Fast‐atom bombardment mass spectrometric analysis of cyclic nucleotide and nucleotide triphosphate analogues used in studies of cyclic nucleotide‐related enzymes

Abstract

Two isomers of adenosine 3',5'-cyclic monophosphate, which show agonist and antagonist activity with cyclic AMP-dependent protein kinase, were found to yield essentially identical positive-ion fast-atom bombardment (FAB) mass spectra, but SI and S2 fragments of differing relative intensities in their collision-induced dissociations, studied using mass-analysed ion kinetic energy (CIDIMIKE) spectra. Halogen-substituted cyclic nucleotides, used in differentiating between protein kinase cyclic nucleotide binding sites, produced FAB mass spectra and CID/MIKE spectra with fragmentations generally analogous to those of the parent cyclic nucleotides; the bromo-derivatives showed a greater propensity for dehalogenation than the chloro-derivatives.The adenosine triphosphate analogues, adenylyl-(l), y-methylenel-diphosphate and adenylyl-imidodiphosphate, alternative substrates for adenylyl cyclase, showed similar fragmentations with the methylene and imido groups blocking cleavage between the l) and y phosphate groups. The fragmentations observed are discussed in the context of the use of these compounds in the assay of protein kinase and adenylyl cyclase activity by quantitative mass spectrometry.Fast-atom bombardment mass spectrometry (FAB-MS) in combination with collisionally induced dissociation and mass-analysed kinetic energy spectrometry (CID/MIKES) of quasi-molecular ions has provided an invaluable means of identifying putative cyclic nucleotides in tissue extracts'-3 and enzyme incubate^,^ and of determining the position and degree of substitution in cell-permeating synthetic derivatives.' The use of quantitative mass spectrometry has also been used to advantage in the study of enzymic reactions which function in the metabolism of cyclic nucleotide~~~' and in their mechanism of action as hormone mediators,8 and this approach has now been extended to the use of a continuous flow FAB which provides the basis for a continuous assay for these enzymes. In addition to enabling a continuous assay, not feasible by the conventional radiometric assays of these enzymes, the major advantage in application of quantitative mass spectrometric analysis to studies of enzyme activity is the ability to monitor several different reaction components simultaneously? and thus it is theoretically feasible to monitor, in the same incubation mixture, substrate(s), product(s), activator(s) and inhibitor(s). In view of the complexity of action of the cyclicnucleotide-related enzymes, the latter facility offers great potential; prior to systematic study of these enzymes by quantitative mass spectrometry, it is necessary first to establish the FAB-MS and CID/MIKES behaviour of compounds, additional to substrate and products previously examined, likely to be included in the enzyme incubates as putative effectors.Cyclic AMP-dependent protein kinase (1) is the enzyme that brings about the intracellular effect of cyclic AMP; it catalyses the phosphorylation of a range

Kinetic analysis of cyclic CMP-specific and multifunctional phosphodiesterases by quantitative positive-ion fast-atom bombardment mass spectrometry

“…Consequently enzymes concerned with the metabolism and action of cyclic nucleotides are important pharmacological targets. Application of qualitative fastatom bombardment (FAB) mass spectrometry (MS) with mass-analysed ion kinetic energy (MIKE) spectrum scanning has proved invaluable in the identification of endogenous cyclic nucleotides [1][2][3] and of their analogues 4,5 and derivatives, [6][7][8] by virtue of the characteristic fragmentations 9,10 observed in the MIKE spectra obtained after collisionally-induced dissociation (CID) of the protonated molecule in the FAB mass spectrum. The advantage of softionization methods such as FAB is the production of cyclic nucleotide mass spectra without a requirement for derivatization and, combined with CID/MIKE analysis, this permits the differentiation of the 3',5'-cyclic nucleotides, 9,10 which are the biochemical second messengers, from the 2',3'-cyclic nucleotide isomers, which are merely intermediates of nucleic acid catabolism.…”