Ion Mobility Mass Spectrometry Research Articles

Accurate Collisional Cross Section Measurement by Multipass Cyclic Ion Mobility Spectrometry.

Ion mobility-mass spectrometry (IM-MS) is a powerful analytical tool for structural characterization. IM measurement provides collision cross section (CCS) values that facilitate analyte identification. While CCS values can be directly calculated from mobility measurements obtained using drift tube ion mobility spectrometry (DT-IMS), this method has limited mobility resolution due to the practical constraints on the length of the ion drift path. Consequently, DT-IMS cannot differentiate analytes with similar mobilities or resolve fine mobility features of individual ions. Cyclic IMS (cIMS) instruments leverage a cyclic path enabled by traveling wave ion mobility (TWIM) technology and offer increased mobility solution to address this challenge. While TWIM devices must first be calibrated to enable CCS measurements, current calibration strategies are primarily tailored for single-pass analyses. This preference is partly attributed to the challenges associated with multipass calibration methods, which require both calibrants and analytes to experience the same number of passes. Achieving this consistency can be complicated due to factors like peak splitting and diffusion, and may not be feasible for online IM-MS analyses. A recent report employed average ion velocities obtained from multiple measurements under different separation pathlengths as a path length-independent metric for CCS calibration. However, the ability to exploit this averaging approach is limited by observed variation in ion drift time/velocity in these measurements. In this study, we introduce a novel calibration strategy designed for multipass cIMS analyses, directly targeting the root cause for the path length- and mobility-dependent variations in ion drift time. With this method, we demonstrate that CCS values derived from multipass measurements closely align with those obtained from single-pass analyses, with an average deviation of 0.1%. We apply this method to characterize four isomeric trisaccharides. Our approach not only results in excellent agreement between our measured cIMSCCS values and the reported DTCCS values, with an average difference of only 0.5%, but also allows us to effectively identify subtle mobility characteristics of each compound and determine their respective CCS values. This level of detail and accuracy was previously unattainable using DT-IMS or single-pass cIMS measurements. We developed an algorithm for reconstructing arrival time distribution in cases where wrap-around has resulted in peak splitting. Collectively, the new calibration strategy and the reconstruction procedure maintain reproducibility and precision in CCS measurements while largely eliminating the need for meticulous selection of separation times. We expect that our method will empower researchers to harness the high mobility resolution offered by multipass cIMS analyses without compromising the accuracy of CCS measurement, making it appropriate for straightforward use across a wide range of applications.

Computational and Experimental IM-MS Determination of the Protonated Structures of Antimalarial Drugs.

A combination of ion mobility-mass spectrometry (IM-MS) measurements and computational methods were used to study structural and physicochemical properties of a range of quinoline-based drugs: amodiaquine (AQ), cinchonine (CIN), chloroquine (CQ), mefloquine (MQ), pamaquine (PQ), primaquine (PR), quinacrine (QR), quinine (QN), and sitamaquine (SQ). In experimental studies, ionization of these compounds using atmospheric pressure chemical ionization (APCI) yields monoprotonated species in the gas phase while electrospray ionization (ESI) also produces diprotonated forms of AQ, CQ, and QR and also for PQ, SQ, and QN in the presence of formic acid as an additive. Comparison of the trajectory-method-calculated and experimental IM-derived collisional cross sections (CCSN2) were used to assign both the protonation sites and conformer geometry of all drugs considered with biases of 0.7-2.8% between calculated and experimental values. It was found that, in solution, AQ and QR are protonated at the ring nitrogen of the quinoline group, whereas the other drugs are protonated at the amine group of the alkyl chain. Finally, the conformers of [M + H]+ and [M + 2H]2+ assigned according to the lowest energies and CCSN2 calculations were used to calculate the pKa values of the antimalarial drugs and the relative abundance of these ions at different pH values that provided validation of the computational and experimental IM-MS results.

Development of a cyclic ion mobility spectrometry-mass spectrometry-based collision cross-section database of permethylated human milk oligosaccharides.

