Electrospray Ion Mobility Mass Spectrometry Research Articles

ESI-IM-MS characterization of cyclodextrin complexes and their chemically cross-linked alpha (α-), beta (β-) and gamma (γ-) cyclodextrin particles as promising drug delivery materials with improved bioavailability.

Cyclodextrins (CDs) are natural cyclic oligosaccharides with a relatively hydrophobic cavity and a hydrophilic outer surface. In this study, alpha (α-), beta (β-) and gamma (γ-) CD particles were prepared by directly using α-, β-, and γ-CDs as monomeric units and divinyl sulfone (DVS) as a crosslinker in a single-step via reverse micelle microemulsion crosslinking technique. Particles of p(α-CD), p(β-CD), and p(γ-CD) were perfectly spherical in sub- 10µm size ranges. The prepared p(CD) particles at 1.0mg/mL concentrations were found biocompatible with >95 % cell viability against L929 fibroblasts. Furthermore, p(α-CD) and p(β-CD) particles were found non-hemolytic with <2 % hemolysis ratios, whereas p(γ-CD) particles were found to be slightly hemolytic with its 2.1±0.4 % hemolysis ratio at 1.0mg/mL concentration. Furthermore, a toxic compound, Bisphenol A (BPA) and a highly antioxidant polyphenol, curcumin (CUR) complexation with α-, β-, and γ-CD molecules was investigated via Electrospray-Ion Mobility-Mass Spectrometry (ESI-IM-MS) and tandem mass spectrometry (MS/MS) analysis. It was determined that the most stable noncovalent complex was in the case of β-CD, but the complex stoichiometry was changed by the hydrophobic nature of the guest molecules. In addition, BPA and CUR were separately loaded into prepared p(CD) particles as active agents. The drug loading and release studies showed that p(CD) particles possess governable loading and releasing profiles.

Thermochemical Studies of Ni(II) and Zn(II) Ternary Complexes using Ion Mobility-Mass Spectrometry.

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry (ES-IM-MS) and energy-resolved threshold collision-induced dissociation (TCID) to measure the thermochemistry of the dissociation of negatively-charged [amb+M(II)+NTA]- ternary complexes into two product channels: [amb+M(II)] + NTA or [NTA+M(II)]-+ amb, where M = Zn or Ni and NTA is nitrilotriacetic acid. The complexes contain one of the alternative metal binding (amb) heptapeptides with the primary structures acetyl-His1-Cys2-Gly3-Pro4-Tyr5-His6-Cys7 or acetyl-Asp1-Cys2-Gly3-Pro4-Tyr5-His6-Cys7, where the amino acids' Aa1,2,6,7 positions are the potential metal-binding sites. Geometry-optimized stationary states of the ternary complexes and their products were selected from quantum chemistry calculations (presently the PM6 semi-empirical Hamiltonian) by comparing their electronic energies and their collision cross-sections (CCS) to those measured by ES-IM-MS. From the PM6 frequency calculations, the molecular parameters of the ternary complex and its products model the energy-dependent intensities of the two product channels using a competitive TCID method to determine the threshold energies of the reactions that relate to the 0 K enthalpies of dissociation (ΔH0). Statistical mechanics thermal and entropy corrections using the PM6 rotational and vibrational frequencies provide the 298 K enthalpies of dissociation (ΔH298). These methods describe an EI-IM-MS routine that can determine thermochemistry and equilibrium constants for a range of ternary metal ion complexes.

Ion mobility-mass spectrometry for the separation and analysis of procyanidins.

Procyanidins are polymeric flavan-3-ones occurring in many plants with antioxidant and other beneficial bioactivities. They are composed of catechin and epicatechin monomeric units connected by single carbon-carbon B-type linkages or A-type linkages containing both carbon-carbon and carbon-oxygen-carbon bonds. Their polymeric structure makes analysis of procyanidin mixtures always difficult. Evaluation of procyanidins according to degree of polymerization (DP) using high-performance liquid chromatography (HPLC) is time-consuming and at best has resolved polymeric families up to DP-17. To expedite studies of procyanidins, the utility of positive ion electrospray ion mobility-mass spectrometry (IM-MS) was investigated for the rapid separation and characterization of procyanidins in mixtures. Applying IM-MS to analyse structurally defined standards containing up to five subunits, procyanidins could be resolved in less than 6ms not only by degree of polymerization but also by linkage type. A-type procyanidins could be resolved from B-type and both could be at least partially resolved from mixed-type procyanidins of the same DP. IM-MS separated higher order procyanidins with DP of at least 24 from extracts of cranberry. As DP increased, the abundances of multiply-charged procyanidins also increased. During IM-MS of ions of similar m/z, the ion drift times decreased inversely with increasing charge state. Therefore, IM-MS was shown to separate mixtures of procyanidins containing at least 24 interconnected subunits in less than 16ms, not only according to DP, but also according to linkage type between subunits and charge state.

