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

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 S1 and S2 fragments of differing relative intensities in their collision-induced dissociations, studied using mass-analysed ion kinetic energy (CID/MIKE) 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-(β,γ-methylene)-diphosphate and adenylyl-imidodiphosphate, alternative substrates for adenylyl cyclase, showed similar fragmentations with the methylene and imido groups blocking cleavage between the β and γ 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.