Quantitation by fast-atom bombardment/mass-analysed ion kinetic energy spectrometry: kinetic analysis of cyclic nucleotide phosphodiesterase activity

Quantitation of cyclic nucleotide phosphodiesterase activity by means of fast-atom bombardment (FAB) mass spectrometry with mass-analysed ion kinetic energy (MIKE) spectrum scanning is described. Characteristic peaks of the substrate, cyclic AMP, and product, AMP, were identified in positive-ion FAB mass spectra and MIKE scans of the protonated molecules. By spiking enzyme incubates with known quantities of cyclic AMP and AMP and measuring peak heights in the MIKE spectra of both spiked and unspiked samples, the concentrations of cyclic AMP and AMP in solution at the end of a series of enzyme incubations have been estimated. From the data obtained the Km and Vmax of the enzymes were calculated as 181 microM and 28.6 nmol/min respectively, showing excellent agreement with values of the Michaelis constant, Km = 205 microM and the maximum velocity Vmax = 33.2 nmol/min obtained by radioactive assay.     

Analysis of tetrabenzyl N-giucosidic,N-mannosidic and N-galactosidic Isomers using fast atom bombardment/mass-analysed ion kinetic energy spectrometry

A series of new synthetic tetrabenzyl N-glucosidic, N-mannosidic and N-galactosidic isomers were investigated by fast atom bombardment (FAB)/mass-analysed ion kinetic energy (MIKE) spectrometry. The [M + H]⁺ ions were obtained with high abundance in the FAB spectra when using 3-nitrobenzyl alcohol as the matrix. The FAB/MIKE spectra provide characteristic daughter ions fragmented from selected molecular parent ions, allowing these isomers to be differentiated. In addition, an interesting rearrangement was found from the MIKE spectra, indicating that the benzyl (Bzl) group on the sugar ring is rearranged on to the N atom of the base (R) group to form [R + Bzl + H]⁺ and [R+ 2Bzl]⁺ ions.     

Differentiation of anomeric C-glycosides by mass spectrometry using fast atom bombardment,mass-analysed ion kinetic energy and collision-activated dissociation

Positive-ion fast atom bombardment mass spectrometry appears to be a useful method for the differentiation of anomeric C-glycosides. The mass-analysed ion kinetic energy (MIKE) and collision-activated dissociation (CAD) MIKE spectra of selected positive ions can be used as fingerprints of the α- or β-anomers. The main fragmentation routes and particularly the formation of the [M ? H]⁺ ion and the [M + H ? PhCH₂OH]⁺ ion were traced for each anomer.     

Identification of butyryl derivatives of cyclic nucleotides by positive ion fast atom bombardment mass spectrometry and mass-analysed ion kinetic energy spectrometry

The syntheses of the dibutyryl derivatives of the 3′,5′-cyclic monophosphates of adenosine, guanosine and cytidine are described. The fast atom bombardment mass spectra of these compounds are discussed, together with the mass-analysed ion kinetic energy spectra of their protonated molecular ions and of diagnostic fragments. A protocol for the identification of the derivatives is reported which includes criteria for confirming retention of the cyclic phosphodiester moiety, substitution of both heterocyclic base and ribose ring, and butyrylation of the 2′-O-position. The origins of significant fragments in the spectra are discussed.     

The structure of the ion: An ion kinetic energy spectrometry study

The kinetic energy release (T) values for the loss of CO from ions from five compounds have been obtained and are consistent with at least two different structures for the ion. This result is supported by the T value obtained for the decomposition of the molecular ions.     

Z-discrimination avoidance in mass-analysed ion kinetic energy spectral studies performed on an unmodified ZAB-E mass spectrometer

A method is introduced by which mass-analysed ion kinetic energy spectra free from Z-discrimination can be obtained for both collisionally activated (CA) and metastable decomposition reactions. The method, performed on a ZAB-E instrument fitted with a collision cell, but applicable also to the ZAB-2F, involves summation of the ‘height resolved’ contributions (formed by beam collimation in the Z-axis and selected by electrostatic deflection of the incident beam) using the signal averaging facility normally available. Representative results (at 8 or 10 keV energy) are given for the CA (Ar target) reactions [CS₂]²⁺ → [CS]⁺; [CS₂]⁺ → S⁺ and [CH₃OH]⁺ → [m/z = 12–31]⁺, and for the metastable reaction [m/z 45]⁺ → [m/z 29]⁺ in ethanol.     