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

Structural characterization of iron azadiethylthiolate complexes: Insights from NMR, crystallography, and ion mobility‐mass spectroscopy

AbstractFe3(CO)12 reacted with azadiethylthiols afforded [Fe2(μ‐S(CH2)2NR(CH2)2S)(CO)6]2 (R = nPr, 1A; iPr, 2A). Structure analysis revealed that 1A features the anti‐(a,e,a,e) configuration, while its iso‐propyl 2A adopts the syn‐(a,e,a,e) arrangement. NMR measurements at room temperature confirmed this structure, showing distinct signals for 2 NCH2 and 2 SCH2 methylene groups. Acid treatment of 1–2 yielded N‐protonated species (1A–H, 2A–H), maintaining the anti‐(a,e,a,e) configuration. Isolation of 1B–H revealed both the syn‐(a,e,e,a) and anti‐(a,e,e,a) configuration by X‐ray single‐crystallography. Protonation of 1A–H induced downfield shifts in methylene proton signals, with NH hydrogen recorded at 6.96 ppm. Furthermore, 1B–H displays similar NMR behavior, indicating the coexistence of two isomeric molecules of 1B–H, syn‐(a,e,e,a) and anti‐(a,e,e,a), which is consistent with the crystallographic results. Moreover, ion mobility‐mass spectrometry (IM‐MS) is harnessed for the swift identification and differentiation of these isomeric complexes in solution. By leveraging accurate mass measurements and MS/MS analyses, the molecular formula and constituents are validated. The sample mobility enables straightforward characterization of 1A, 1B, and 2A in relation to their stereo‐ and regio‐configurations, obviating the need for extensive time or sample quantities. Integration of findings from X‐ray crystallography, NMR, and IM‐MS furnishes precise and comprehensive structural insights into the coordination complexes.

Time-Resolved Ion Mobility-Mass Spectrometry Reveals Structural Transitions in the Disassembly of Modular Polyketide Syntheses.

The type 1 polyketide synthase (PKS) assembly line uses its modular structure to produce polyketide natural products that form the basis of many pharmaceuticals. Currently, several cryoelectron microscopy (cryo-EM) structures of a multidomain PKS module have been constructed, but much remains to be learned. Here we utilize ion-mobility mass spectrometry (IM-MS) to record size and shape information and detect different conformational states of a 207 kDa didomain dimer comprised of ketosynthase (KS) and acyl transferase (AT), excised from full-length module. Furthermore, gas-phase stability differences between these different conformations are captured by collision induced unfolding (CIU) technology. Additionally, through tracking these forms as a function of time, we elucidate a detailed disassembly pathway for KS-AT dimers for the first time.

The rise and fall of adenine clusters in the gas phase: a glimpse into crystal growth and nucleation.

The emergence of a crystal nucleus from disordered states is a critical and challenging aspect of the crystallization process, primarily due to the extremely short length and timescales involved. Methods such as liquid-cell or low-dose focal-series transmission electron microscopy (TEM) are often employed to probe these events. In this study, we demonstrate that ion mobility spectrometry-mass spectrometry (IMS-MS) offers a complementary and insightful perspective on the nucleation process by examining the sizes and shapes of small clusters, specifically those ranging from n = 2 to 40. Our findings reveal the significant role of sulfate ions in the growth of adeninediium sulfate clusters, which are the precursors to the formation of single crystals. Specifically, sulfate ions stabilize adenine clusters at the 1:1 ratio. In contrast, guanine sulfate forms smaller clusters with varied ratios, which become stable as they approach the 1:2 ratio. The nucleation size is predicted to be between n = 8 and 14, correlating well with the unit cell dimensions of adenine crystals. This correlation suggests that IMS-MS can identify critical nucleation sizes and provide valuable structural information consistent with established crystallographic data. We also discuss the strengths and limitations of IMS-MS in this context. IMS-MS offers rapid and robust experimental protocols, making it a valuable tool for studying the effects of various additives on the assembly of small molecules. Additionally, it aids in elucidating nucleation processes and the growth of different crystal polymorphs.

Full Native timsTOF PASEF-Enabled Quantitative Proteomics with the i2MassChroQ Software Package.

