RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. 6,601-607 (1992)

Quantitation by Fast-atom Bombardment Mass Spectrometry: Assay of Cytidine 3’,5’-cyclic Monophosphate-responsive Protein Kinase Russell P. Newton,*? Jalal A. Khan,? A. Gareth Brenton,*$ James I. Langridge,? Frank M. Harris$. and Terence J. Walton? tBiochemistry Research Group, School of Biological Sciences, and $Mass Spectrometry Research Unit, Department of Chemistry, University College of Swansea, Swansea SA2 SPP, UK

A protein kinase, stimulated by cytidine 3’,5’-cyclic monophosphate, is conventionally assayed by monitoring the incorporation of radiolabelled phosphate from adenosine triphosphate into a histone substrate. Here the assay of the protein kinase is carried out by positive-ion fast-atom bombardment mass spectrometric analysis of the enzyme incubation mixture after the reaction has been terminated. The data so obtained show good agreement with data obtained by the conventional radiometric assay: the intrinsic advantage of the mass spectrometric assay is the capacity for multiple component monitoring; the ability of the kinase to bind competing cyclic nucleotides together with integral adenosine triphosphatase (ATPase) and phosphodiesterase activity can also be assessed.

Cyclic nucleotides are naturally occurring molecules which are composed of a cyclic phosphate group, a ribose ring and a heterocyclic base. They perform a vital role in the regulation of cellular metabolism, for example acting as the mediators of action of many mammalian hormones; consequently the enzyme systems which catalyze the biosynthesis and degradation of cyclic nucleotides are currently subjects of intense research activity in view of their potential value as pharmacological targets. The advent of fast-atom bombardment (FAB) as a means of ‘soft’ ionization’ enabled the production of mass spectra containing large quasi-molecular ion peaks from underivatized cyclic nucleotides;’ such peaks had been absent from previous electron ionization (EI) and chemical ionization (CI) mass spectra of the trimethylsilyl (TMS) derivatives of cyclic nucleotide~.~The selection of this quasimolecular ion and generation of a mass-analysed ion kinetic energy (MIKE) spectrum after its collisionally induced dissociation (CID) has been used successfully to differentiate cyclic nucleotide isomers4 and to identify putative cyclic nucleotides in tissue extracts.’-’ Mass spectrometric identification of cyclic nucleotides is based upon the presence in the spectrum of characteristic fragments corresponding to protonated base, protonated base + 28, and protonated base 42 mass units4 and changes in the relative intensity of the characteristic peaks have been used in the assignment of positions of substitution in synthetic cyclic nucleotide derivatives.’ The application of FABMS to studies of cyclic nucleotide biochemistry has been extended from qualitative to quantitative analysis by the examination of the kinetics of cyclic adenosine monophosphate (AMP) phosphodiesterase’ and cyclic cytidine monophosphate (CMP) phosphodiesterase” activities. Quantitation was achieved by determining the peak heights of the diagnostic fragments in the CID-MIKE spectra of the protonated molecules of substrate and product, both before and after spiking with known quantities of substrate and product standard, then cal-

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Authors to whom correspondence should be addressed. 0951-41 98/92/ 100601-07 $08.50

01992 by John Wiley & Sons, Ltd.

culating the relative concentrations of substrate and product by a proportionation c o m p ~ t a t i o nlo. ~The ~ data so obtained showed excellent agreement with data produced by the conventional radiometric assay, and the mass spectrometric method possesses inherent advantages relative to the orthodox phosphodiesterase assays in that it does not require the use of radioisotopically labelled substrate, it permits monitoring of the turnover of several incubation components simultaneously, and with the implementation of a continuous-flow FAB system,” it offers the potential of a continuous assay for this reaction.’* The successful kinetic analysis of the phosphodiesterase led to an attempt to quantitate cyclic nucleotideresponsive protein kinase activity by mass spectrometric methods. Protein kinase activation is the major means by which cyclic nucleotides induce changes in intracellular metabolism; the protein kinase is either inactive, or operating at a basal level, in the absence of the cyclic nucleotide, but the presence of the latter brings about a stimulation of the protein kinase a ~ t i v i t y ,leading ’~ to the phosphorylation of a numer of protein kinase substrates. This change in phosphorylation state causes an alteration in the substrate protein conformation, leading in turn to an increase or decrease in the protein’s activity, the latter change constituting the physiological response to the original extracellular signalling molecule, the hormone or neurotransmitter.l4 The routine assay of cyclic nucleotide-responsive protein kinase activity is to monitor the incorporation of [32P]-P04into the protein substrate from [y3’P]-ATP in the resence of the protein kinase and cyclic nucleotide; 19 Protein + [ y 3 ’ P ] - A T P S P r ~ t e i n - ~ ~ P +OADP , PK

cNMP

This procedure requires a separation step, to remove unconsumed [y3’P]-ATP from radiolabelled protein, and has the disadvantage of providing only a single datum from each enzyme incubation. Mass spectroReceived 11 August I992 Accepted I September 1992

