Structural Identification of Eicosanoids with Ring Structures Using Differential Mobility Spectrometry-Electron Impact Excitation of Ions from Organics Mass Spectrometry.

We developed a structural identification method for eicosanoids with various ring structures using mass spectrometry. We discovered that an electron beam with a kinetic energy of 10 eV, which is in the Electron Impact Excitation of Ions from Organics (EIEIO) regime, cleaved the fatty acids enough to distinguish constitutional and cis/trans isomers. In addition to EIEIO, a comparison to authentic standards using differential mobility spectrometry (DMS) can identify diastereomers, which was difficult by EIEIO. The combination of EIEIO and DMS can provide a high-throughput method to identify complete structures of eicosanoids in mixed samples, which is not allowed with conventional analytical methods though eicosanoids are important signaling molecules in biosystems.

Electronic spectroscopy of differential mobility-selected prototropic isomers of protonated para-aminobenzoic acid.

para-Aminobenzoic acid (PABA) was electrosprayed from mixtures of protic and aprotic solvents, leading to formation of two prototropic isomers in the gas phase whose relative populations depended on the composition of the electrospray solvent. The two ion populations were separated in the gas phase using differential mobility spectrometry (DMS) within a nitrogen-only environment at atmospheric pressure. Under high-field conditions, the two prototropic isomers eluted with baseline signal separation with the N-protonated isomer having a more negative CV shift than the O-protonated isomer, in accord with previous DMS studies. The conditions most favorable for formation and separation of each tautomer were used to trap each prototropic isomer in a quadrupole ion trap for photodissociation action spectroscopy experiments. Spectral interrogation of each prototropic isomer in the UV region (3-6 eV) showed good agreement with previously recorded spectra, although a previously reported band (4.8-5.4 eV) was less intense for the O-protonated isomer in our measured spectrum. Without DMS selection, the measured spectra contained features corresponding to both protonated isomers even when solvent conditions were optimised for formation of a single isomer. Interconversion between protonated isomers within the ion trap was observed when protic ESI solvents were employed, leading to spectral cross contamination even with mobility selection. CCSD vertical excitation energies and vertical gradient (VG) Franck-Condon simulations are presented and reproduce the measured spectral features with near-quantitative agreement, providing supporting evidence for spectral assignments.

UVPD spectroscopy of differential mobility-selected prototropic isomers of protonated adenine.

Two prototropic isomers of adenine are formed in an electrospray ion source and are resolved spatially in a differential mobility spectrometer before detection in a triple quadrupole mass spectrometer. Each isomer is gated in CV space before being trapped in the linear ion trap of the modified mass spectrometer, where they are irradiated by the tuneable output of an optical parametric oscillator and undergo photodissociation to form charged fragments with m/z 119, 109, and 94. The photon-normalised intensity of each fragmentation channel is measured and the action spectra for each DMS-gated tautomer are obtained. Our analysis of the action spectra, aided by calculated vibronic spectra and thermochemical data, allow us to assign the two signals in our measured ionograms to specific tautomers of protonated adenine.

UVPD Spectroscopy of Differential Mobility-Selected Prototropic Isomers of Rivaroxaban.

Two ion populations of protonated Rivaroxaban, [C19H18ClN3O5S + H]+, are separated under pure N2 conditions using differential mobility spectrometry prior to characterization in a hybrid triple quadrupole linear ion trap mass spectrometer. These populations are attributed to bare protonated Rivaroxaban and to a proton-bound Rivaroxaban-ammonia complex, which dissociates prior to mass-selecting the parent ion. Ultraviolet photodissociation (UVPD) and collision-induced dissociation (CID) studies indicate that both protonated Rivaroxaban ion populations are comprised of the computed global minimum prototropic isomer. Two ion populations are also observed when the collision environment is modified with 1.5% (v/v) acetonitrile. In this case, the protonated Rivaroxaban ion populations are produced by the dissociation of the ammonium complex and by the dissociation of a proton-bound Rivaroxaban-acetonitrile complex prior to mass selection. Again, both populations exhibit a similar CID behavior; however, UVPD spectra indicate that the two ion populations are associated with different prototropic isomers. The experimentally acquired spectra are compared with computed spectra and are assigned to two prototropic isomers that exhibit proton sharing between distal oxygen centers.