Ion mobility mass spectrometry has become popular in proteomics lately, in particular because the Bruker timsTOF instruments have found significant adoption in proteomics facilities. The Bruker's implementation of the ion mobility dimension generates massive amounts of mass spectrometric data that require carefully designed software both to extract meaningful information and to perform processing tasks at reasonable speed. In a historical move, the Bruker company decided to harness the skills of the scientific software development community by releasing to the public the timsTOF data file format specification. As a proteomics facility that has been developing Free Open Source Software (FOSS) solutions since decades, we took advantage of this opportunity to implement the very first FOSS proteomics complete solution to natively read the timsTOF data, low-level process them, and explore them in an integrated quantitative proteomics software environment. We dubbed our software i2MassChroQ because it implements a (peptide)identification-(protein)inference-mass-chromatogram-quantification processing workflow. The software benchmarking results reported in this paper show that i2MassChroQ performed better than competing software on two critical characteristics: (1) feature extraction capability and (2) protein quantitative dynamic range. Altogether, i2MassChroQ yielded better quantified protein numbers, both in a technical replicate MS runs setting and in a differential protein abundance analysis setting.

Quantitative Online Monitoring of an Immobilized Enzymatic Network by Ion Mobility-Mass Spectrometry.

The forward design of in vitro enzymatic reaction networks (ERNs) requires a detailed analysis of network kinetics and potentially hidden interactions between the substrates and enzymes. Although flow chemistry allows for a systematic exploration of how the networks adapt to continuously changing conditions, the analysis of the reaction products is often a bottleneck. Here, we report on the interface between a continuous stirred-tank reactor, in which an immobilized enzymatic network made of 12 enzymes is compartmentalized, and an ion mobility-mass spectrometer. Feeding uniformly 13C-labeled inputs to the enzymatic network generates all isotopically labeled reaction intermediates and products, which are individually detected by ion mobility-mass spectrometry (IMS-MS) based on their mass-to-charge ratios and inverse ion mobilities. The metabolic flux can be continuously and quantitatively monitored by diluting the ERN output with nonlabeled standards of known concentrations. The real-time quantitative data obtained by IMS-MS are then harnessed to train a model of network kinetics, which proves sufficiently predictive to control the ERN output after a single optimally designed experiment. The high resolution of the time-course data provided by this approach is an important stepping stone to design and control sizable and intricate ERNs.

Ion Mobility-Mass Spectrometry Strategies to Elucidate the Anhydrous Structure of Noncovalent Guest/Host Complexes.

Structural mass spectrometry (MS) techniques are fast and sensitive analytical methods to identify noncovalent guest/host complexation phenomena for desirable solution-phase properties. Current MS-based studies on guest/host complexes of drug and drug-like molecules are sparse, and there is limited guidance on how to interpret MS information in the context of host nanoencapsulation and inclusion. Here, we use structural MS strategies, combining energy-resolved MS (ERMS), ion mobility-MS (IM-MS), and computational modeling, to characterize 14 chemically distinct drug and drug-like compounds for their propensity to form guest/host complexes with the widely used excipient, beta-cyclodextrin (βCD). The majority (11/14) yielded a 1:1 guest/host complex, and ion mobility collision cross section (CCS) analysis provided subtle evidence of gas-phase compaction of complexes in both polarities. The three distinct dissociation channels observed in ERMS (i.e., charged βCD, charged guest, and partial guest loss) were used to direct charge-site assignments for computational modeling, and structural candidates were prioritized using helium-derived CCS measurements combined with root-mean-square distance analysis. The combined analytical information from ERMS, IM-MS, and computational modeling suggested that the majority of anhydrous complexes are inclusion complexes with βCD. Taken together, this work demonstrates a roadmap for how multiple MS-based analytical measurements can be combined to interpret the structures that guest/host complexes adopt in the absence of water.

Differentiation of regioisomers of sulfobenzoic acid by traveling-wave ion mobility mass spectrometry.