How to measure the kinetic energy distribution of the precursor ion main beam in mass-analyzed ion kinetic energy spectrometry experiments

Scans of the electrostatic analyzer (ESA) across the precursor ion beam in reverse-geometry (BE) mass spectrometers that are operated under double-focusing conditions do not measure the “energy resolution of the main beam”: They only measure double-focusing resolution. The only way that ESA scans can measure the kinetic energy distribution of the main beam is to operate the instrument so that angular (directional) focusing is not achieved. Thus, the mass spectrometer is no longer double-focusing. Under double-focusing conditions, however, scans of the accelerating voltage while the magnetic field and ESA are held constant can be used to measure either the kinetic energy distribution of the main beam that enters the magnet or the energy-resolving power of the instrument. Scans at a constant ratio of B²/E can be used similarly. The energy-resolving power of any ESA is defined by its dispersion and the widths of the energy-resolving object and image slits that immediately precede and follow the ESA, respectively. The use of BE, EB, and triple-sector instruments to measure energy-resolving power and the kinetic energy distribution of the precursor ion main beam is compared and discussed.     

Kinetic energy release in metastable ion transitions. A study by ion kinetic energy spectrometry and rrkm theory

An approach to energy partitioning using experimental data in conjunction with theoretical calculations is presented, with the McLafferty and related rearrangements occurring in the molecular ions of ketones, esters, amides and acid chlorides representing the specific reactions examined. The kinetic energy release associated with statistical partitioning of the nonfixed energy of the activated complex was calculated from unimolecular reaction theory and compared with experimental data. Calculations of the energy dependence of the rate constant and the average statistically released kinetic energy were made for 24 activated complexes corresponding to the two basic structures, cyclic and linear. The frequencies used in the calculations were all modeled upon the 2-pentanone case with sufficient variation in the frequencies used to allow for any reasonable type of perturbation of the linear and cyclic structures. To further test that the complexes chosen were reasonable, A-factors were determined. From a comparison of the calculated and experimental results, the contributions of the nonfixed energy of the activated complex and of the reverse activation energy to the energy release were separated. This appears to be applicable, not only to the 2-pentanone case, but across the series of reactions studied. It is also shown that, despite the fact that these reactions release relatively small kinetic energies, the fraction due to the reverse activation energies may be large. The consequences of these observations for ion enthalpy determinations are emphasized.     

Influence of instrumental parameters and sample preparation on mass-analysed ion kinetic energy spectra with fast atom bombardment exemplified with the oxonium ion of peracetylated ribopyranose

When mass-analysed ion kinetic energy (MIKE) spectra are required to discriminate between isomeric ions formed under conditons of fast atom bombardment (FAB) in the ion source, severe interference may be observed. The interfering peaks in MIKE spectra obtained with a reversed-geometry instrument can arise from different sources. Moreover, the intensity distribution of the true ions from the selected precursor ion may depend strongly on the instrument being used. This means that the FAB–MIKE or collisionally induced dissociation (CID) spectrum is not an absolute characteristic of a particular ion. The [M + H ? HOAc]⁺ ion in the spectrum of peracetylated ribopyranose is used as an example to illustrate this and to trace and discuss the origin of the phenomena observed.     

Identification of cyclic nucleotide derivatives synthesized for radioimmunoassay development by fast atom bombardment with collision-induced dissociation and mass-analysed ion kinetic energy spectroscopy

The syntheses of 2′-O-succinyl, 2′-O-succinyltyrosinyl methyl ester and 2′-O-succinyliodotyrosinyl methyl ester derivatives of a cyclic nucleotide, derivatives necessary for the successful development of a specific radioimmuno-assay, are described. Fast atom bombardment with collision-induced dissociation and mass-analysed ion kinetic energy spectroscopy were used to verify the positions of substitution and the retention of the 3′,5′-cyclic phosphate moiety. Comparison of spectra produced after different iodination times permitted the optimization of the reaction conditions.     

Identification of aminophospholipid stereomers by positive-ion fast atom bombardment combined with collisional activation mass-analysed ion kinetic energy analysis and high-performance liquid chromatography

Two methods for the identification of aminophospholipid stereomers are described. After a chemical derivatization, 9-fluorenylmethoxycarbonyl derivatives of diacyl-sn-2- and diacyl-sn-3-phosphoserine and diacyl-sn-2- and diacyl-sn-3-phosphoethanolamine can be characterized by positive-ion fast atom bombardment combined with collisional activation mass-analysed ion kinetic energy analysis based on the differentiation of relative abundances [M + H + diethanolamine ? 89]⁺ and [M + H]⁺ fragments derived from [M + H + diethanolamine]⁺ ions, the protonated solvated molecules, and normal phase high-performance liquid chromatography on the basis of different elution times of the derivatives of the aminophospholipid stereomers on an aminopropyl-bonded column.     