Predicting differential ion mobility behaviour in silico using machine learning.

Although there has been a surge in popularity of differential mobility spectrometry (DMS) within analytical workflows, determining separation conditions within the DMS parameter space still requires manual optimization. A means of accurately predicting differential ion mobility would benefit practitioners by significantly reducing the time associated with method development. Here, we report a machine learning (ML) approach that predicts dispersion curves in an N2 environment, which are the compensation voltages (CVs) required for optimal ion transmission across a range of separation voltages (SVs) between 1500 to 4000 V. After training a random-forest based model using the DMS information of 409 cationic analytes, dispersion curves were reproduced with a mean absolute error (MAE) of ≤ 2.4 V, approaching typical experimental peak FWHMs of ±1.5 V. The predictive ML model was trained using only m/z and ion-neutral collision cross section (CCS) as inputs, both of which can be obtained from experimental databases before being extensively validated. By updating the model via inclusion of two CV datapoints at lower SVs (1500 V and 2000 V) accuracy was further improved to MAE ≤ 1.2 V. This improvement stems from the ability of the "guided" ML routine to accurately capture Type A and B behaviour, which was exhibited by only 2% and 17% of ions, respectively, within the dataset. Dispersion curve predictions of the database's most common Type C ions (81%) using the unguided and guided approaches exhibited average errors of 0.6 V and 0.1 V, respectively.

Determining Collision Cross Sections from Differential Ion Mobility Spectrometry.

The experimental determination of ion-neutral collision cross sections (CCSs) is generally confined to ion mobility spectrometry (IMS) technologies that operate under the so-called low-field limit or those that enable empirical calibration strategies (e.g., traveling wave IMS; TWIMS). Correlation of ion trajectories to CCS in other non-linear IMS techniques that employ dynamic electric fields, such as differential mobility spectrometry (DMS), has remained a challenge since its inception. Here, we describe how an ion's CCS can be measured from DMS experiments using a machine learning (ML)-based calibration. The differential mobility of 409 molecular cations (m/z: 86-683 Da and CCS 110-236 Å2) was measured in a N2 environment to train the ML framework. Several open-source ML routines were tested and trained using DMS-MS data in the form of the parent ion's m/z and the compensation voltage required for elution at specific separation voltages between 1500 and 4000 V. The best performing ML model, random forest regression, predicted CCSs with a mean absolute percent error of 2.6 ± 0.4% for analytes excluded from the training set (i.e., out-of-the-bag external validation). This accuracy approaches the inherent statistical error of ∼2.2% for the MobCal-MPI CCS calculations employed for training purposes and the <2% threshold for matching literature CCSs with those obtained on a TWIMS platform.

The Charge-State and Structural Stability of Peptides Conferred by Microsolvating Environments in Differential Mobility Spectrometry.

