A. Gareth Brenton

Cerebrospinal Fluid Steroidomics: Are Bioactive Bile Acids Present in Brain?

In this study we have profiled the free sterol content of cerebrospinal fluid by a combination of charge tagging and liquid chromatography-tandem mass spectrometry. Surprisingly, the most abundant cholesterol metabolites were found to be C27 and C24 intermediates of the bile acid biosynthetic pathways with structures corresponding to 7α-hydroxy-3-oxocholest-4-en-26-oic acid (7.170 ± 2.826 ng/ml, mean ± S.D., six subjects), 3β-hydroxycholest-5-en-26-oic acid (0.416 ± 0.193 ng/ml), 7α,x-dihydroxy-3-oxocholest-4-en-26-oic acid (1.330 ± 0.543 ng/ml), and 7α-hydroxy-3-oxochol-4-en-24-oic acid (0.172 ± 0.085 ng/ml), and the C26 sterol 7α-hydroxy-26-norcholest-4-ene-3,x-dione (0.204 ± 0.083 ng/ml), where x is an oxygen atom either on the CD rings or more likely on the C-17 side chain. The ability of intermediates of the bile acid biosynthetic pathways to activate the liver X receptors (LXRs) and the farnesoid X receptor was also evaluated. The acidic cholesterol metabolites 3β-hydroxycholest-5-en-26-oic acid and 3β,7α-dihydroxycholest-5-en-26-oic acid were found to activate LXR in a luciferase assay, but the major metabolite identified in this study, i.e. 7α-hydroxy-3-oxocholest-4-en-26-oic acid, was not an LXR ligand. 7α-Hydroxy-3-oxocholest-4-en-26-oic acid is formed from 3β,7α-dihydroxycholest-5-en-26-oic acid in a reaction catalyzed by 3β-hydroxy-Δ5-C27-steroid dehydrogenase (HSD3B7), which may thus represent a deactivation pathway of LXR ligands in brain. Significantly, LXR activation has been found to reduce the symptoms of Alzheimer disease (Fan, J., Donkin, J., and Wellington C. (2009) Biofactors 35, 239–248); thus, cholesterol metabolites may play an important role in the etiology of Alzheimer disease.

Identification of cyclic nucleotide constituents of meristematic and non-meristematic tissue of Pisum sativum roots

Abstract The extraction of endogenous 3′,5′-cyclic-AMP, 3′,5′-cyclie-GMP, 3′,5′-cyclic-CMP, 3′,5′-cyclic-UMP, 3′,5′-cyclic-IMP and 3′,5′-cyclic-deoxyTMP from meristematic and non-meristematic regions of Pisum roots is described. Extracts were subjected to a sequential purification procedure involving adsorption chromatography on alumina, ion-exchange chromatography, and preparative electrophoresis. Purified samples were compared with cyclic nucleotide standards by HPLC and UV absorbance spectrophotometry; further analysis was carried out by fast atom bombardment mass spectrometry and mass-analysed ion kinetic energy spectrometry. 3′,5′-Cyclic-AMP, -GMP, -CMP and -UMP were found to be present in Pisum meristematic tissue and 3′,5′-cyclic-AMP, -GMP, -CMP, -IMP and -DTMP are present in the non-meristematic regions. Data indicate that there is a significantly higher concentration of cyclic CMP in meristematic tissue relative to that in non-meristematic regions.

The resolution of charge-stripping peaks using charge exchange in a multi-sector mass spectrometer

Abstract The charge-stripping process, when studied in sector mass spectrometers, is often masked by the presence of interfering collision-induced fragmentation processes. This presents problems, for example, in interpreting E/2 spectra which are meant to be representative of the doubly charged ions formed by charge-stripping singly charged ions issuing from the ion source. A method is described for completely resolving the spectrum of doubly charged ions from interferences, which uses charge exchange and a further stage of energy(-to-charge) analysis. A simple modification to a double-focusing mass spectrometer is described for achieving this. Examples of fully “resolved” E/2 and charge-stripping spectra are given.

Qualitative and quantitative mass spectrometric analysis of cyclic nucleotides and related enzymes

Abstract The application of fast atom bombardment mass spectrometry (MS) and collisionally induced dissociation with mass-analysed kinetic energy spectrum scanning to biochemical studies of cyclic nucleotides is reviewed. MS analysis of partially purified tissue extracts has enabled the demonstration of the natural occurrence in mammals and higher plants of five cyclic nucleotides in addition to the previously established adenosine 3′,5′-cyclic monophosphate; analysis of the products of a putative cytidylate cyclase enzyme-catalyzed reaction has demonstrated the biosynthesis of cytidine 3′,5′-cyclic monophosphate and four novel analogues as side products, thereby enabling the development of specific assays. Quantitative applications have included kinetic analysis of phosphodiesterase and cyclic nucleotide-responsive protein kinase activity, while further quantitative applications have included the structural elucidation of synthetic cyclic nucleotide derivatives used in pharmacological studies. The impact of MS upon cyclic nucleotide research and its future potential are discussed.