An ion mobility mass spectrometry (IM-MS) investigation using a Synapt G2 mass spectrometer was conducted to separate anions generated from the three regioisomers of sulfobenzoic acid. The results revealed that the differences in arrival time distributions (ATDs) were inadequate to differentiate the isomers unambiguously. However, the ATD profiles of the product ions, generated by fragmenting the respective mass-selected m/z 201 precursor ions in the Trap region of the three-compartment traveling-wave ion guide of the Synapt G2 mass spectrometer, were distinctly different, enabling definitive differentiation of the isomers. An arrival-time peak for an ion of m/z 157 resulting from the loss of CO2 from the respective precursors was common to all three mobilograms. However, only the profile recorded from the para-isomer exhibited a unique arrival-time peak for an ion of m/z 137, originating from an SO2 loss. Such a peak corresponding to an SO2 loss was absent in the ATD profiles of the ortho- and meta-isomers. Additionally, the mobilogram of the meta-isomer displayed a unique peak at 3.42 ms. Based on its product ion spectrum, this peak was attributed to the bisulfite anion (m/z 81; HSO3-). Previously, this meta-isomer specific m/z 81 ion had been proposed to originate from a two-step process involving the intermediacy of an m/z 157 ion formed by CO2 loss. However, our detailed tandem mass spectrometric experiments suggest that the m/z 81 is not a secondary product but rather an ion that originated from a direct elimination of a benzyne derivative from the m/z 201 precursor ion.

Dependency of fentanyl analogue protomer ratios on solvent conditions as measured by ion mobility-mass spectrometry.

Recently, our group has shown that fentanyl and many of its analogues form prototropic isomers ("protomers") during electrospray ionization. These different protomers can be resolved using ion mobility spectrometry and annotated using mobility-aligned tandem mass spectrometry fragmentation. However, their formation and the extent to which experimental variables contribute to their relative ratio remain poorly understood. In the present study, we systematically investigated the effects of mixtures of common chromatographic solvents (water, methanol, and acetonitrile) and pH on the ratio of previously observed protomers for 23 fentanyl analogues. Interestingly, these ratios (N-piperidine protonation vs. secondary amine/O = protonation) decreased significantly for many analogues (e.g., despropionyl ortho-, meta-, and para-methyl fentanyl), increased significantly for others (e.g., cis-isofentanyl), and remained relatively constant for the others as solvent conditions changed from 100% organic solvent (methanol or acetonitrile) to 100% water. Interestingly, pH also had significant effects on this ratio, causing the change in ratio to switch in many cases. Lastly, increasing conditions to pH ≥ 4.0 also prompted the appearance of new mobility peaks for ortho- and para-methyl acetyl fentanyl, where all previous studies had only showed one single distribution. Because these ratios have promise to be used qualitatively for identification of these (and emerging) fentanyl analogues, understanding how various conditions (i.e., mobile phase selection and/or chromatographic gradient) affect their ratios is critically important to the development of advanced ion mobility and mass spectrometry methodologies to identify fentanyl analogues.

Leveraging High-Resolution Ion Mobility-Mass Spectrometry for Cyclic Peptide Soft Spot Identification.

Cyclic peptides are an important class of molecules that gained significant attention in the field of drug discovery due to their unique pharmacological characteristics and enhanced proteolytic stability. Yet, gastrointestinal degradation remains a major hurdle in the discovery of orally bioavailable cyclic peptides. Soft spot identification (SSID) of the regions in the cyclic peptide sequence susceptible to amide hydrolysis by proteases is used in the discovery stage to guide medicinal chemistry design. SSID can be an arduous task, traditionally performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), often resulting in complex and time-consuming manual analysis, particularly when isomeric linear peptide metabolites chromatographically coelute. Here, we present an alternative orthogonal approach that entails a high-resolution ion mobility (HRIM) system based on Structures for Lossless Ion Manipulation (SLIM) technology interfaced with quadrupole time-of-flight (QTOF) mass spectrometry to address some of the challenges associated with SSID. Two strategies were used to resolve linear isomeric peptide metabolites: labeled and label-free, both utilizing the HRIM platform. The label-free strategy leverages negative polarity to ionize the isomers which achieves better separation of the gas phase ions in the ion mobility (IM) dimension as compared to positive polarity, which is a more conventional approach when studying proteins and peptides. The second approach uses an isotope-labeled dimethyl tag on the terminal amine group, acting as a "shift reagent" to influence the mobility of isomers in the positive mode. This method resulted in baseline separation for the isomers of interest and produced unique product ions in the fragmentation spectra for unambiguous soft spot identification. Both label-free and labeled strategies demonstrated the ability to solve the challenges associated with SSID for cyclic peptides.

