Neuronal metabolomics by ion mobility mass spectrometry: cocaine effects on glucose and selected biogenic amine metabolites in the frontal cortex, striatum, and thalamus of the rat

We report results of studies of global and targeted neuronal metabolomes by ambient pressure ion mobility mass spectrometry. The rat frontal cortex, striatum, and thalamus were sampled from control nontreated rats and those treated with acute cocaine or pargyline. Quantitative evaluations were made by standard additions or isotopic dilution. The mass detection limit was ～100 pmol varying with the analyte. Targeted metabolites of dopamine, serotonin, and glucose followed the rank order of distribution expected between the anatomical areas. Data was evaluated by principal component analysis on 764 common metabolites (identified by m/z and reduced mobility). Differences between anatomical areas and treatment groups were observed for 53 % of these metabolites using principal component analysis. Global and targeted metabolic differences were observed between the three anatomical areas with contralateral differences between some areas. Following drug treatments, global and targeted metabolomes were found to shift relative to controls and still maintained anatomical differences. Pargyline reduced 3,4-dihydroxyphenylacetic acid below detection limits, and 5-HIAA varied between anatomical regions. Notable findings were: (1) global metabolomes were different between anatomical areas and were altered by acute cocaine providing a broad but targeted window of discovery for metabolic changes produced by drugs of abuse; (2) quantitative analysis was demonstrated using isotope dilution and standard addition; (3) cocaine changed glucose and biogenic amine metabolism in the anatomical areas tested; and (4) the largest effect of cocaine was on the glycolysis metabolome in the thalamus confirming inferences from previous positron emission tomography studies using 2-deoxyglucose.

Evaluation of ion mobility in capillary electrophoresis coupled to mass spectrometry for the identification in metabolomics

Currently, feature annotation remains one of the main challenges in untargeted metabolomics. In this context, the information provided by high-resolution mass spectrometry (HRMS) in addition to accurate mass can improve the quality of metabolite annotation, and MS/MS fragmentation patterns are widely used. Accurate mass and a separation index, such as retention time or effective mobility (μ_eff), in chromatographic and electrophoretic approaches, respectively, must be used for unequivocal metabolite identification. The possibility of measuring collision cross-section (CCS) values by using ion mobility (IM) is becoming increasingly popular in metabolomic studies thanks to the new generation of IM mass spectrometers. Based on their similar separation mechanisms involving electric field and the size of the compounds, the complementarity of ^DTCCS_N2 and μ_eff needs to be evaluated. In this study, a comparison of ^DTCCS_N2 and μ_eff was achieved in the context of feature identification ability in untargeted metabolomics by capillary zone electrophoresis (CZE) coupled with HRMS. This study confirms the high correlation of ^DTCCS_N2 with the mass of the studied metabolites as well as the orthogonality between accurate mass and μ_eff, making this combination particularly interesting for the identification of several endogenous metabolites. The use of IM-MS remains of great interest for facilitating the annotation of neutral metabolites present in the electroosmotic flow (EOF) that are poorly or not separated by CZE.     

Ion characterisation by comparison of ion mobility spectrometry and mass spectrometry data

The major uncertainty related to ion mobility spectrometry is the lack of knowledge about the characteristics of the ions detected. When using a radioactive atmospheric pressure ionisation source (e.g. ⁶³Ni), from theory proton bound water clusters are expected as reactant ions. When analyte ions occur, proton transfer should lead to proton-bound monomer and dimer ions. To increase the knowledge about those ionisation processes in an ion mobility spectrometer (IMS), a ß-radiation ionisation source was coupled to a mass spectrometer (MS) and an identical one to an IMS. Exemplarily, acetone, limonene and 2- and 5-nonanone were introduced into both instruments in varying concentrations. By correlating the MS and IMS spectra, conclusions about the identities of the ions detected by IMS could be drawn. Proton-bound monomer, dimer and even trimer ions (MH⁺, 2MH⁺, 3MH⁺) could be observed in the MS spectra for acetone and 5-nonanone and could be assigned to the related signals detected by IMS. The oligomers could be expected from theory for increasing concentration. Limonene and 2-nonanone yielded in a variety of different ions and fragments indicating complex gas phase ion chemistry. Those findings on the obviously different behaviour of different analytes require further research focussed on the ion chemistry in IMS including the comparison of different ionisation sources.     