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metric analysis offers the potential advantage of monitoring not only the incorporation of the y-phosphate moiety from ATP into the protein, but also the consumption of ATP by any ATPase activity present in the protein kinase preparation, the extent of cyclic nucleotide hydrolysis by endogenous phosphodiesterase activity, and the capacity of the protein kinase to bind several cyclic nucleotides simultaneously. To examine this potential, a cyclic CMP-responsive protein kinase preparation was examined both by the established radiometric method and by a quantitative mass spectrometric method; in order to utilize the latter without recourse to either spiking samples or MIKE scanning an extensive series of controls was included in order to carry out fully comparative analyses.

EXPERIMENTAL Materials. Radiochemicals were purchased from Amersham International plc (Amersham, UK). Nucleotides were obtained either from Sigma Chemical Co. (Poole, UK) or from the Boehringer Corp. (London, UK). All other biochemicals were either from Sigma Chemical Co. or BDH Chemicals (Poole, UK), and all other chemicals either from BDH Chemicals or the Aldrich Chemical Co. (Poole, UK) unless otherwise specified. All items were of the highest purity available commercially. Preparation of cyclic CMP-responsive protein kinase. 15 g of rat liver was homogenized in 135 mL of 50 nM Tris-HC1 buffer, pH 7.4, containing 6 nM mercaptoethanol then centrifuged at 10 000 x g for 5 min at 4 "C and the precipitate discarded. The supernatant liquid was dialysed overnight at 4 "C then subjected to (NH4),S04 fractionational precipitation; the 15-45% saturated precipitate fraction was dialysed and applied to a Sephacryl S-200 column ( 7 0 c m x 2 S c m ) and eluted with 50 mM Tris-HC1 buffer, p H 7.4, at 15 mL/h and 15mL fractions collected. Fractions 18- 24 were retained, pooled, dialysed then concentrated by freezedrying and resuspension in minimal volume of 5 0 y ~ Tris-HCI buffer, pH 7.4. Estimation of cyclic CMP-responsive protein kinase by radiometric assay. Assay was carried out by a modified version of that developed for cyclic AMP-dependent protein kinase. l5 The basic protocol contained the following components, added to the incubation tubes at 37 "C and incubated for 10 min: 50 pL of 2.5 mM magnesium acetate, 0-100 nmol cCMP in 50 pL, 50 yg histone IIA in lOOpL, 1OOp.L of protein kinase preparation, and 15 nmol ATP containing [ y3*P]ATP (6 x lo5dpm) in 100 pL, added in this order and made up to 500 yL with 50 ~ L MTris-HCL buffer, pH 7.4. A boiled enzyme set of controls was utilized and the reaction terminated by heating in a water bath at 90 "C for 2min. The radioactive content of the precipitated protein was determined as previously described.6 Analysis of cyclic CMP-responsive protein kinuse by FABMS. A series of incubations was carried out in the same manner as described for the radiometric assay but with the absence of radiolabelled ATP: Series I comprised 100 yL protein kinase preparation plus a series of concentrations of cyclic CMP (0100 nmol), together with a set of controls identical to

