FAST-ATOM-BOMBARDMENT MASS-SPECTROMETRIC ANALYSIS OF CYCLIC-NUCLEOTIDE AND NUCLEOTIDE TRIPHOSPHATE ANALOGS 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-(beta,gamma-methylene)-diphosphate and adenylyl-imidodiphosphate, alternative substrates for adenylyl cyclase, showed similar fragmentations with the methylene and imido groups blocking cleavage between the beta and gamma 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.