Stereoselective Assembly of Gigantic Chiral Molybdenum Blue Wheels Using Lanthanide Ions and Amino Acids.

The synthesis of chiral polyoxometalates (POMs) is a challenge because of the difficulty to induce the formation of intrinsically chiral metal-oxo frameworks. Herein we report the stereoselective synthesis of a series of gigantic chiral Mo Blue (MB) POM clusters 1–5 that are formed by exploiting the synergy between coordinating lanthanides ions as symmetry breakers to produce MBs with chiral frameworks decorated with amino acids ligands; these promote the selective formation of enantiopure MBs. All the compounds share the same framework archetype, based on {Mo124Ce4}, which forms an intrinsically chiral Δ or Λ configurations, controlled by the configurations of functionalized chiral amino acids. The chirality and stability of 1–5 in solution are confirmed by circular dichroism, 1H NMR, and electrospray ion mobility–mass spectrometry studies. In addition, the framework of the {Mo124Ce4} MB not only behaves as a host able to trap a chiral {Mo8} cluster that is not accessible by traditional synthesis but also promotes the transformation of tryptophan to kynurenine in situ. This work demonstrates the potential and applicability of our synthetic strategy to produce gigantic chiral POM clusters capable of host–guest chemistry and selective synthetic transformations.

Entrapment of amino acids in gas phase surfactant assemblies: The case of tryptophan confined in positively charged (1R,2S)-dodecyl (2-hydroxy-1-methyl-2-phenylethyl) dimethylammonium bromide aggregates.

The ability of positively charged aggregates of the surfactant (1R,2S)-dodecyl(2-hydroxy-1-methyl-2-phenylethyl)dimethylammonium bromide (DMEB) to incorporate D-tryptophan or L-tryptophan in the gas phase has been investigated by electrospray ion mobility mass spectrometry (ESI-IM-MS). Strongly impacted by the pH of the electrosprayed solutions, both protonated (T+ ) and deprotonated (T- ) tryptophan are effectively included into the aggregates, whereas, tryptophan in zwitterionic (T0 ) form is practically absent in singly charged DMEB aggregates but can be found in multiply charged ones. The ability to incorporate tryptophan increases with the aggregation number and charge state of aggregates. More than 1 tryptophan species can be entrapped (aggregates including up to 5 tryptophan are observed). Collision induced dissociation experiments performed on the positively singly charged DMEB hexamer containing 1 T- show that at low collision energies the loss of a DMEB molecule is preferred with respect to the loss of the DMEB cation plus T- species which, in turn, is preferred with respect to the loss of mere tryptophan, suggesting that the deprotonated amino acid is preferentially located in proximity of a DMEB head group and with the ionic moiety pointing towards the core of the aggregate. The analysis of the collision cross sections (CCS) of bare and tryptophan containing aggregates allowed evaluating the contributions of tryptophan and bromide ions to the total aggregate CCS. No significant discrimination between D-tryptophan and L-tryptophan by the chiral DMEB aggregates has been evidenced by mass spectra data, CID experiments, and CCS values.

Gas-phase protein conformation/multimer ion formation by electrospray ion mobility-mass spectrometry: bovine insulin and ubiquitin.