The presence of solvent vapor in a differential mobility spectrometry (DMS) cell creates a microsolvating environment that can mitigate complications associated with field-induced heating. In the case of peptides, the microsolvation of protonation sites results in a stabilization of charge density through localized solvent clustering, sheltering the ion from collisional activation. Seeding the DMS carrier gas (N2) with a solvent vapor prevented nearly all field-induced fragmentation of the protonated peptides GGG, AAA, and the Lys-rich Polybia-MP1 (IDWKKLLDAAKQIL-NH2). Modeling the microsolvation propensity of protonated n-propylamine [PrNH3]+, a mimic of the Lys side chain and N-terminus, with common gas-phase modifiers (H2O, MeOH, EtOH, iPrOH, acetone, and MeCN) confirms that all solvent molecules form stable clusters at the site of protonation. Moreover, modeling populations of microsolvated clusters indicates that species containing protonated amine moieties exist as microsolvated species with one to six solvent ligands at all effective ion temperatures (Teff) accessible during a DMS experiment (ca. 375-600 K). Calculated Teff of protonated GGG, AAA, and Polybia-MPI using a modified two-temperature theory approach were up to 86 K cooler in DMS environments seeded with solvent vapor compared to pure N2 environments. Stabilizing effects were largely driven by an increase in the ion's apparent collision cross section and by evaporative cooling processes induced by the dynamic evaporation/condensation cycles incurred in the presence of an oscillating electric separation field. When the microsolvating partner was a protic solvent, abstraction of a proton from [MP1 + 3H]3+ to yield [MP1 + 2H]2+ was observed. This result was attributed to the proclivity of protic solvents to form hydrogen-bond networks with enhanced gas-phase basicity. Collectively, microsolvation provides analytes with a solvent "air bag," whereby charge reduction and microsolvation-induced stabilization were shown to shelter peptides from the fragmentation induced by field heating and may play a role in preserving native-like ion configurations.

Separating Isomers, Conformers, and Analogues of Cyclosporin using Differential Mobility Spectroscopy, Mass Spectrometry, and Hydrogen-Deuterium Exchange.

Cyclosporins are an invaluable class of drug used to prevent the rejection of transplanted tissue. While the most popular drug in this group is cyclosporin A, several other analogues are available, including some enantiomeric and structurally isomeric forms. Unfortunately, the presence of such isomers can make the detection and identification of these drugs by mass spectrometry (MS) alone quite challenging. Here, we demonstrate the separation and analysis of six cyclosporin analogues using liquid chromatography (LC) and differential mobility spectroscopy (DMS) coupled to MS. Using DMS, we demonstrate the separation of three isomers: CycA and CycH (cyclosporin H), which are enantiomers, and isocyclosporin A (a structural isomer of CycA and CycH). For several of the cyclosporins, we can separate different conformers for each isomeric form. After DMS separation, tandem mass spectrometry (MS/MS) analyses of the separated isomers also distinguish these isomeric forms of cyclosporin. In addition, we have probed differences between each isomer by using gas-phase hydrogen-deuterium exchange (HDX) immediately after DMS separation, which reveals differences in the levels of intramolecular hydrogen bonding between each of the cyclosporins.

Measuring Electronic Spectra of Differential Mobility-Selected Ions in the Gas Phase.

We describe the modification of a commercially available tandem differential mobility mass spectrometer (DMS) that has been retrofitted to facilitate photodissociation (PD) of differential mobility-separated, mass-selected molecular ions. We first show that a mixture of protonated quinoline/isoquinoline (QH+/iQH+) can be separated using differential mobility spectrometry. Efficient separation is facilitated by addition of methanol to the DMS environment and increased residence time within the DMS. In action spectroscopy experiments, we gate each isomer using appropriate DMS settings, trap the ions in the third quadrupole of a triple quadrupole mass spectrometer, and irradiate them with tunable light from an optical parametric oscillator (OPO). The resulting mass spectra are recorded as the OPO wavelength is scanned, giving PD action spectra. We compare our PD spectra with previously recorded spectra for the same species and show that our instrument reproduces previous works faithfully.

How Hot Are Your Ions in Differential Mobility Spectrometry?