Evaluating Solid Phase Adsorption Toxin Tracking (SPATT) for passive monitoring of per- and polyfluoroalkyl substances (PFAS) with Ion Mobility Spectrometry-Mass Spectrometry (IMS-MS)

Detection and monitoring of per- and polyfluoroalkyl substances (PFAS) in aquatic environments has become an increasingly higher priority of regulatory agencies as public concern for human intake of these chemicals continues to grow. While many methods utilize active sampling strategies (“grab samples”) for precise PFAS quantitation, here we evaluate the efficacy of low-cost passive sampling devices (Solid Phase Adsorption Toxin Tracking, or SPATTs) for spatial and temporal PFAS assessment of aquatic systems. For this study, passive samplers were initially deployed in North Carolina along the Cape Fear River during the summer and fall of 2016 and 2017. These were originally intended for the detection of microcystins and monitoring potentially harmful algal blooms, though this period also coincided with occurrences of PFAS discharge from a local fluorochemical manufacturer into the river. Additional samplers were then deployed in 2022 to evaluate changes in PFAS fingerprint and abundances. Assessment of PFAS showed legacy compounds were observed across almost all sampling sites over all 3 years (PFHxS, PFOS, PFHxA, etc.), while emerging replacement PFAS (e.g., Nafion byproducts) were predominantly localized downstream from the manufacturer. Furthermore, samplers deployed downstream from the manufacturer in 2022 noted sharp decreases in observed signal for replacement PFAS in comparison to samplers deployed in 2016 and 2017, indicating mitigation and remediation efforts in the area were able to reduce localized fluorochemical contamination.

CIUSuite 3: Next-Generation CCS Calibration and Automated Data Analysis Tools for Gas-Phase Protein Unfolding Data.

Ion mobility-mass spectrometry (IM-MS) has become a technology deployed across a wide range of structural biology applications despite the challenges in characterizing closely related protein structures. Collision-induced unfolding (CIU) has emerged as a valuable technique for distinguishing closely related, iso-cross-sectional protein and protein complex ions through their distinct unfolding pathways in the gas phase. With the speed and sensitivity of CIU analyses, there has been a rapid growth of CIU-based assays, especially regarding biomolecular targets that remain challenging to assess and characterize with other structural biology tools. With information-rich CIU data, many software tools have been developed to automate laborious data analysis. However, with the recent development of new IM-MS technologies, such as cyclic IM-MS, CIU continues to evolve, necessitating improved data analysis tools to keep pace with new technologies and facilitating the automation of various data processing tasks. Here, we present CIUSuite 3, a software package that contains updated algorithms that support various IM-MS platforms and supports the automation of various data analysis tasks such as peak detection, multidimensional classification, and collision cross section (CCS) calibration. CIUSuite 3 uses local maxima searches along with peak width and prominence filters to detect peaks to automate CIU data extraction. To support both the primary CIU (CIU1) and secondary CIU (CIU2) experiments enabled by cyclic IM-MS, two-dimensional data preprocessing is deployed, which allows multidimensional classification. Our data suggest that additional dimensions in classification improve the overall accuracy of class assignments. CIUSuite 3 also supports CCS calibration for both traveling wave and drift tube IM-MS, and we demonstrate the accuracy of a new single-field CCS calibration method designed for drift tube IM-MS leveraging calibrant CIU data. Overall, CIUSuite 3 is positioned to support current and next-generation IM-MS and CIU assay development deployed in an automated format.

Distinguishing Isomeric Cyclobutane Thymidine Dimers by Ion Mobility and Tandem Mass Spectrometry.