Ion mobility-mass spectrometry (IMMS) is a very attractive method for studies in structural biology because of the ability of rapid isolation by nearly simultaneous m/z characterization and size separation, leading to an emergence of IMMS as a complimentary biochemical tool. Earlier, we developed a method based on varying the protein concentration in solution prior to electrospray ionization (ESI) with subsequent m/z selection and dissociation of protein multimers by IMMS of cytochrome c. The focus of this work will be to correctly distinguish truly different ion conformations formed by ESI versus homomultimeric complexes with the same m/z for well-studied proteins bovine ubiquitin and insulin. These proteins were chosen due to their large difference in solution phase structures: insulin tightly bound by disulfide linkages, and ubiquitin-a protein that may adopt a range of states from compact to extended. Our preliminary results, as with cytochrome c reveal false negatives for protein oligomer formation and false positives for protein conformational states. In addition, these results will be couched in terms of the need for quantification of IMMS analysis of proteins given the total area under IMMS peaks can also distinguish conformation versus aggregation as higher order oligomers have more mass per ion.This article is part of the themed issue 'Quantitative mass spectrometry'.

ATP and autophosphorylation driven conformational changes of HipA kinase revealed by ion mobility and crosslinking mass spectrometry.

Toxin-antitoxin systems are genetic modules involved in a broad range of bacterial cellular processes including persistence, multidrug resistance and tolerance, biofilm formation, and pathogenesis. In type II toxin-antitoxin systems, both the toxin and antitoxin are proteins. In the prototypic Escherichia coli HipA-HipB module, the antitoxin HipB forms a complex with the protein kinase HipA and sequesters it in the nucleoid. HipA is then no longer able to phosphorylate glutamyl-tRNA-synthetase and this prevents the initiation of the forthcoming stringent response. Here we investigated the assembly of the Shewanella oneidensis MR-1 HipA-HipB complex using native electrospray ion mobility-mass spectrometry and chemical crosslinking combined with mass spectrometry. We revealed that the HipA autophosphorylation was accompanied by a large conformational change, and confirmed structural evidence that S. oneidensis MR-1 HipA-HipB assembly was distinct from the prototypic E. coli HipA-HipB complex. Graphical abstract Ion mobility mass spectrometry shows a two phase transition from unstructured HipA to a compact folded phosphorylated protein.

Electrospray ion mobility mass spectrometry of positively and negatively charged (1R,2S)-dodecyl(2-hydroxy-1-methyl-2-phenylethyl)dimethylammonium bromide aggregates.

Self-assembling processes of surfactants in the gas phase constitute a developing research field of interest since they allow information to be gained on the peculiar structural organization of these aggregates, on their ability to incorporate from small molecules up to proteins and on their possible use as carriers of drugs in the gas phase or as cleaning agents and exotic reaction media. The mass spectra of charged aggregates of the chiral surfactant (1R,2S)-dodecyl(2-hydroxy-1-methyl-2-phenylethyl)dimethylammonium bromide (DMEB) in the gas phase have been recorded using a Synapt G2-Si mass spectrometer in the positive and negative ion mode. For comparison purposes, the mass spectra of sodium bis(2-ethylhexyl)sulfosuccinate and sodium octane sulfonate aggregates have also been recorded under the same experimental conditions. The collisional cross sections of positively and negatively charged DMEB aggregates were obtained through an appropriate calibration of the measured drift times. For all the surfactants investigated, it has been found that there is a lowest and a highest limit of the aggregation number at each charge state: no aggregates are found outside this range. Moreover, the occurrence at each aggregation number and extra charge of a unique value of drift time points toward aggregates whose conformations do not show discernible shape change in the experiment time scale. The analysis of the collisional cross sections emphasizes that the DMEB aggregates are nearly spherical clusters somewhat affected by the charge state and constituted by interlaced polar and apolar domains. The analysis of all the experimental findings indicates that in the gas phase DMEB forms supramolecular aggregates characterized by an internal organization whose stability is triggered by the charge state. The comparison of the behavior of DMEB aggregates with that of sodium bis(2-ethylhexyl)sulfosuccinate and sodium octane sulfonate aggregates allows us to highlight the effects on the aggregate organization in gas phase due to nature of the head group and alkyl chain steric hindrance.

Differentiation of prototropic ions in regioisomeric caffeoyl quinic acids by electrospray ion mobility mass spectrometry.