the experimental incubations except that the protein kinase had been denatured by boiling. Series I1 was identical to series I except that 15 nmol ATP was included in both experimental and controls. Series I11 was identical to series I1 except that 100 yg/ mL histone was included in both experimental and controls. Series IV was identical to series I11 except that 15 nmol cyclic AMP was included in both experimental and controls. Series V was identical to series I11 except that 15 nmol cyclic GMP was included in both experimental and controls. At the end of the incubation the reaction was terminated as described above and precipitated protein removed by centrifugation at 15 000 X g for 15 min. The supernatant liquid was removed, freeze-dried and decationized as previously described,6 then resuspended in the minimum quantity of deionized water and immediately frozen until utilized for mass spectrometric analysis. FAB mass spectrometry. Positive-ion FAB mass spectra were obtained on a ZAB-2F mass spectrometer (VG Analytical Ltd., Manchester, UK) under conditions previously ~pecified.~5 yL samples made up in glycerol +water, 1:l v/v, were placed on the FAB target; sample lifetime was between 90 s and 6 min. Quantitation of FA B mass spectra. The series I incubations were designed to examine interaction between the protein kinase and cyclic CMP in the absence of either protein kinase substrate, this interaction being indicated by differences in the spectra obtained from active and inactive protein kinase samples. The series I1 incubations were to examine the same interactions in the presence of one protein kinase substrate, ATP, and to examine the turpover of the latter. Series I11 was designed to monitor these effects again, this time in the presence of both protein kinase substrates, ATP and histone. Finally, series IV and V were set up to monitor the above in the presence of cyclic AMP and cyclic GMP, respectively. Peak heights corresponding to ions corresponding to protonated molecules and their adducts, together with characteristic fragments as previously identified4were determined in each spectrum and a series of computational formulae incorporating these peak heights based on the relationship Aconcent ration of component

sum of relevant peak intensities of characteristic sample ions in expt.

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RESULTS A single representative pair of mass spectra for each of I-V from the series of six pairs obtained is shown in Fig. 1. In Fig. l(a) and (b) the mass spectra for control and experimental runs of I are respectively reproduced.

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Figure 1. Positive-ion fast-atom bombardment mass spectra of protein kinase incubates. (a) Control I; denatured protein kinase preparation incubated with cyclic CMP. (b) Experimental I; active protein kinase preparation incubated with cyclic CMP. (c) Control 11; denatured protein kinase preparation incubated with cyclic CMP and ATP. (d) Experimental 11; active protein kinase preparation incubated with cyclic CMP and ATP. (e) Control 111; denatured protein kinase preparation incubated with cyclic CMP, ATP and histone. (f) Experimental 111; active protein kinase preparation incubated with cyclic CMP, ATP and histone. (g) Control IV; denatured protein kinase preparation incubated with cyclic CMP, ATP, histone and cyclic AMP. (h) Experimental IV; active protein kinase preparation incubated with cyclic CMP, ATP, histone and cyclic AMP. (i) Control V; denatured protein kinase preparation incubated with cyclic CMP, ATP, histone and cyclic GMP. (j) Experimental V; active protein kinase preparation incubated with cyclic CMP, ATP, histone and cyclic GMP. See text for further details.

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In the control (Fig. l(a)) with the denatured protein kinase, strong peaks at mlz 306, 328, 398 and 420, corresponding to MH+, MNa+, MGroH+ and MGroNa' (where Gro represents glycerol) for cyclic CMP are apparent, together with matrix-derived peaks at mlz 369,391,461 and 483. In the spectrum from the incubate containing the active protein kinase (Experimental), the relative intensity of the cyclic CMP-derived peaks at mlz 306, 328, 398 and 420 has decreased, consistent with the binding of this molecule to the now precipitated protein. Interestingly there is a larger peak at rnlz 324, corresponding to [MH]' for CMP in the experimental sample than in the control, suggesting that some cyclic CMP is being hydrolysed to CMP by the protein kinase preparation. In Fig. l(c) and (d) the mass spectra are reproduced from series I1 in which ATP has been added. Again a difference between relative intensities of the cyclic CMP-derived peaks at rnlz 306 and 328 in the experimental and control is apparent, indicating the binding of cyclic CMP to the protein in the non-denatured preparation. Peaks arising from ATP are present at rnlz 508 and 530 in both spectra corresponding to MH' and MNa', with peaks at mlz 428 and 450 present arising from ATP hydrolysis to ADP. The effect of the addition of histone, the substrate for phosphorylation, to the incubations can be gauged from Fig. l(e) and (f) obtained from series I11 incubations. These contain the same pattern as that observed in the spectra from series I and 11, relating to the cyclic CMP peaks at rnlz 306 and 328, with these peaks being of a lower relative intensity in the experimental when compared to the control, again indicative of cyclic CMP binding to the substrate; in Fig. l(c) and (d) however, the relative intensities of the ATP peaks at rnlz 508 and 530 were similar in experimental and control, but while still apparent in the spectrum of the control (Fig. l(e)), the relative intensities of the ATP-derived ions have markedly decreased in the experimental (Fig. l(f)), due to the activity of the protein kinase preparation. The addition of cyclic AMP to the incubation (series IV) is clearly reflected in the mass spectra shown in Fig. l(g) and (h) with characteristic peaks for cyclic AMP at mlz 330, 352, 422 and 444, corresponding to MH+, MNa' , MGroH' and MGroNa+, respectively, apparent in both experimental and control. The difference between the relative intensities of the ATP peaks at mlz 508 and 530 in the experimental and control in the presence of cyclic AMP (Fig. l(g) and (h)), is small compared to that observed in the absence of cyclic AMP (Fig. l(e) and (f)), indicating that the presence of cyclic AMP reduces the protein kinase activity of this preparation. No such effect is observed in the presence of cyclic GMP (Fig. l(i) and (j)) obtained from series V incubations. In these spectra cyclic GMP-derived peaks are apparent at mlz 346, 368, 438 and 460, corresponding to MH+, MNa', MGroH+ and MGroNa+, respectively, in both control and experimental spectra. However, while peaks at mlz 508 and 530, corresponding to MH+ and MNa' for ATP, are readily apparent in the spectrum from the control incubation (Fig. l(i)), they are much smaller in comparison to adjacent peaks relative to those in experimental incubation (Fig. l(j)), when compared to the spectra in Fig. l(g) and (h), indicating that protein kinase activity is taking place at

