and broad-coverage lipidomics method using the LC-IM-MS technology. The method required only 8 minutes for separation and detected over 1000 lipid molecules in a single analysis of common biological samples. The high reproducibility and accurate quantification of this high-throughput lipidomics method were systematically characterized. We then applied the method to comprehensively measure dysregulated lipid metabolism in patients with colorectal cancer (CRC). Our results revealed 115 significantly changed lipid species between preoperative and postoperative plasma of patients with CRC and also disclosed associated differences in lipid classes such as phosphatidylcholines (PC), sphingomyelins (SM), and triglycerides (TG) regarding carbon number and double bond. Together, a fast and broad-coverage lipidomics method was developed using ion mobility-mass spectrometry. This method is feasible for large-scale clinical lipidomic studies, as it effectively balances the requirements of high-throughput and broad-coverage in clinical studies.

Streamlining Phenotype Classification and Highlighting Feature Candidates: A Screening Method for Non-Targeted Ion Mobility Spectrometry-Mass Spectrometry (IMS-MS) Data.

Nontargeted analysis (NTA) is increasingly utilized for its ability to identify key molecular features beyond known targets in complex samples. NTA is particularly advantageous in exploratory studies aimed at identifying phenotype-associated features or molecules able to classify various sample types. However, implementing NTA involves extensive data analyses and labor-intensive annotations. To address these limitations, we developed a rapid data screening capability compatible with NTA data collected on a liquid chromatography, ion mobility spectrometry, and mass spectrometry (LC-IMS-MS) platform that allows for sample classification while highlighting potential features of interest. Specifically, this method aggregates the thousands of IMS-MS spectra collected across the LC space for each sample and collapses the LC dimension, resulting in a single summed IMS-MS spectrum for screening. The summed IMS-MS spectra are then analyzed with a bootstrapped Lasso technique to identify key regions or coordinates for phenotype classification via support vector machines. Molecular annotations are then performed by examining the features present in the selected coordinates, highlighting potential molecular candidates. To demonstrate this summed IMS-MS screening approach, we applied it to clinical plasma lipidomic NTA data and exposomic NTA data from water sites with varying contaminant levels. Distinguishing coordinates were observed in both studies, enabling the evaluation of phenotypic molecular annotations and resulting in screening models capable of classifying samples with up to a 25% increase in accuracy compared to models using annotated data.

Investigating Anion Effects on Metal Ion Binding Interactions With Amyloid ß Peptide by Ion Mobility Mass Spectrometry.

The study of metal ion's role in the biological processes of Alzheimer's disease has spurred investigations into the coordination chemistry of amyloid beta peptide and its fragments. Nano-electrospray ionization mass spectrometry (nESI-MS) has been utilized to examine the stabilization of bound anions on multiprotein complexes without bulk solvent. However, the effects of anions on metal ion binding interactions with amyloid beta peptide have not been explored. This study directly examined metal-peptide complexes using nESI-MS and investigated the effects of various anions on the binding ratio and stability of these complexes from ammonium salt solutions. The results indicate that different anions have distinct effects on the binding ratio and stability of various metal-peptide complexes. Of these, the bicarbonate ion exhibits the highest binding ratios for metal-peptide complexes, while binding ratios for these complexes in phosphate are comparatively low. Our results suggest that acetate, formate, bicarbonate, and phosphate have weak affinities and act as weak stabilizers of the metal-peptide complex structure in the gas phase. Intriguingly, chloride and sulfate act as stabilizers of the metal-peptide complex in the gas phase. The rank order determined from these data is substantially different from the Hofmeister salt series in solution. Although this outcome was anticipated due to the reduced influence of anions and water solvation, our findings correlate well with expected anion binding in solution and emphasize the importance of both hydration layer and anion-metal-peptide binding effects for Hofmeister-type stabilization in solution. This approach proved useful in examining the interactions between metal ions and amyloid beta peptide, which are relevant to Alzheimer's disease, using direct ESI-MS.

Metal Polycation Adduction to Lipids Enables Superior Ion Mobility Separations with Ultrafast Ozone-Induced Dissociation.

Specific lipid isomers are functionally critical, but their structural rigidity and usually minute geometry differences make separating them harder than other biomolecules. Such separations by ion mobility spectrometry (IMS) were recently enabled by new high-definition methods using dynamic electric fields, but major resolution gains are needed. Another problem of identifying many isomers with no unique fragments in ergodic collision-induced dissociation (CID) was partly addressed by the direct ozone-induced dissociation (OzID) that localizes the double bonds, but a low reaction efficiency has limited the sensitivity, dynamic range, throughput, and compatibility with other tools. Typically lipids are analyzed by MS as singly charged protonated, deprotonated, or ammoniated ions. Here, we explore the differential IMS (FAIMS) separations with OzID for exemplary lipids cationized by polyvalent metals. These multiply charged adducts have much greater FAIMS compensation voltages (UC) than the 1+ ions, with up to 10-fold resolution gain enabling baseline isomer separations even at a moderate resolving power of the SelexION stage. Concomitantly OzID speeds up by many orders of magnitude, producing a high yield of diagnostic fragments already in 1 ms. These capabilities can be ported to the superior high-definition FAIMS and high-pressure OzID systems to take lipidomic analyses to the next level.