A series of dietary important regioisomeric chlorogenic acids were investigated by ion mobility mass spectrometry (IM-MS). The existence of prototropic isomers separated in the drift dimension was observed and investigated further using tandem mass spectrometry (MS/MS) and compared with suitable synthetic analogues. Using a quadrupole ion mobility time-of-flight mass spectrometer, the IM-MS and IM-MS/MS spectra of selected chlorogenic acids were recorded in the negative ion mode and compared with synthetic analogues. Regioisomeric di- and monocaffeoyl quinic acids can be readily separated and investigated using IM-MS. Comparison of drift times allows assignment of the regiochemistry of precursor ions as well as for fragment ions. For 5-caffeoyl quinic acid the existence of prototropic ions was suggested and probed using synthetic analogues, unable to show this type of isomerism. These investigations suggest the presence of prototropic isomers with carboxylate and phenolate sites, respectively. We report on IM-MS measurements on regioisomeric mono- and dicaffeoyl quinic acids, which are important dietary natural products. Both classes of compounds can be readily separated by IM-MS in the drift time dimension and, following MS(2) experiments, fragment ion regiochemistry unambiguously determined. 5-Caffeoyl quinic acid shows two IM-MS signals, which we assign to prototropic isomers after comparison with suitable synthetic analogues, with a negative charge located at the carboxalate or phenolate functionality, respectively.

Structural and biophysical characterization of Bacillus thuringiensis insecticidal proteins Cry34Ab1 and Cry35Ab1.

Bacillus thuringiensis strains are well known for the production of insecticidal proteins upon sporulation and these proteins are deposited in parasporal crystalline inclusions. The majority of these insect-specific toxins exhibit three domains in the mature toxin sequence. However, other Cry toxins are structurally and evolutionarily unrelated to this three-domain family and little is known of their three dimensional structures, limiting our understanding of their mechanisms of action and our ability to engineer the proteins to enhance their function. Among the non-three domain Cry toxins, the Cry34Ab1 and Cry35Ab1 proteins from B. thuringiensis strain PS149B1 are required to act together to produce toxicity to the western corn rootworm (WCR) Diabrotica virgifera virgifera Le Conte via a pore forming mechanism of action. Cry34Ab1 is a protein of ∼14 kDa with features of the aegerolysin family (Pfam06355) of proteins that have known membrane disrupting activity, while Cry35Ab1 is a ∼44 kDa member of the toxin_10 family (Pfam05431) that includes other insecticidal proteins such as the binary toxin BinA/BinB. The Cry34Ab1/Cry35Ab1 proteins represent an important seed trait technology having been developed as insect resistance traits in commercialized corn hybrids for control of WCR. The structures of Cry34Ab1 and Cry35Ab1 have been elucidated to 2.15 Å and 1.80 Å resolution, respectively. The solution structures of the toxins were further studied by small angle X-ray scattering and native electrospray ion mobility mass spectrometry. We present here the first published structure from the aegerolysin protein domain family and the structural comparisons of Cry34Ab1 and Cry35Ab1 with other pore forming toxins.

Speciation effects of solvent chemistry on the analysis of drugs and explosives by electrospray ion mobility mass spectrometry

The present work highlights the effect that solvent chemistry has on analytes being detected when utilizing electrospray ionization in ion mobility mass spectrometers. Various studies were conducted involving both positive and negative mode ionization. The enhancements observed by utilizing different levels of acid modifiers as well as the different ionic species of analytes being formed and detected are presented. The makeup of the electrospray solvent determined whether the ionic species would be protonated, sodium adducted, ammonium adducted, proton abstracted or chlorinated. Addition of solvent modifiers such as acids in the positive mode, and organic halides in the negative mode improved signal strength of analytes as well as improved spray characteristics that also decreased signal variability. The ability to perform simultaneous ionization of complex mixtures was also demonstrated through the use of solvent modifiers thereby allowing electrospray ionization ion mobility mass spectrometry the ability to simultaneously analyze complicated mixtures very quickly (millisecond separations).