a higher rate in the presence of cyclic GMP than cyclic AMP. The spectra shown are representative pairs of each of the five series for the six concentrations of cyclic CMP utilized. Observations such as those made above from direct visual interpretation of the spectra, whilst indicative, are to a considerable extent necessarily subjective, and in complex mixtures of this type, single peak heights are not a reliable indication of concentration and cannot be used for simple direct quantitation;'.'" in order to quantitate the level of each component of interest, even purely on a comparative basis, it is necessary to sum the relative intensities of all the major peaks arising from the component of interest. Furthermore we have found it necessary to carry out the same summation of relative intensities for matrixderived peaks as a basis for comparison since, although the concentration of glycerol is held constant in determining each spectrum, the relative abundance for a particular glycerol-derived peak is not constant, even in the controls. In the spectra above, the intensities of all peaks are expressed as a percentage of the largest peak above rnlz 300, i.e., the [4GroH]+ peak at rnlz 369. The variation in a glycerol-derived peak can be gauged from the height of mlz 461, corresponding to [5GroH]+. In order to quantitate the components relative to one another, in theory every peak that can be produced by fragmentation or adduct formation should be included in the calculation; not only is this impracticable, but it would also include many peaks with contributions from more than one of the incubation mixture components. Thus, it was necessary to select the major ions arising from each component and sum these as follows: C I, sample &om

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Z I, sample

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where I, = sum of peak intensities relative to rnlz 369 for characteristic ions for sample component or matrix. For cyclic nucleotides and ATP the peak relative intensities utilized were those corresponding to [MH]+, [MNa]', [MNa+Na]+, [M+Gro]+, [MNa+Gro]+, [MNa + GroNa]+, plus each of these less 17 mass units and less 18 mass units, corresponding to loss of NH3 and loss of H 2 0 , respectively. For the matrix, the peak relative intensities used were at [3Gro 2Na]', [4GroH]+, [4GroNa]+, [4GroNa + Na]+, [SGroH]+, [5Gro+Na]+, [SGroNa+Na]+, [M+Gro]+, [MNa + Gro]+ plus each of these minus 18, corresponding to a loss of H 2 0 . Kinetics data obtained for the protein kinase preparations from the application of this formula are depicted in Fig. 2. When cyclic AMP alone (series I) was added, an increase in cyclic CMP binding is observed until saturation is reached upon addition of 10nmol cyclic CMP (Fig. 2(a)). In Fig. 2(b), while binding saturation was again reached at 10 nmol cyclic CMP, the inclusion of ATP reduced the binding of cyclic CMP by ca 25%. Previous experiments (Newton and Hakeem, unpublished observation) have suggested that cyclic CMP may bind at the ATP binding site of

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some cyclic nucleotide-sensitive protein kinases in the absence of ATP; a similar process here may be the explanation of the change observed in the current study. Addition of histone in addition to ATP should not, in theory, alter binding of cyclic CMP to the protein kinase; consistent with this there was little difference between the spectra obtained from incubation in the presence (Fig. 2(c)) and absence (2(b)) of histone. The presence of cyclic AMP, on the other hand, produced a marked change in the binding profile of cyclic CMP (Fig. 2(d)) pushing the curve to the right and reducing the binding of cyclic CMP until it was present in excess of the added cyclic AMP. This is in agreement with the observation that cyclic AMP is able to bind to this protein kinase. Cyclic GMP however, previously shown not to bind to the protein kinase, has little or no effect on cyclic CMP binding (Fig. 2(e)). The ability to monitor several compounds simultaneously, a major potential advantage of a mass spectrometric approach compared with conventional assays la1