Shapes of supramolecular cages by ion mobility mass spectrometry

Mass spectrometry and drift tube ion mobility mass spectrometry have been used to analyse several isobaric, multicomponent cages yielding information on three dimensional structure, interactions and dynamics of assembly in the gas phase.     

Structure-drift time relationships in ion mobility mass spectrometry

Ion mobility spectrometry (IMS) separates ions while they travel through a buffer gas under the influence of an electrical field. The separation is affected by mass and charge but most particularly by shape (collision cross section). When coupled to MS, IMS-MS offers therefore a powerful tool for structural elucidation and isomer separation. Systematic studies aimed to compare and quantitate the effects of structural changes on drift time such as length and ramification of carbon chain, unsaturation, geometrical isomerism (cis/trans isomers for instance), cyclization and ring size are, however, scarce. Herein we used traveling wave ion mobility mass spectrometry (TWIM-MS) to systematically evaluate the relationship between structure and drift time. For that, a series of deprotonated carboxylic acids were used as model ions with a carboxylate “charge tag” for gas phase MS manipulation. Carboxylic acids showed a near linear correlation between the increase of carbon number and the increase of collision cross section (CCS). The number of double bonds changes slightly the CCS of unsaturated acids. No differences in drift time and no significant differences in CCS of cis- and trans-double bond of oleic and elaidic acids were observed. Cyclization considerably reduces the CCS. In cyclic carboxylic acids, the increase of double bonds and aromatization significantly reduces the CCS and the drift times. The use of a more polarizable drift gas, CO_2, improved in some cases the separation, as for biomarker isomers of steranoic acids. The β-isomer (cis-decaline) has smaller CCS and therefore displayed lower drift time compared to the α-isomer (trans-decaline). Structural changes revealed by calculations were correlated with trends in drift times.     

Conformation of glycosaminoglycans by ion mobility mass spectrometry and molecular modelling

We have performed conformational analyses of heparin-derived oligosaccharide ions in the gas phase using a combination of ion-mobility mass spectrometry and molecular modelling. Negative mode electrospray ionisation was used to generate singly (disaccharide, [C12H15NO19S3Na3]-) and doubly charged (tetrasaccharides, [C24H30N2O38S6Na6]2- and [C24H31N2O35S5Na5]2-) ions containing three and six Na+ ions, respectively. Good agreement was obtained between the experimental and theoretical cross sections. The latter were obtained using modelled structures generated by the AMBER-based force field. Analysis of the conformations of the oligosaccharide ions shows that sodium cations play a major role in stabilizing these ions in the gas phase. This was seen in the formation of oligomers of the disaccharide ion and "compact" structures of tetrasaccharide ions. Interestingly, the gas phase conformations of the three tetrasaccharide ions with different primary structures were significantly different.     

Diastereomer assignment of an olefin-linked bis-paracyclophane by ion mobility mass spectrometry

trans-1,2-Bis([2.2]paracyclophanyl)ethene (1) exists as a pair of diastereomers whose conformations, and thus effective collision cross sections, are quite different. The two forms can be obtained by different transition metal-catalyzed reactions. To assign meso and racemic structures, a novel method is reported in which experimental gas-phase ion mobility data are compared with theoretical structures obtained from molecular mechanics calculations.     

Separation of catechin epimers by complexation using ion mobility mass spectrometry

Ion mobility coupled with mass spectrometry provides a fast and repeatable method to separate catechin epimers by previous complexation with selected chiral modifiers and transition metals. Several combinations with chiral ligands such as D‐ and L‐amino acids and/or additional metal cations, chiral crown ethers, tartaric acid and heptakis(2,6‐di‐O‐methyl)‐β‐cyclodextrin were screened for their ability to affect the separation efficiency. The clusters having the form of [2M + D‐amino acid + Cu²⁺ ? 3H]^? (M stands for (?)‐epicatechin or (+)‐catechin) showed improvement in stereodifferentiation between two epimeric catechins in comparison to the analysis of pure epimers, where no separation was observed or the separation was hampered by the formation of mixed dimer complexes. Among various examined D‐amino acids only those possessing hydrophobic side chains induced the improvement of separation efficiency. The best peak‐to‐peak resolution (R_p–p) was determined to be 0.71 for [2M + D‐Leucine + Cu²⁺ ? 3H]^? clusters. Copyright © 2015 John Wiley & Sons, Ltd.     

