BIOLOGICAL MASS SPECTROMETRY, VOL. 20, 759-762 (1991)
Thermospray Tandem Mass Spectrometric Analysis of Oxygen Incorporation into Citrulline by Nitric Oxide Synthase A. M. Leone,? P. L. Francis, R. M. J. Palmer, D. S. Ashton and S. Moncada Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS, UK
Citrulline is formed as a co-product in the biosynthesis of nitric oxide from barginine by the action of either constitutive or inducible nitric oxide synthase which is present in a variety of cells. We have previously shown that the oxygen atom incorporated into both nitric oxide and citrulline derives from molecular oxygen and not water. Tbii paper describes the tandem mass spectrometric analysis of citrulline synthesized by the macrophage cell line 5774 in the presence of native or guanidino-labelled arginine and air or isotopically enriched oxygen. The results confirm that oxygen is incorporated into the ureido position of citrulline.
INTRODUCTION
Citrulline is formed as a co-product in the biosynthesis of nitric oxide (NO) from L-arginine by nitric oxide synthase. Two forms of this enzyme are now recognized. A constitutive Ca2+-dependent form has been demonstrated in the vascular endothelium,'*2brain,3 platelets4 and adrenal gland,5 where NO acts as a transduction mechanism for the stimulation of soluble guanylate cyclase to regulate vascular tone, platelet activation and other function^.'*^-^ The enzyme has also been found in a Ca2+-independent form, which is induced by immunological stimuli,'-12 in activated macrophages,'.' the vascular endothelium' and vascular smooth muscle' and the liver"*" and lung;" in this case, NO contributes to the cytotoxic effect of activated macrophages against tumour cells and micro- organism^'^ and may contribute to other functions including pathological vasodilation. The mechanism by which N O and citrulline are synthesized from L-arginine has been the subject of considerable spe~ulation.~*'.'~ Recent studies have shown that dioxygen is the source of oxygen incorporated into and the citrulline and NO by both the con~titutive'~ inducible' 5*16 enzymes. In our study,' the pseudomolecular ion of intact, derivatized citrulline was monitored to allow us to distinguish between water and dioxygen as the source of the oxygen in citrulline. In the present study we wished to confirm the ureido position as the site of incorporation of the oxygen atom into citrulline. We have, therefore, modified our method of analysis' to allow us to examine diagnostic fragment ions arising from the induced fragmentation of the pseudo-molecular ion of derivatized citrulline. MATERIALS A N D METHOD
The methods of cell preparation and the extraction and derivatization of citrulline have been described in detail
t Author to whom correspondence should be addressed. 1052-9306/91/12075944 $05.00
0 1991 by John Wiley & Sons, Ltd.
elsewhere." Briefly, 5774 cells were cultured for 24 h in the presence of endotoxin (10 pg ml-') and interferon y (150 u ml-') and for a further 24 h under an atmoor sphere of air or "O,, with either (14NG)~-arginine ( l 5NG)~-arginine as substrate. At the end of the incubation period 1 ml of the incubation medium was passed through an SCX Bond-Elut column containing 300 mg of sorbent. The column was successively washed with water, methanol, 2% ammonia in methanol, 5% ammonia in methanol and the citrulline eluted with 15% ammonia in methanol. The eluant was evaporated to dryness under oxygen-free nitrogen (OFN) and the residue derivatized at 135°C for 15 min with a mixture of dimethylformamide dimethylacetalacetonitrile-methanol (3 :2 : 1 v/v). Excess reagent was removed under a stream of OFN at 40°C and the residue dissolved in 50 p1 of methanol-water (60 :40 v/v) containing 0.1 M ammonium acetate. Tandem mass spectrometric analysis was performed on a Quattro 2000MD triple-quadrupole mass spectrometer (VG Biotech, Altrincham, UK). Samples were introduced into the source in methanol-water (60 :40 v/v) containing 0.1 M ammonium acetate by loop injection through a thermospray interface. Mass spectrometer operating conditions were optimized by tuning on the pseudo-molecular ion of authentic derivatized citrulline continuously introduced into the ion source at a concentration of 6 pg ml-' as described above. Daughter ion spectra were acquired over the mass range 70-320 u by collisionally activated dissociation (CAD) of the relevant pseudo-molecular ion at 75 eV with argon as the collision gas at a pressure of 1 mtorr. RESULTS
The pseudo-molecular ion of authentic derivatized citrulline (m/z 300) showed two CAD daughter ions at m/z 99 and 228 (Fig. 1). A similar spectrum was obtained from the pseudo-molecular ion at m/z 300 of citrulline extracted from cells incubated in the presence of (14NG)~-arginineand under an atmosphere of air Received 25 June 1991 Accepted (Revised) 26 August I991
A.
760
M. LEONE ET AL.
228
1
80
100
I
120 140
I
160
I
I
180 200
I
I
I
I
220
240
260
280
1
300
320
m/z Figure 1. The daughter ion spectrum of the dimethylaminomethylene methyl ester of authentic citrulline.
loo
3 I0
II
99
80
100
120
140
160
180 200
220
240
260
280
c,
300 320
mtz Figure 2. The daughter ion spectrum of derivatized citrulline extracted from 5774 cells incubated with ('4NG)~-argininein air.