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for analysis of protein kinase activity, is apparent from Fig. 2(f). In addition to monitoring cyclic CMP binding, at varied concentrations, in the presence of a fixed concentration of cyclic AMP or cyclic GMP, the binding of cyclic AMP or cyclic GMP in the presence of a varied concentration of cyclic CMP can also be determined. In Fig. 2(f) it can be seen that, at low cyclic CMP concentrations, cyclic AMP is bound but this binding decreases as cyclic CMP levels increase, the profile that would be anticipated in view of that seen in Fig. 2(d). Similarly cyclic GMP provides the predicted binding profile (Fig. 2(f)), i.e., little or no binding of cyclic GMP is observed irrespective of cyclic CMP concentration. In addition to cyclic nucleotide levels the ATP levels were also determined (Fig. 3). The change observed in ATP levels in the absence of the histone substrate (series 11) is at least partially due to ATPase activity. I11 represents apparent protein kinase activity in response to increase in cyclic CMP concentration, reaching a maximum at 25 nmol cyclic CMP, a higher concentraIbl

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Figure 2. Cyclic nucleotide-binding capacity of the protein kinase preparation calculated from the mass spectrometric data. (a) Binding profile of cyclic CMP in the absence of other nucleotides. (b) Binding profile of cyclic CMP in the presence of ATP. (c) Binding profile of cyclic CMP in the presence of ATP and histone. (d) Binding profile of cyclic CMP in the presence of ATP, histone and cyclic AMP. (e) Binding profile of cyclic CMP in the presence of ATP, histone and cyclic GMP. (f) Binding of cyclic AMP and cyclic GMP (+) at varied concentrations of cyclic CMP. See text for further details.

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tion than that observed for cyclic CMP binding (Fig. 2(a)). Cyclic AMP, a competitor for cyclic CMP binding and a weaker stimulator of the protein kinase activity, depresses the peak of activity but reduces the subsequent tailing off of activity at higher cCMP concentrations (IV, Fig. 3). Cyclic GMP, shown to bind to the enzyme only to a much smaller extent (Fig. 2(e) and (f)) slightly reduces the maximal protein kinase activity (V, Fig. 3) but again reduces the tailing off of activity at higher concentrations of cyclic CMP. The most logical explanation of this is that cyclic AMP and cyclic GMP are acting as competitive inhibitors of cyclic CMP phosphodiesterase activity present in the protein kinase preparation; such a hypothesis is supported by the lower relative intensities of mlz 324, indicating CMP levels in the mass spectra arising from experimental incubates not containing cyclic AMP and cyclic GMP compared to those from which they were included (Fig. 1(W7(h) and (j)). Figure 4(a) represents protein kinase activity plotted against cyclic CMP concentration calculated by both (a1

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the mass spectrometric quantitation and the conventional radiometric technique. For the mass spectrometric assay each datum point is effectively that from series I11 representing apparent protein kinase activity, minus that from series 11, representing ATPase activity. A second mass spectrometric calculation, involving relative peak intensities of ADP- and AMP-related peaks to calculate the ATPase activity before subtraction of I1 and 111produced an essentially identical plot. The close correlation between the plots verifies the validity of the mass spectrometric assay, as does comparison between the protein kinase data in the presence of both cCMP and cGMP obtained by both assay methods (Fig. 4(b)). DISCUSSION The data reported above indicate the success of the quantitative mass spectrometric analysis of cyclic CMP-responsive protein kinase. The use of the positive-ion FAB mass spectra from the enzyme incubates, compared to our previous use of the MIKE scans for quantitation of phosphodiesterase a c t i ~ i t y lo , ~ ,is encouraging since the advantages discussed for quantitative MIKE are retained but this procedure is more sensitive and can be carried out more rapidly. In terms of sensitivity, the quantitative FABMS protein kinase assay has no inherent advantage or disadvantage over the conventional radiometric assay: while the former has the advantage that no radioisotopically labelled substrate and consequent safe handling facility is required, the latter has the advantage that several assays can be handled simultaneously by a single operator. The major advantage of the mass spectrometric procedure developed, lies in its potential for continuous assay via continuous flow FAB, and its capacity for multiple compound monitoring. The data obtained confirm the existence of a protein kinase responsive to, and with specificity for, cyclic CMP, adding credence to the concept of a key regulatory role for cyclic CMP in cellular metab~lism.'~ The explanation that cyclic GMP enhances cyclic CMP stimulation of the protein kinase by 'protecting the cyclic CMP from phosphodiesterase activity can be deduced from single spectra: such a deduction could not have been reached from a single incubation by the lbl