Probing hemoglobin structure by means of traveling-wave ion mobility mass spectrometry

Hemoglobin (Hb) is a tetrameric noncovalent complex consisting of two α- and two β-globin chains each associated with a heme group. Its exact assembly pathway is a matter of debate. Disorders of hemoglobin are the most common inherited disorders and subsequently the molecule has been extensively studied. This work attempts to further elucidate the structural properties of the hemoglobin tetramer and its components. Gas-phase conformations of hemoglobin tetramers and their constituents were investigated by means of traveling-wave ion mobility mass spectrometry. Sickle (HbS) and normal (HbA) hemoglobin molecules were analyzed to determine whether conformational differences in their quaternary structure could be observed. Rotationally averaged collision cross sections were estimated for tetramer, dimer, apo-, and holo-monomers with reference to a protein standard with known cross sections. Estimates of cross section obtained for the tetramers were compared to values calculated from X-ray crystallographic structures. HbS was consistently estimated to have a larger cross section than that of HbA, comparable with values obtained from X-ray crystallographic structures. Nontetrameric species observed included apo- and holo- forms of α- and β-monomers and heterodimers; α- and β-monomers in both apo- and holo- forms were found to have similar cross sections, suggesting they maintain a similar fold in the gas phase in both the presence and the absence of heme. Heme-deficient dimer, observed in the spectrum when analyzing commercially prepared Hb, was not observed when analyzing fresh blood. This implies that holo-α-apo-β is not an essential intermediate within the Hb assembly pathway, as previously proposed.     

Evaluation of micro-electrospray ionization with ion mobility spectrometry/mass spectrometry

In recent years, the resolving power of ion mobility instruments has been increased significantly, enabling ion mobility spectrometry (IMS) to be utilized as an analytical separation technique for complex mixtures. In theory, decreasing the drift tube temperature results in increased resolution due to decreased ion diffusion. However, the heat requirements for complete ion desolvation with electrospray ionization (ESI) have limited the reduction of temperatures in atmospheric pressure ion mobility instruments. Micro-electrospray conditions were investigated in this study to enable more efficient droplet formation and ionization with the objective of reducing drift tube temperatures and increasing IMS resolution. For small molecules (peptides), the drift tube temperature was reduced to ambient temperature with good resolution by employing reduced capillary diameters and flow rates. By employing micro-spray conditions, experimental resolution values approaching theoretically predicted resolution were achieved over a wide temperature range (30 to 250 °C). The historical heat requirements of atmospheric pressure IMS due to ESI desolvation were eliminated due to the use of micro-spray conditions and the high-resolution IMS spectra of GLY-HIS-LYS was obtained at ambient temperature. The desolvation of proteins (cytochrome c) was found to achieve optimal resolution at temperatures greater than 125 °C. This is significantly improved from earlier IMS studies that required drift tube temperatures of 250°C for protein desolvation.     

Gated trapped ion mobility spectrometry coupled to fourier transform ion cyclotron resonance mass spectrometry

Analysis of molecules by ion mobility spectrometry coupled with mass spectrometry (IMS-MS) provides chemical information on the three dimensional structure and mass of the molecules. The coupling of ion mobility to trapping mass spectrometers has historically been challenging due to the large differences in analysis time between the two devices. In this paper we present a modification of the trapped ion mobility (TIMS) analysis scheme termed “Gated TIMS” that allows efficient coupling to a Fourier Transform Ion Cyclotron Resonance (FT-ICR) analyzer. Analyses of standard compounds and the influence of source conditions on the TIMS distributions produced by ion mobility spectra of labile ubiquitin protein ions are presented. Ion mobility resolving powers up to 100 are observed. Measured collisional cross sections of ubiquitin ions are in excellent qualitative and quantitative agreement to previous measurements. Gated TIMS FT-ICR produces results comparable to those acquired using TIMS/time-of-flight MS instrument platforms as well as numerous drift tube IMS-MS studies published in the literature.     