Ions can experience significant field-induced heating in a differential mobility cell. To investigate this phenomenon, the fragmentation of several para-substituted benzylpyridinium "thermometer" ions (R = OMe, Me, F, Cl, H, CN) was monitored in a commercial differential mobility spectrometer (DMS). The internal energy of each benzylpyridinium derivative was characterized by monitoring the degree of fragmentation to obtain an effective temperature, Teff, which corresponds to a temperature consistent with treating the observed fragmentation ratio using a unimolecular dissociation rate weighted by a Boltzmann distribution at a temperature T. It was found that ions are sufficiently thermalized after initial activation from the ESI process to the temperature of the bath gas, Tbath. Once a critical field strength was surpassed, significant fragmentation of the benzylpyridinium ions was detected. At the maximum bath gas temperature (450 K) and separation voltage (SV; 4400 V) for our instrument, Teff for the benzylpyridinium derivatives ranged from 664 ± 9 K (p-OMe) to 759 ± 17 K (p-H). The extent of activation at a given SV depends on the ion's mass, degrees of freedom, (NDoF), and collision frequency as represented by the ion's collision cross section. Plots of Teff vs the product of ion mass and NDoF and the inverse of collision cross section produce strong linear relationships. This provides an attractive avenue to estimate ion temperatures at a given SV using only intrinsic properties. Moreover, experimentally determined Teff correlate with theoretically predicted Teff using with a self-consistent method based on two-temperature theory. The various instrumental and external parameters that influence Teff are additionally discussed.

Separating chiral isomers of amphetamine and methamphetamine using chemical derivatization and differential mobility spectrometry.

The separation and analysis of chiral compounds, especially enantiomers, presents a great challenge to modern analytical chemistry, particularly to mass spectrometry (MS). As a result, integrated orthogonal separations, such as chiral liquid chromatography (chiral LC), gas chromatography (GC), or capillary electrophoresis (CE), are often employed to separate enantiomers prior to MS analysis. Here, we combine chemical derivatization with differential mobility spectrometry (DMS) and MS to separate and quantitate the transformed enantiomeric pairs R- and S-amphetamine, as well as R- and S-methamphetamine. We also demonstrate separation of these drugs by using reverse-phase LC. However, while the LC method requires ∼5 min to provide separation, we have developed a flow-injection analysis (FIA) method using DMS as the exclusive mode of separation (FIA-DMS), requiring only ∼1.5 min with equivalent quantitative metrics (1-1000 ng/mL range) to the LC method. The DMS-based separation of each diastereomeric pair is driven by differences in binding energies between the analyte ions and the chemical modifier molecules (acetonitrile) added to the DMS environment.

Preferential Ion Microsolvation in Mixed-Modifier Environments Observed Using Differential Mobility Spectrometry.

The preferential solvation behavior for eight different derivatives of protonated quinoline was measured in a tandem differential mobility spectrometer mass spectrometer (DMS-MS). Ion-solvent cluster formation was induced in the DMS by the addition of chemical modifiers (i.e., solvent vapors) to the N2 buffer gas. To determine the effect of more than one modifier in the DMS environment, we performed DMS experiments with varying mixtures of water, acetonitrile, and isopropyl alcohol solvent vapors. The results show that doping the buffer gas with a binary mixture of modifiers leads to the ions binding preferentially to one modifier over another. We used density functional theory to calculate the ion-solvent binding energies, and in all cases, calculations show that the quinolinium ions bind most strongly with acetonitrile, then isopropyl alcohol, and most weakly with water. Computational results support the hypothesis that the quinolinium ions bind exclusively to whichever solvent they have the strongest interaction with, regardless of the presence of other modifier gases.

Ultraviolet irradiation of trypsin, lysozyme and ß-galactosidase: how does UVC affect these enzymes when used as a secondary barrier against adventitious agents?

Trypsin is one of the essential raw materials used in the manufacturing of biopharmaceutical products. As an animal derived product, it can potentially carry a serious risk of contamination with adventitious agents that can result in production shut down and lost product. To mitigate these risks, several methods are currently being used in the industry to remove contamination including physical and chemical methods. Ultraviolet-C (UVC) light is known to inactivate adventitious agents that are resistant to physical and chemical methods and could be a secondary barrier strategy. In this study, we investigated the effect of UVC irradiation on the activity and structure of trypsin. Extreme doses of UVC light were applied to trypsin using a collimated beam apparatus. The effect of UVC light on trypsin enzymatic activity was measured using a colorimetric activity assay and the effect on structure was analyzed by spectrophotometry, gel electrophoresis, and mass spectrometry. To broaden the scope, the effect of UVC light on the activity of two additional enzymes, lysozyme and ß-galactosidase, was also examined. At high doses of UVC light, changes to protein structure and protein fragmentation resulted in decreased trypsin activity. However, minimal damage was observed at doses applicable to inactivating adventitious agents, making UVC a feasible treatment for viral inactivation of trypsin products.