Differentiation of Isomeric Purine and Pyrimidine Mononucleotides by Fast Atom Bombardment Tandem Mass Spectrometry

Abstract The use of positive ion fast atom bombardment mass-analysed ion kinetic energy (FAB/MIKE) spectroscopy to differentiate the 2′, 3′-and 5′-monophosphate isomers of adenosine, guanosine and cytidine is described.

Application of continuous-flow fast atom bombardment mass spectrometry to cyclic nucleotide biochemistry

Abstract The modification and optimization of a continuous-flow fast atom bombardment system for the mass spectrometric analysis of cyclic nucleotides is described. The most effective solvent matrix was water-methanol-glycerol (70: 20: 10, v/v/v) and the optimum probe tip operating temperature was 40 °C. Modification of the probe tip was necessary to facilitate solvent evaporation and a solvent degassing and filter system were found to be essential. Comparison of the theoretical sensitivies of the continuous and static systems with cytidine 3′,5′-cyclic monophosphate as analyte indicated a increase of between one and two orders of magnitude, with a significant increase in signal-to-noise ratio also observed. Analysis of sample mixtures simulating enzyme incubates has proved successful and the potential application of dynamic fast atom bombardment systems in quantitative analysis of enzymes is further discussed here.

He22+, the experimental detection of a remarkable molecule

The smallest possible doubly charged molecule, He22+, has been detected spectroscopically by charge-stripping mass spectrometry.

Some applications of collision spectroscopy of gas-phase ions

Three inelastic processes that can occur during collisions of high-velocity gas-phase ions with gas molecules are considered. These are: charge exchange (sometimes called electron transfer), in which, typically, one or more electrons from the gas are transferred to the ion; charge stripping, in which one or more free electrons leave the ion; and excitation, in which the ion and/or the gas become excited but neither changes its charge state. Deductions that can be made concerning the states populated in these processes and the critical energies involved from measurements of the translational energy loss (or gain) of the fast-moving product are outlined. Novel equipment designed to improve the attainable energy resolution in the third processes is described. For brevity, the discussion is restricted to consideration of positively charged ions.

Observation of rotational predissociation in 20NeH+, 22NeH+, 20NeD+, 40ArH+ and D2+ formed from various precursors

Abstract Translational energy spectroscopy has been used to investigate the unimolecular dissociation, at keV translational energies, of NeH+ → H+, ArH+ → H+ and D+2 → D+. For D+2 in its ground electronic state (X2Σ+g), fragmentation is known to proceed solely by the mechanism of rotational predissociation. This study indicates the same mechanism to account for the unimolecular fragmentation of NeH+ and possibly for ArH+. For D+2 five quasibound states have been previously detected using a mass spectrometric technique. In this experiment, D+2 was formed from a number of deuterated organic molecules, by dissociative electron impact ionization CD4 was found to produce the largest D+2 signal, where a total of at least ten and possibly more quasibound states have been identified. The energy releases have been measured for all the observed predissociating states. For D+2, where theoretical calculations exist, the initial relative populations have been determined and found to lie in a range differing by no more than a factor of four. Populations of rovibronic states were found to depend on the precursor used to form D+2 and were estimated from the translational energy spectra. For 20NeH+, 22NeH+ and 20NeD+, five, three and five predissociating states were observed, respectively, whilst for 40ArH+ the very low signal to noise ratio of the H+ product allowed only a tentative assignment of the fragmentation mechanism and estimation of a single energy release.

Accurate mass measurement by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. I. Measurement of positive radical ions using porphyrin standard reference materials: Accurate mass MALDI of positive radical ions using porphyrins

A method for the accurate mass measurement of positive radical ions by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) is described. Initial use of a conjugated oligomeric calibration material was rejected in favour of a series of meso-tetraalkyl/tetraalkylaryl-functionalised porphyrins, from which the two calibrants required for a particular accurate mass measurement were chosen. While all measurements of monoisotopic species were within +/-5 ppm, and the method was rigorously validated using chemometrics, mean values of five measurements were used for extra confidence in the generation of potential elemental formulae. Potential difficulties encountered when measuring compounds containing multi-isotopic elements are discussed, where the monoisotopic peak is no longer the lowest mass peak, and a simple mass-correction solution can be applied. The method requires no significant expertise to implement, but care and attention is required to obtain valid measurements. The method is operationally simple and will prove useful to the analytical chemistry community.