Characterizing ion mobility and collision cross section of fatty acids using electrospray ion mobility mass spectrometry

This study investigated the ion mobility (IM) and the collision cross section (CCS) of fatty acids (FAs) using electrospray IM MS. The IM analysis of 18 FA ions showed intriguing differences among the saturated FAs, monounsaturated FAs, multi‐unsaturated FAs, and cis‐isomer/trans‐isomer with respect to the aliphatic tail chains. The length of aliphatic tail chain present in the ion structures had a strong influence on the differentiation of drift, while the number of double bond showed a weaker influence. The tiny drift differences between cis‐isomer and trans‐isomer were also observed. In the CCS measurements, two internal standards were involved in the mobility calibration and accuracy estimation. It insured our empirical CCS values were of high experimental precision (±0.35% or better) and accuracy (±0.25% or better). Moreover, the mass‐to‐charge ratio (m/z) – mobility plots obtained by ion mobility spectrometry with mass spectrometry analysis of FAs – was used to investigate the structural relationship between the molecules. Each series of FAs sharing a similar structure was aligned in the linear plot. Finally, the developed procedure was applied to the determination of FAs in rat adipose tissues, and it allowed the presence of 13 FAs to be confirmed with their exact masses and CCS values. These studies reveal the direct relationship between the behaviors in IM and the molecular structures and thus may provide further validations to the FA identification process. Copyright © 2015 John Wiley & Sons, Ltd.     

Investigation of neuroleptics and other aromatic compounds by laser-based ion mobility mass spectrometry

Laser-based ion mobility (IM) spectrometry was used for the detection of neuroleptics and PAH. A gas chromatograph was connected to the IM spectrometer in order to investigate compounds with low vapour pressure. The substances were ionized by resonant two-photon ionization at the wavelengths λ?=?213 and 266 nm and pulse energies between 50 and 300 μJ. Ion mobilities, linear ranges, limits of detection and response factors are reported. Limits of detection for the substances are in the range of 1–50 fmol. Additionally, the mechanism of laser ionization at atmospheric pressure was investigated. First, the primary product ions were determined by a laser-based time-of-flight mass spectrometer with effusive sample introduction. Then, a combination of a laser-based IM spectrometer and an ion trap mass spectrometer was developed and characterized to elucidate secondary ion–molecule reactions that can occur at atmospheric pressure. Some substances, namely naphthalene, anthracene, promazine and thioridazine, could be detected as primary ions (radical cations), while other substances, in particular acridine, phenothiazine and chlorprothixene, are detected as secondary ions (protonated molecules). The results are interpreted on the basis of quantum chemical calculations, and an ionization mechanism is proposed.     

Resolution of isomeric multi-ruthenated porphyrins by travelling wave ion mobility mass spectrometry

The ability of travelling wave ion mobility mass spectrometry (TWIM-MS) to resolve cationic meta/para and cis/trans isomers of mono-, di-, tri- and tetra-ruthenated supramolecular porphyrins was investigated. All meta isomers were found to be more compact than the para isomers and therefore mixtures of all isomeric pairs could be properly resolved with baseline or close to baseline peak-to-peak resolution (R(p-p)). Di-substituted cis/trans isomers were found, however, to present very similar drift times and could not be resolved. N(2) and CO(2) were tested as the drift gas, and similar α but considerably better values of R(p) and R(p-p) were always observed for CO(2).     

Sizing large proteins and protein complexes by electrospray ionization mass spectrometry and ion mobility

Mass spectrometry (MS) and ion mobility with electrospray ionization (ESI) have the capability to measure and detect large noncovalent protein-ligand and protein-protein complexes. Using an ion mobility method of gas-phase electrophoretic mobility molecular analysis (GEMMA), protein particles representing a range of sizes can be separated by their electrophoretic mobility in air. Highly charged particles produced from a protein complex solution using electrospray can be manipulated to produce singly charged ions, which can be separated and quantified by their electrophoretic mobility. Results from ESI-GEMMA analysis from our laboratory and others were compared with other experimental and theoretically determined parameters, such as molecular mass and cryoelectron microscopy and X-ray crystal structure dimensions. There is a strong correlation between the electrophoretic mobility diameter determined from GEMMA analysis and the molecular mass for protein complexes up to 12 MDa, including the 93 kDa enolase dimer, the 480 kDa ferritin 24-mer complex, the 4.6 MDa cowpea chlorotic mottle virus (CCMV), and the 9 MDa MVP-vault assembly. ESI-GEMMA is used to differentiate a number of similarly sized vault complexes that are composed of different N-terminal protein tags on the MVP subunit. The average effective density of the proteins and protein complexes studied was 0.6 g/cm(3). Moreover, there is evidence that proteins and protein complexes collapse or become more compact in the gas phase in the absence of water.