Separation of Sialylated Glycan Isomers by Differential Mobility Spectrometry.

Mass spectrometry has proven itself to be an important technology for characterizing intact glycoproteins, glycopeptides, and released glycans. However, these molecules often present significant challenges during analysis. For example, glycans of identical molecular weights can be present in many isomeric forms, with one form having dramatically more biological activity than the others. Discriminating among these isomeric forms using mass spectrometry alone can be daunting, which is why orthogonal techniques, such as ion mobility spectrometry, have been explored. Here, we demonstrate the use of differential mobility spectrometry (DMS) to separate isomeric glycans differing only in the linkages of sialic acid groups (e.g., α 2,3 versus α 2,6). This ability extends from a small trisaccharide species to larger biantennary systems and is driven, in part, by the role of intramolecular solvation of the charge site(s) on these ions within the DMS environment.

Combining Charge-Switch Derivatization with Ozone-Induced Dissociation for Fatty Acid Analysis.

The specific positions of carbon-carbon double bond(s) within an unsaturated fatty acid exert a significant effect on the physical and chemical properties of the lipid that ultimately inform its biological function(s). Contemporary liquid chromatography-mass spectrometry (MS) strategies based on electrospray ionization coupled to tandem MS can easily detect fatty acyl lipids but generally cannot reveal those specific site(s) of unsaturation. Herein, we describe a novel and versatile workflow whereby fatty acids are first converted to fixed charge N-(4-aminomethylphenyl)pyridinium (AMPP) derivatives and subsequently subjected to ozone-induced dissociation (OzID) on a modified triple quadrupole mass spectrometer. The AMPP modification enhances the detection of fatty acids introduced by direct infusion. Fragmentation of the derivatized fatty acids also provides diagnostic fragment ions upon collision-induced dissociation that can be targeted in precursor ion scans to subsequently trigger OzID analyses in an automated data-dependent workflow. It is these OzID analyses that provide unambiguous assignment of carbon-carbon double bond locations in the AMPP-derivatized fatty acids. The performance of this analysis pipeline is assessed in profiling the patterns of unsaturation in fatty acids within the complex biological secretion vernix caseosa. This analysis uncovers significant isomeric diversity within the fatty acid pool of this sample, including a number of hitherto unreported double bond positional isomers that hint at the activity of potentially new metabolic pathways.

Recommendations for reporting ion mobility Mass Spectrometry measurements.

Here we present a guide to ion mobility mass spectrometry experiments, which covers both linear and nonlinear methods: what is measured, how the measurements are done, and how to report the results, including the uncertainties of mobility and collision cross section values. The guide aims to clarify some possibly confusing concepts, and the reporting recommendations should help researchers, authors and reviewers to contribute comprehensive reports, so that the ion mobility data can be reused more confidently. Starting from the concept of the definition of the measurand, we emphasize that (i) mobility values (K0 ) depend intrinsically on ion structure, the nature of the bath gas, temperature, and E/N; (ii) ion mobility does not measure molecular surfaces directly, but collision cross section (CCS) values are derived from mobility values using a physical model; (iii) methods relying on calibration are empirical (and thus may provide method-dependent results) only if the gas nature, temperature or E/N cannot match those of the primary method. Our analysis highlights the urgency of a community effort toward establishing primary standards and reference materials for ion mobility, and provides recommendations to do so. © 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc.

A parallelized molecular collision cross section package with optimized accuracy and efficiency.