Irradiation of the major conformation of duplex DNA found in cells (B form) produces cyclobutane pyrimidine dimers (CPDs) from adjacent pyrimidines in a head-to-head orientation (syn) with the C5 substituents in a cis stereochemistry. These CPDs have crucial implications in skin cancer. Irradiation of G-quadruplexes and other non-B DNA conformations in vitro produces, however, CPDs between nonadjacent pyrimidines in nearby loops with syn and head-to-tail orientations (anti) with both cis and trans stereochemistry to yield a mixture of six possible isomers of the T=T dimer. This outcome is further complicated by formation of mixtures of nonadjacent CPDs of C=T, T=C, and C=C, and successful analysis depends on development of specific and sensitive methods. Toward meeting this need, we investigated whether ion mobility mass spectrometry (IMMS) and MS/MS can distinguish the cis,syn and trans,anti T=T CPDs. Ion mobility can afford baseline separation and give relative mobilities that are in accord with predicted cross sections. Complementing this ability to distinguish isomers is MS/MS collisional activation where fragmentation also distinguishes the two isomers and confirms conclusions drawn from ion mobility analysis. The observations offer early support that ion mobility and MS/MS can enable the distinction of DNA photoproduct isomers.

Improvement of Tumor Neoantigen Detection by High-Field Asymmetric Waveform Ion Mobility Mass Spectrometry.

Cancer neoantigens have been shown to elicit cancer-specific T-cell responses and have garnered much attention for their roles in both spontaneous and therapeutically induced antitumor responses. Mass spectrometry (MS) profiling of tumor immunopeptidomes has been used, in part, to identify MHC-bound mutant neoantigen ligands. However, under standard conditions, MS-based detection of such rare but clinically relevant neoantigens is relatively insensitive, requiring 300 million cells or more. Here, to quantitatively define the minimum detectable amounts of therapeutically relevant MHC-I and MHC-II neoantigen peptides, we analyzed different dilutions of immunopeptidomes isolated from the well-characterized T3 mouse methylcholanthrene (MCA)-induced cell line by MS. Using either data-dependent acquisition or parallel reaction monitoring (PRM), we established the minimum amount of material required to detect the major T3 neoantigens in the presence or absence of high field asymmetric waveform ion mobility spectrometry (FAIMS). This analysis yielded a 14-fold enhancement of sensitivity in detecting the major T3 MHC-I neoantigen (mLama4) with FAIMS-PRM compared with PRM without FAIMS, allowing ex vivo detection of this neoantigen from an individual 100 mg T3 tumor. These findings were then extended to two other independent MCA-sarcoma lines (1956 and F244). This study demonstrates that FAIMS substantially increases the sensitivity of MS-based characterization of validated neoantigens from tumors.

Cyclic Ion Mobility of Isomeric New Psychoactive Substances Employing Characteristic Arrival Time Distribution Profiles and Adduct Separation.

Analysis of new psychoactive substances (NPS), which is essential for toxicological and forensic reasons, can be made complicated by the presence of isomers. Ion mobility has been used as a standalone technique or coupled to mass spectrometry to detect and identify NPS. However, isomer separation has so far chiefly relied on chromatography. Here we report on the determination of isomeric ratios using cyclic ion mobility-mass spectrometry without any chromatographic separation. Isomers were distinguished by mobility separation of lithium adducts. Alternatively, we used arrival time distribution (ATD) profiles that were characteristic of individual isomers and were acquired for protonated molecules or fragment ions. Both approaches provided comparable results. Calculations were used to determine the structures and collision cross sections of both protonated and lithiated isomers that accurately characterized their ion mobility properties. The applicability of ATD profiles to isomer differentiation was demonstrated using direct infusion and flow injection analysis with electrospray of solutions, as well as desorption electrospray of solid samples. Data processing was performed by applying multiple linear regression to the ATD profiles. Using the proposed ATD profile-based approach, the relationships between the determined and given content of isomers showed good linearity with coefficients of determination typically greater than 0.99. Flow injection analysis using an autosampler allowed us to rapidly determine isomeric ratios in a sample containing two isomeric pairs with a minor isomer of 10% (determined 9.3% of 3-MMC and 11.0% of 3-FMC in a mixture with buphedrone and 4-FMC). The proposed approach is not only useful for NPS, but also may be applicable to small isomeric molecules analyzed by ion mobility when complete separation of isomers is not achieved.