(Fig. 2; n = 3), where n = the number of replicate incubates. When the cells were incubated with (14NG)-~arginine but under an atmosphere of "0, , the pseudomolecular ion shifted to m/z 302 and gave rise to ions at m/z 230 and 101 (Fig. 3; n = 3). When the cells were incubated with (' 'NG)~-arginineunder an atmosphere of "O,, the pseudo-molecular ion shifted to m/z 303 and gave rise to ions at m/z 230 and 102 (Fig. 4; n = 2).
DISCUSSION We have recently shown15 that N O synthase is a dioxygenase. In that study we monitored the pseudomolecular ion of intact derivatized citrulline to minimize background interference. Although this conferred a high degree of specificity on the analysis, allowing us to demonstrate that oxygen was incorporated from dioxygen and not water, it did not allow us to determine the site of incorporation. To do this it is necessary to analyze fragment ions diagnostic of the possible sites of incorporation for a mass shift due to the presence of
"0. As the derivatized extracts give rise to a complex low-mass background these mass shifts could not be easily determined from the full mass spectra. Consequently, to reduce this chemical noise, we used tandem mass spectrometry, in which the pseudo-molecular ion was selected by MS1, fragmented in the collision cell and the daugher ions analysed by MS2. The CAD spectrum of the pseudo-molecular ion of citrulline showed fragments at m/z 99 and 228 which have been assigned the structures shown in Scheme 1. If "0 were to be incorporated into the ureido position,
L228
Scheme 1.
N
II
MS/MS ANALYSIS OF OXYGEN INCORPORATION INTO CITRULLINE
80
100
120
140
160
180 200
220
240
260
280 300
761
320
mlz Figure 3. The daughter ion spectrum of derivatized citrulline extracted from J774 cells incubated in ''0,and arginine.
in the presence of ( ' * N C ) ~ -
3 3
7 230
80
100 120
140
160
180 200 220 m/z
240
260
280
30
1 320
Figure 4. The daughter ion spectrum of derivatized citrulline extracted from J774 cells incubated in ''0,and arginine.
then both the pseudo-molecular ion and the m/z 99 fragment would increase by two mass units. However, if the oxygen were to be incorporated into the a-carboxyl then the m/z 99 fragment would remain unchanged. The m/z 228 fragment would increase by two mass units in either case. When the cells were incubated in " O , , the m/z 99 fragment increased by two mass units, indicating that the ureido position is the site of oxygen incorporation. Furthermore, when (15NG)~-arginine was used as substrate in an incubation under the m/z 99 fragment increased by 3 u; i.e. an additional one mass unit,
in the presence of
(15NG)~-
due to the presence of a guanido "N atom. This observed mass shift clearly shows that the " 0 and 15N atoms are present in the same fragment (m/z 99) and this confirms both the assigned structure and the ureido position as the site of oxygen incorporation into citrulline by NO synthase. In conclusion, we have confirmed our previous finding that di-oxygen is the source of the oxygen incorporated into citrulline by inducible N O synthase and have further shown unambiguously that it is incorporated into the ureido position.
REFERENCES 1. R. M. J. Palmer, D. S. Ashton & S. Moncada, Nature (Lond.) 317, 524-526 (1988). 2. R. M. J. Palmer & S. Moncada, Biochem. Biophys. Res. Commun. 159,348 (1989). 3. R. G. Knowles, M. Palacios, R. M. J. Palmer & S. Moncada, Proc. Natl. Acad. Sci. USA 86, 5159 (1989). 4. M. W. Radomski, R. M. J. Palmer & S. Moncada, Proc. Nati. Acad. Sci. USA 87,5193 (1990). 5. M. Palacios, R. G. Knowles. R. M. J. Palmer & S. Moncada, Biochem. Biophys. Res. Commun. 165,802 (1989). 6. J. €3. Hibbs. R. R. Taintor and 2. Vazrin, Science 253, 473 (1987).
7. M. A. Marletta, P. S. Yoon, R. lyengar, C. D. Leaf and J. S. Wishnok, Biochemistry 27,8706 (1988). 8. M. W. Radomski, R. M. J. Palmer and S. Moncada, Proc. Natl. Acad. Sci. USA 87, 10043 (1990). 9. D. D. Rees, S. Cellek, R. M. J. Palmer and S. Moncada, Biochem. Biophys. Res. Commun. 173, 541 (1990). 10. T. R. Billiar, R. 0.Curran, 0.J. Stuehr, K. Hofmann and R. L. Simons, J. Exp. Med 169, 1467 (1989). 11. R. G. Knowles, M. Merrett, M. Salter and S. Moncada. Biochem.J. 270. 833 (1990). 12. T. 6. McCall, N. K. Boughton-Smith. R. M. J. Palmer, 6. J. R. Whittle and S. Moncada, Biochem.J. 262, 293 (1989).
162
A.