A study of some cations formed in the ammonia chemical ionization of ketones using mass analysed ion kinetic energy spectrometry

The structures of the major adduct ions formed in ammonia chemical ionization of thirteen aliphatic and aromatic ketones have been studied by mass analysed ion kinetic energy spectrometry. The [M+NH₃+H]⁺ ion is shown to have a protonated carbinolamine structure, [M+2NH₃+H]⁺ to be a protonated carbinolamine with hydrogen-bonded ammonia and [2M+NH₃+H]⁺ to be, at least in part, a protonated carbinolamine with hydrogen-bonded ketone. These structures may imply a nucleophilic addition of ammonia at the carbonyl of the ketone-ammonium ion complex. An unusual hydroxy migration is seen in the internal rearrangement of the [2M+NH₃+H]⁺ ion leading to the formation of a protonated imine.     

Ion kinetic energy modulation for improved ion trapping in electrospray ionization fourier transform ion cyclotron resonance mass spectrometry

A new technique for manipulating the kinetic energy distribution of electrospray ions that arrive at a Fourier transform ion cyclotron resonance trapped-ion cell is presented. Narrow kinetic energy distributions can complicate the selection of appropriate trapping conditions for electrospray ions and introduce charge discrimination in resulting mass spectra. Modulation of the applied skimmer potential controllably broadens the kinetic energy distribution, which improves the reproducibility of acquired spectra and eliminates charge discrimination. Mass spectra of horse heart cytochrome c are presented to demonstrate the utility of the technique. For example, applied static skimmer potentials of 12 and 9 V yield charge state distributions ranging from [M+19H]⁺¹⁹ to [M+12H]⁺¹² and [M+15H]⁺¹⁵ to [M+7H]⁺⁷, respectively. A 12 ± 2 V, 100-Hz modulation of the skimmer potential yields an electrospray spectrum with charge states that range from [M+19H]⁺¹⁹ to [M+7H]⁺⁷, which is more representative of the source distribution.     

The ion kinetic energy spectra of some chlorinated insecticides

The ion kinetic energy (IKE) spectra of the p,p′ and o,p′ DDT, DDD and DDE isomers have been examined. The results indicated that the decomposing [M-CCl₃] ions (b) (m/e 235) for the p,p′ and o,p′ DDT isomers were not energetically similar, suggesting retention of the positional identity of the aromatic chlorine substituents. Similar results were obtained for the [M – CHCl₂] ions (b) for the p,p′ and o,p′ DDD isomers. The IKE spectra of p,p′ and o,p′ DDE were indistinguishable, indicating loss of substituent identity of the aromatic chlorine group. The mechanistic and analytical consequences of these data are discussed. The IKE spectra of the four hexachlorocyclohexane isomers (V to VIII) are different and these spectra are obviously useful in the analysis and characterization of these structurally similar compounds. The differences in their reactivity must in some part be due to their different stereochemical orientations of the chlorine substituents.     

The ion kinetic energy spectra of some aromatic hydrocarbons

The technique of ion kinetic energy spectroscopy has been applied to the study of the aromatic hydrocarbons benzene, toluene, naphthalene, 2-methyl naphthalene, biphenyl and anthracene. The method is illustrated by a complete study of naphthalene in which transitions of metastable doubly- and singly-charged ions are listed, including reactions in which singly-charged ions are formed by collision induced charge-exchange reactions of doubly-charged ions and by the double process of charge-exchange and metastable decomposition with loss of one or two hydrogen atoms. Decompositions of doubly-charged ions into two singly-charged ions, together with the kinetic energies released in these decompositions, are also given for all the compounds studied.     

The benzoyl ion. Thermochemistry and kinetic energy release

The thermochemistry of benzoyl ion formation from a variety of sources has been examined by using the measured kinetic energy release for metastable ions to estimate the excess energy of the activated complex. A correlation is observed between this estimated excess energy and literature values of the heat of formation of the benzoyl ion. From this relationship and the observed correlation between the uncorrected heat of formation and the difference between the appearance potential of [C6H5CO]⁺ and the ionization potential of the parent compound, the large range of reported values for ΔHf[C6H5CO]⁺ is seen to be due, at least in part, to variation in the kinetic shift with the critical energy of the reaction. With the exception of the ion generated from trifluoroacetophenone and possibly that from benzaldehyde, the fragmenting [C7H5O]⁺ ions are shown, from kinetic energy release data, to be structurally identical. The approach adopted here may have general merit in improving or testing the accuracy of thermochemical data based on appearance potential measurements.     

Translational energy release and stereochemistry of steroids. 14. Epimeric dihydroxy steroids of the androstane series

The stereochemistry of dihydroxy steroids, both the mode of the A/B ring junctions and the configuration of OH groups, may be determined from translational energy (T50%) measurements for the loss of a CH.3 radical, from the ratios of metastable-ion peak heights to those of the main beam (determined for the dehydration reactions), and by comparing unimolecular and collision-induced, mass-analysed ion kinetic energy spectra of the new main beam of [M-H2O]+ ions (i.e. those formed via dehydration of metastable molecular ions of epimeric hydroxy steroids in the first field-free region of a double-focusing mass spectrometer.