Hyphenation of supercritical fluid chromatography and trapped ion mobility-mass spectrometry for quantitative lipidomics

BackgroundLipidomics studies require rapid separations with accurate and reliable quantification results to further elucidate the role of lipids in biological processes and their biological functions. Supercritical fluid chromatography (SFC), in particular, can provide this rapid and high-resolution separation. The combination with trapped ion mobility spectrometry (TIMS) has not yet been applied, although the post-ionization separation method in combination with liquid chromatography or imaging techniques has already proven itself in resolving isomeric and isobaric lipids and preventing false identifications. However, a multidimensional separation method should not only allow confident identification but also provide quantitative results to substantiate studies with absolute concentrations. ResultsA SFC method was developed and the hyphenation of SFC and TIMS was further explored towards the separation of different isobaric overlaps. Furthermore, lipid identification was performed using mass spectrometry (MS) and parallel accumulation serial fragmentation (PASEF) MS/MS experiments in addition to retention time and collision cross section (CCS). Quantification was further investigated with short TIMS ramps and performed based on the ion mobility signal of lipids, since TIMS increases the sensitivity by noise filtering. The final method was, as an exemplary study, applied to investigate the function of different ceramide synthases (CerS) in the nematode and model organism Caenorhabditis elegans (C. elegans). Loss of three known CerS hyl-1, hyl-2 and lagr-1 demonstrated different influences on and alterations in the sphingolipidome. SignificanceThis method describes for the first time the combination of SFC and TIMS-MS/MS, which enables a fast and sensitive quantification of lipids. The results of the application to C. elegans samples prove the functionality of the method and support research on the metabolism of sphingolipids in nematodes.

µPhos: a scalable and sensitive platform for high-dimensional phosphoproteomics

Mass spectrometry has revolutionized cell signaling research by vastly simplifying the analysis of many thousands of phosphorylation sites in the human proteome. Defining the cellular response to perturbations is crucial for further illuminating the functionality of the phosphoproteome. Here we describe µPhos (‘microPhos’), an accessible phosphoproteomics platform that permits phosphopeptide enrichment from 96-well cell culture and small tissue amounts in <8 h total processing time. By greatly minimizing transfer steps and liquid volumes, we demonstrate increased sensitivity, >90% selectivity, and excellent quantitative reproducibility. Employing highly sensitive trapped ion mobility mass spectrometry, we quantify ~17,000 Class I phosphosites in a human cancer cell line using 20 µg starting material, and confidently localize ~6200 phosphosites from 1 µg. This depth covers key signaling pathways, rendering sample-limited applications and perturbation experiments with hundreds of samples viable. We employ µPhos to study drug- and time-dependent response signatures in a leukemia cell line, and by quantifying 30,000 Class I phosphosites in the mouse brain we reveal distinct spatial kinase activities in subregions of the hippocampal formation.

Carboxylated pillar[5]arene cavity accommodates organic cations in the host-guest complexes

The electron-rich cavity of carboxylated pillar[5]arene efficiently encapsulates cationic guest molecules within supramolecular assemblies. In this study we provide evidence of the host-guest complexation between carboxylated pillar[5]arene and benzamidine, a competitive inhibitor of serine proteases. We show the unexpected inclusion of the polar amidinium functionality within the central cavity of the macrocyclic host sustained by N+-H···π and cation···π interactions. In addition to the in-cavity complexation, simultaneous external binding of bipyridinium guests with varying stoichiometries has been observed. The interesting aspect is the inclusion of a hydrogen bonded pair of 1,2-bis(4-pyridyl)ethylene guests into pillar[5]arene cavity. Additional insights into inclusion characteristics are provided by NMR and ion mobility mass spectrometry (IM-MS) studies, offering information for the solution/gas phase and enabling a comprehensive description of the complexation process across different phases. This work expands the portfolio of cationic guest molecules capable of being encapsulated by carboxylated pillar[5]arene, showing new promises and directions for their inclusion and self-assembly behavior.