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Figure 4. Protein kinase kinetics derived from mass spectrometric data and radiometric data. (a) Cyclic CMP-responsive protein kinase activity, plotted against cyclic CMP concentration, calculated by both mass spectroand radiometric assay (+). (b) Cyclic CMP-responsive protein kinase activity in the metric quantitation (a) presence of cyclic GMP, plotted against cyclic CMP ion concentration, calculated by both mass spectrometric quantitation (+) and radiometric assay(+).

QUANTITATION OF A PROTEIN KINASE BY FABMS

radiometric assay and clearly illustrates the great advantage of quantitative mass spectrometry in its capacity for simultaneous monitoring of the turnover of several compounds. The use of 8-chlorocyclic AMP as a cancer therapy by virtue of a mechanism which remains to be elucidated, but which is deduced to involve binding of the analogue at one of two sites on a cyclic AMP-dependent protein kinase,'* provides an example of another protein kinase system, the study of which will be greatly facilitated by quantitative mass spectrometry; the successful implementation of a continuousflow system will make a further significant contribution to the elucidation of the chlorocyclic AMP-protein kinase interaction.

REFERENCES 1. M. Barber, R. S. Bordoli, R. D. Sedgewick and E. T. Whalley, Biomed. Mass Spectrom. 8, 325 (1981). 2. E. E. Kingston, J. H. Beynon and R. P. Newton, Biomed. Mass Spectrom. 11, 367 (1984). 3. A. M. Lawson, R. N. Stillwell, M. M. Tacker, K. Tsubogarna and J. A. McCloskey, J. A m . Chem. SOC.93, 1014 (1971).

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4. E. E. Kingston, J. H. Beynon, R. P. Newton and J. G . Liehr, Biomed. Mass Spectrom. 12, 525 (1985). 5. R. P. Newton, S. C. Salih, B. J . Salvage and E. E. Kingston, Biochem. J. 221, 665 (1984). 6. R. P. Newton, E. E. Kingston, N. A. Hakeern, S. G . Salih, J. H. Beynon and C. D. Moyse, Biochem. J . 236,431 (1986). 7. R. P. Newton, D. Chiatante, D. Ghosh, A. G. Brenton, T. J. Walton, F. M. Harris and E. G. Brown, Phytochemisfry 28,2243 (1989). 8. R. P. Newton, T. J. Walton, S. A. Basaif, A. M. Jenkins, A. G. Brenton, D. Ghosh and F. M. Harris, Org. Mass Spectrom. 24, 679 (1989). 9. R. P. Newton, T. J. Walton, A. G. Brenton, E. E. Kingston and F. M. Harris, Rapid Commun. Mass Spectrom. 3 , 178 (1989). 10. R. P. Newton, J. A. Khan, D. Ghosh, J. I. Langridge, A. G. Brenton, F. M. Harris and T. J. Walton, Org. Mass Spectrom. 26, 447 (1991). 11. R. M. Caprioli, Mass Spectrom. Reu. 6, 237 (1987). 12. J. I. Langridge, A. M. Evans, D. Ghosh, T. J. Walton, A. G. Brenton, F. M. Harris and R. P. Newton, Analytica Chimica Acfa 247, 177 (1991). 13. D. A. Flockhart and J. D. Corbin, CRC Critical Reu. Biochem. 12, 133 (1982). 14. T. M. Lincoln and J. D. Corbin, Proc. NatlAcad. Sci. (USA) 74, 3293 (1977). 15. J. F. Kuo and P. Greengard, Methods Enzymol. 38, 329 (1992). 16. T. J. Walton, D. E. Ghosh, R . P. Newton, A. G. Brenton and F. M. Harris, Nucleotides and Nucleosides 9, 967 (1990). 17. R. P. Newton, Biochem. SOC.Trans. 20, 469 (1992). 18. Y. S. Cho-Chung, Biochem. SOC.Trans. 20,425 (1992).

Quantitation by fast-atom bombardment mass spectrometry: assay of cytidine 3',5'-cyclic monophosphate-responsive protein kinase.

A protein kinase, stimulated by cytidine 3',5'-cyclic monophosphate, is conventionally assayed by monitoring the incorporation of radiolabelled phosph...
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