Ion mobility-based separation prior to mass spectrometry has become an invaluable tool in the structural elucidation of gas-phase ions and in the characterization of complex mixtures. Application of ion mobility to structural studies requires an accurate methodology to bridge theoretical modelling of chemical structure with experimental determination of an ion's collision cross section (CCS). Herein, we present a refined methodology for calculating ion CCS using parallel computing architectures that makes use of atom specific parameters, which we have called MobCal-MPI. Tuning of ion-nitrogen van der Waals potentials on a diverse calibration set of 162 molecules returned a RMSE of 2.60% in CCS calculations of molecules containing the elements C, H, O, N, F, P, S, Cl, Br, and I. External validation of the ion-nitrogen potential was performed on an additional 50 compounds not present in the validation set, returning a RMSE of 2.31% for the CCSs of these compounds. Owing to the use of parameters from the MMFF94 forcefield, the calibration of the van der Waals potential can be extended to additional atoms defined in the MMFF94 forcefield (i.e., Li, Na, K, Si, Mg, Ca, Fe, Cu, Zn). We expect that the work presented here will serve as a foundation for facile determination of molecular CCSs, as MobCal-MPI boasts up to 64-fold speedups over traditional calculation packages.

Determining molecular properties with differential mobility spectrometry and machine learning.

The fast and accurate determination of molecular properties is highly desirable for many facets of chemical research, particularly in drug discovery where pre-clinical assays play an important role in paring down large sets of drug candidates. Here, we present the use of supervised machine learning to treat differential mobility spectrometry - mass spectrometry data for ten topological classes of drug candidates. We demonstrate that the gas-phase clustering behavior probed in our experiments can be used to predict the candidates' condensed phase molecular properties, such as cell permeability, solubility, polar surface area, and water/octanol distribution coefficient. All of these measurements are performed in minutes and require mere nanograms of each drug examined. Moreover, by tuning gas temperature within the differential mobility spectrometer, one can fine tune the extent of ion-solvent clustering to separate subtly different molecular geometries and to discriminate molecules of very similar physicochemical properties.

Quantitative structural multiclass lipidomics using differential mobility: electron impact excitation of ions from organics (EIEIO) mass spectrometry.

We report a method for comprehensive structural characterization of lipids in animal tissues using a combination of differential ion mobility spectrometry (DMS) with electron-impact excitation of ions from organics (EIEIO) mass spectrometry. Singly charged lipid ions in protonated or sodiated forms were dissociated by an electron beam having a kinetic energy of 10 eV in a branched radio-frequency ion trap. We established a comprehensive set of diagnostics to characterize the structures of glycerophospholipids, sphingolipids, and acylglycerols, including glycosylated, plasmalogen, and ester forms. This EIEIO mass spectrometer was combined with DMS as a separation tool to analyze complex lipid extracts. Deuterated quantitative standards, which were added during extraction, allowed for the quantitative analysis of the lipid molecular species in various lipid classes. We applied this technique to the total lipids extracted from porcine brain, and we structurally characterized over 300 lipids (with the exception of cis/trans double-bond isomerism in the acyl chains). The structural dataset of the lipidomes, whose regioisomers were distinguished, exhibit a uniquely defined distribution of acyl chains within each lipid class; that is, sn-1 and sn-2 in the cases of glycerophospholipids or sn-2 and (sn-1, sn-3) in the cases of triacylglycerols.

Identifying Fenton-Reacted Trimethoprim Transformation Products Using Differential Mobility Spectrometry.

A transformation product of trimethoprim, a contaminant of emerging concern in the environment, is generated using an electro-assisted Fenton reaction and analyzed using differential mobility spectrometry (DMS) in combination with MS/MS techniques and quantum chemical calculations to develop a rapid method for identification. DMS is used as a prefilter to separate positional isomers prior to subsequent identification by mass spectrometric analyses. Collision induced dissociation of each DMS separated species is used to reveal fragmentation patterns that can be correlated to specific isomer structures. Analysis of the experimental data and supporting quantum chemical calculations show that methylene-hydroxylated and methoxy-containing phenyl ring hydroxylated transformation products are observed. The proposed methodology outlines a high-throughput technique to determine transformation products of small molecules accurately, in a short time and requiring minimal sample concentrations (<25 ng/mL).