Critical Examination of Gas-Phase Protein Conformation/Multimer Ion Formation by Electrospray Ion Mobility-Mass Spectrometry

The generally accepted view of protein structure in the gas-phase is that protein ions produced by electrospray ionization (ESI) exist in a number of different states, and the resulting charge state distribution (CSD) and ion mobility spectrum is interpreted as evidence for protein ions retaining some memory of solution-phase conformation. Even with the inclusion of ion mobility information, reports of protein ion structure in the gas-phase are oftentimes in disagreement not only within the discipline but also as interpreted by other gas-phase techniques. The focus of this work will be to correctly distinguish truly different ion conformations formed by ESI versus homomultimeric complexes with the same m/z. The concentration of cytochrome c in solution was varied over a wide range, and the multiply charged multimers (MCMs) present in the ion mobility/mass spectrum were unambiguously assigned by m/z selection and dissociation prior to ion mobility/mass spectrometry analysis. The results revealed false negatives for protein oligomer formation and false positives for protein conformational states and no evidence that gas-phase cytochrome c ions retain memory of solution-phase conformation, characteristics of great importance for structural biology. The results also suggest that the total IM-MS distribution for a protein is the complex result of individual MCMs either surviving until detection (undissociated) or dissociating into lower order multimers or a number of product ions for each m/z.

Real time pharmaceutical reaction monitoring by electrospray ion mobility-mass spectrometry

Reaction monitoring is a critical step in the synthesis of new drug entities, and many pharmaceutical companies spend significant resources on equipment and new methods to perform this task. The potential of electrospray ion mobility-mass spectrometry (ESI-IMMS) as a method for monitoring pharmaceutical reactions in real time is demonstrated with a reductive amination reaction that is commonly used in the synthesis of amines for drug discovery but poses problems for detection with current analytical instrumentation. Data were collected on the timescale of 300s and reaction completion time was determined by monitoring the starting, intermediate and product materials during the reaction. The detection limits for nicotinaldehyde (starting material 1), 4-picolylamine (starting material 2), and di-(2-picolyl)amine (the product) were 0.32, 0.10 and 0.05μM, respectively, with a linear dynamic range of more than 2 orders of magnitude for each analyte. Starting materials were not separated by their mobility in a nitrogen buffer gas, but could be separated in a buffer gas of carbon dioxide. In the nitrogen buffer gas, the reduced mobility (Ko) values for the reaction starting materials (nicotinaldehyde, 4-picolylamine, 4-pyridinementhanol) were 1.86cm2/Vs and the reduced mobility value for the product (di-(2-picolyl)amine) was 1.69cm2/Vs. In carbon dioxide the Ko values for the reaction starting materials were 1.43cm2/Vs and 1.38cm2/Vs for nicotinaldehyde and 4-picolylamine, respectively. The coupling of IMS to MS enabled the discovery of a hemiacetal complex forming between nicotinaldehyde and methanol, limiting the availability of nicotinaldehyde for the reaction. Thus use of IMS in conjunction with MS provided information about the reaction mechanism that would not have been possible with MS alone. The complexation prevented the formation of the intermediate species, presumably due to the formation of the hemiacetal from SM1; however, the intermediate species could be formed when the nicotinaldehyde concentration was increased by 10-fold. While either IMS or MS alone could be used to monitor this and similar reactions, the combined ESI-IMMS approach offered more complete information about the reaction, demonstrating potential as a rapid and selective technique to aid pharmaceutical process control.

The novel analysis of uranyl compounds by electrospray-ion mobility-mass spectrometry

This study relates the first mass identification of mobility peaks associated with uranyl species. These uranyl species were introduced into the gas phase by electrospray ionization and detected by ion mobility-mass spectrometry (IM-MS) to obtain rapid chemical information from uranyl compounds. Uranyl compound analysis in nuclear forensic science is typically performed using alpha, gamma, and mass spectrometry after extensive sample preparation and purification. Although providing highly sensitive isotopic and concentration information, these methods do not provide chemical information during the initial stages of analysis. Ion mobility spectrometry, when coupled with mass spectrometry, provides chemical information, including mass-identified mobility values, for analyte identification. In this study, uranyl compounds were detected in both the positive and negative ionization modes by electrospray-ion mobility-time of flight mass spectrometry (ESI-IM-TOFMS). The results showed that the sample type influenced the analyte ions that formed in the negative mode and that ESI solvent composition was the main factor that influenced analyte ion formation in the positive mode analysis. These results indicate that ESI-IM-TOFMS can be used to obtain rapid, chemical information for the initial analysis of a sample containing uranyl compounds.

