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 agonis t and antagonist activity with cyclic AMP-dependent protein kinase, we re found to yield essentially identical positive-ion fast-atom bombard ment (FAB) mass spectra, but S1 and S2 fragments of differing relative intensities in their collision-induced dissociations, studied using m ass-analysed ion kinetic energy (CID/MIKE) spectra. Halogen-substitute d cyclic nucleotides, used in differentiating between protein kinase c yclic nucleotide binding sites, produced FAB mass spectra and CID/MIKE spectra with fragmentations generally analogous to those of the paren t cyclic nucleotides; the bromo-derivatives showed a greater propensit y for dehalogenation than the chloro-derivatives. The adenosine tripho sphate analogues, adenylyl-(beta,gamma-methylene)-diphosphate and aden ylyl-imidodiphosphate, alternative substrates for adenylyl cyclase, sh owed similar fragmentations with the methylene and imido groups blocki ng cleavage between the beta and gamma phosphate groups. The fragmenta tions observed are discussed in the context of the use of these compou nds in the assay of protein kinase and adenylyl cyclase activity by qu antitative mass spectrometry.