Assay of adenylyl cyclase activity by quantitative mass spectrometry. 'Heba El-Shazley, 'Russell P. Newton, 'Frank M. Harris, 'A. Gareth Brenton, 'Terence J.
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Biochemical Society Transactions ( 1 996)24
Assay of adenylyl cyclase activity by quantitative mass spectrometry. 'Heba El-Shazley, 'Russell P. Newton, 'Frank M. Harris, 'A. Gareth Brenton, 'Terence J. Walton, 'Colin J. Restall, 'Mark Bayliss, 'Adam Wilkins, 'David E. Games and 'James I. Langridge. 'Biochemistry Research Group, School of Biological Sciences, and %ass Spectrometry Research Unit, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, U.K. Fast atom bombardment mass spectrometry (FABMS), in concert with mass-analysed ion kinetic energy spectrum scanning (CIDflIKES) or with an alternative form of tandem MS, has successfully been used qualitatively in cyclic nucleotide studies, primarily for the identification of putative cyclic nucleotides in tissue extracts or enzyme incubates [l-41. Quantitative FABMS was first utilized in studies of cyclic nucleotide-related enzymes to follow the kinetics of phosphodiesterases [5,6] by monitoring the peak heights in the CIDflIKE spectra obtained from the protonated molecules in enzyme incubates: cyclic nucleotide-responsive kinases have been monitored by similar use of the primary FAB mass spectra [ 7 , 8 ] . The procedures and rationale used to study the kinases have now been extended to quantitative FABMS studies of adenylyl cyclase. Although both ATP and cyclic AMP, the adenylyl cyclase substrate and product respectively, display readily measurable peaks corresponding to their protonated molecules in the FAB mass spectrum, measurement of a sinqle analyte-derived peak height does not provide a realistic estimate of that analytes concentration [ 9 ] . Thus in order to quantitate adenylyl cyclase activities from the mass spectra obtained from enzyme incubates, it is necessary to compute the differences between the contol and experimental peak heights of a series of diagnostic ions:
A conc. .=
together with peaks at the m/z values above minus 17 and 18 mass units, corresponding to loss of H,O and NH, respectively. Application of the computation detailed above provides quantitative data with good correlation to those obtained by the conentional radiometric assay monltoring [32P]-ATP conversion into ["PI-CAMP. For and example in this preliminary study the ,,V y for an adenylyl cyclase preparation were 438 nmol min-'mg-' and 23 uM by the radiometric assay, and 429 nmol min-'mg-' and 27 uM by quantitative FABMS. One advantage of this MS assay is to circumvent a requirement for radiolabelled substrates inherent in alternative assays: this advantage is however offset by the greater intricacy of the data acquisition and the requirement for expensive, complex, instrumentation. The major motivation for our use of quantitative MS for adenylyl cyclase determinations is the facility, unavailable in other procedures, of simultaneously monitoring several components in a single assay. Thus by this novel method it has been possible to estimate not only the adenylyl cyclase activity of enriched membrane preparations but also ATPase, GTPase and phosphodiesterase activities, GTP-binding activity and the turnover of effectors simultaneously in the same sample. A further advantage is the scope to unequivocally identify the putative cyclic AMP product by its CID/MIKE spectrum. Acknowledgements: the authors gratefully acknowledge support from The Wellcome Trust, Science and Engineering Research Council, Smith Kline Beecham and A.P.K. Nd. 1. Kingston E.E., Beynon J.H. and Newton R.P. (1984) Biomed. Mass Spectrom. 11, 367-374. 2. Newton R.P., Salih S.G., Salvage B.J. and Kingston E.E. (1984) Biochem. J. 221, 665673. 3. Kingston E.E., Beynon J.H., Newton R.P. and Liehr J.G. (1985) Biomed. Mass Spectrom. 12, 525-535. 4. Newton R.P., Kingston E.E., Hakeem N.A., Salih S.G., Beynon J.H. and Moyse C.D. (1986) Biochem. J. 236, 431-439. 5. Newton R.P., Walton T.J., Brenton A.G., Kingston E.E. and Harris F.M. (1989) Rapid Commun. Mass Spectrom. 3, 178-183. 6. Newton R.P., Khan J.A., Ghosh D.,
Langridge J. I. , Brenton A.G. , Harris F.M. and Walton T.J. (1991) Organic Mass Spectrom. 26, 447-453. 7. Newton
Forthe glycerol matrix, ATPandcyclic AMP m/z values below, the peaks at the corresponding to the protonated molecules plus significant adducts, were monitored : GrO ATP CMIP 277 299 321 369 391 413 422 453 475 545 600
508 530 552 574 596 600 622
330 352 374 422 444
R.P., Khan J.A., Brenton A.G., Langridge J.I., Harris F.M. and Walton T.J. (1992) Rapid Commun. Mass Spectrom. 6, 601607. 8 . Newton R.P., Evans A.M., Langridge J.I., Walton T.J., Harris F.M. and Brenton A.G. (1995) Analyt. Biochem. 224, 32-38. 9. Newton R.P. (1993) Rapid Commun. Mass Spectrom. 7, 528-537.