Resolving Structural Isomers of Monosaccharide Methyl Glycosides Using Drift Tube and Traveling Wave Ion Mobility Mass Spectrometry

Monosaccharide structural isomers including sixteen methyl-D-glycopyranosides and four methyl-N-acetylhexosamines were subjected to ion mobility measurements by electrospray ion mobility mass spectrometry. Two ion mobility-MS systems were employed: atmospheric pressure drift tube ion mobility time-of-flight mass spectrometry and a Synapt G2 HDMS system which incorporates a low pressure traveling wave ion mobility separator. All the compounds were investigated as [M + Na](+) ions in the positive mode. A majority of the monosaccharide structural isomers exhibited different mobility drift times in either system, depending on differences in their anomeric and stereochemical configurations. In general, drift time patterns (relative drift times of isomers) matched between the two instruments. Higher resolving power was observed using the atmospheric pressure drift tube. Collision cross section values of monosaccharide structural isomers were directly calculated from the atmospheric pressure ion mobility experiments, and a collision cross section calibration curve was made for the traveling wave ion mobility instrument. Overall, it was demonstrated that ion mobility-mass spectrometry using either drift tube or traveling wave ion mobility is a valuable technique for resolving subtle variations in stereochemistry among the sodium adducts of monosaccharide methyl glycosides.

Metabolic differences among melanoma and two prostate cancer cell lines by electrospray ion mobility mass spectrometry

Detecting early stages of cancer has been a challenge for analytical methods. Even with the advances in mammography and prostate specific antigen (PSA) testing the number of false positives and negatives is still significant. Cancer cell lines provide realistic simulations to assess detection technologies for different stages and types of cancer. Metabolomics, the measure of end products of all biological processes, is a recent addition to the field of systems biology and can be targeted towards early disease detection. For this study, the metabolomes of three cancer cell lines, two prostate (DU145 and PC3) and one melanoma (A375), were investigated by an ion mobility mass spectrometer (IMMS) for both positive and negative ion detection mode. IMMS produced multidimensional data that includes m/z, mobility and intensity for each ion detected in the sample. In order to see differences between the cancer cell lines, principal components analysis (PCA), a multi-variant technique used to pull patterns out of the data, was used in conjunction with the IMMS data. The sample patterns from the PCA were observed in the score plot and the individual metabolites that contributed the most to the model were shown in the loadings plot. For positive mode 127 metabolite ions were statistically analyzed and in the negative mode 115 metabolite ions were analyzed. PCA score plots were able to model 98% and 87% of the original data for positive and negative mode, respectively. The metabolite ions from the loadings plot were tentatively identified based on m/z.

Conformation and Self-Assembly of the Transmembrane Peptide Gramicidin A: Insights from ion Mobility Spectrometry and Molecular Dynamics

Gramicidin A which is composed of alternating L- and D-amino acids is a naturally occurring pentadecapeptide from Bacillus brevis known to form monovalent metal ion channels in lipid membranes. The active form is a noncovalently bound dimer. The conformation and self-assembly behavior of gramicidin A highly depends on the solution environment. In this presentation, we report the use of electrospray-ion mobility-mass spectrometry to study the conformation of alkali metals adducts of gramicidin A monomer, as well as the monomerization and conformer interconversion equilibrium of gramicidin A dimer as a function of the solvent. The conformation of gramicidin A monomer vary significantly upon binding different metal adducts. Enhanced sampling molecular dynamics simulations are performed on alkali metals adducts to provide thermodynamics information of different conformers and gain insights of the interaction of different metal ions with the monomer. The kinetics of the monomerization and conformer interconversion processes of dimer in various alcohol solutions (Ethanol, 1-Propanol and, Isobutanol) are monitored by using the ion mobility profile of the monomer and the dimer. The rate constants and the temperature dependence of the rate constants of the monomer compare well with literature values which were obtained by using fluorescence. Furthermore, we found that the water content in the alcohol solution greatly influences the self-assembly process significantly. The role of water in catalyzing the conformer interconversion is being investigated further. Ion mobility spectrometry (IMS) combined with molecular dynamics simulations is a merging technique for conformational analysis of gas-phase low-lying energy level structures of biomolecules. In this study, we will demonstrate that this gas phase technique can also be of utility in studying a solution phase structural dynamics problem.