M. LEONE ET AL.
13. J. B. Hibbs, R. R. Taintor, Z. Vavrin. D. L. Granger, J.-C. Drapier, 1. J. Amber & J. R. Lancaster, in Nitric Oxide from L-Aginine: A Bioregulatory Mechanism, ed by s. Moncada and E. A. Higgs, p. 189. Excerpta Medica 897 (1 990). 14. E. G. De Master, L. Raij. S. L. Archer and E. K. Weir, Biochem. Biophys. Res. Commun. 163, 527 (1989).
15. A. M. Leone, R. M. J. Palmer, R. G. Knowles, P. L. Francis, D. S. Ashton and S. Moncada, J . Biol. Chem. (in press). 16. N. S. Kwon, C. F. Nathan, C. Gilker, 0. W. Griffith, D. E. Matthews and D. J. Stuehr, J. Biol. Chem. 265, 13442 (1 990).
Thermospray Tandem Mass Spectrometric Analysis of Oxygen Incorporation into Citrulline by Nitric Oxide Synthase A. M. Leone,? P. L. Francis, R. M. J. Palmer, D. S. Ashton and S. Moncada Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS, UK
Citrulline is formed as a co-product in the biosynthesis of nitric oxide from barginine by the action of either constitutive or inducible nitric oxide synthase which is present in a variety of cells. We have previously shown that the oxygen atom incorporated into both nitric oxide and citrulline derives from molecular oxygen and not water. Tbii paper describes the tandem mass spectrometric analysis of citrulline synthesized by the macrophage cell line 5774 in the presence of native or guanidino-labelled arginine and air or isotopically enriched oxygen. The results confirm that oxygen is incorporated into the ureido position of citrulline.
INTRODUCTION
Citrulline is formed as a co-product in the biosynthesis of nitric oxide (NO) from L-arginine by nitric oxide synthase. Two forms of this enzyme are now recognized. A constitutive Ca2+-dependent form has been demonstrated in the vascular endothelium,'*2brain,3 platelets4 and adrenal gland,5 where NO acts as a transduction mechanism for the stimulation of soluble guanylate cyclase to regulate vascular tone, platelet activation and other function^.'*^-^ The enzyme has also been found in a Ca2+-independent form, which is induced by immunological stimuli,'-12 in activated macrophages,'.' the vascular endothelium' and vascular smooth muscle' and the liver"*" and lung;" in this case, NO contributes to the cytotoxic effect of activated macrophages against tumour cells and micro- organism^'^ and may contribute to other functions including pathological vasodilation. The mechanism by which N O and citrulline are synthesized from L-arginine has been the subject of considerable spe~ulation.~*'.'~ Recent studies have shown that dioxygen is the source of oxygen incorporated into and the citrulline and NO by both the con~titutive'~ inducible' 5*16 enzymes. In our study,' the pseudomolecular ion of intact, derivatized citrulline was monitored to allow us to distinguish between water and dioxygen as the source of the oxygen in citrulline. In the present study we wished to confirm the ureido position as the site of incorporation of the oxygen atom into citrulline. We have, therefore, modified our method of analysis' to allow us to examine diagnostic fragment ions arising from the induced fragmentation of the pseudo-molecular ion of derivatized citrulline. MATERIALS A N D METHOD
The methods of cell preparation and the extraction and derivatization of citrulline have been described in detail
t Author to whom correspondence should be addressed. 1052-9306/91/12075944 $05.00
0 1991 by John Wiley & Sons, Ltd.
elsewhere." Briefly, 5774 cells were cultured for 24 h in the presence of endotoxin (10 pg ml-') and interferon y (150 u ml-') and for a further 24 h under an atmoor sphere of air or "O,, with either (14NG)~-arginine ( l 5NG)~-arginine as substrate. At the end of the incubation period 1 ml of the incubation medium was passed through an SCX Bond-Elut column containing 300 mg of sorbent. The column was successively washed with water, methanol, 2% ammonia in methanol, 5% ammonia in methanol and the citrulline eluted with 15% ammonia in methanol. The eluant was evaporated to dryness under oxygen-free nitrogen (OFN) and the residue derivatized at 135°C for 15 min with a mixture of dimethylformamide dimethylacetalacetonitrile-methanol (3 :2 : 1 v/v). Excess reagent was removed under a stream of OFN at 40°C and the residue dissolved in 50 p1 of methanol-water (60 :40 v/v) containing 0.1 M ammonium acetate. Tandem mass spectrometric analysis was performed on a Quattro 2000MD triple-quadrupole mass spectrometer (VG Biotech, Altrincham, UK). Samples were introduced into the source in methanol-water (60 :40 v/v) containing 0.1 M ammonium acetate by loop injection through a thermospray interface. Mass spectrometer operating conditions were optimized by tuning on the pseudo-molecular ion of authentic derivatized citrulline continuously introduced into the ion source at a concentration of 6 pg ml-' as described above. Daughter ion spectra were acquired over the mass range 70-320 u by collisionally activated dissociation (CAD) of the relevant pseudo-molecular ion at 75 eV with argon as the collision gas at a pressure of 1 mtorr. RESULTS
The pseudo-molecular ion of authentic derivatized citrulline (m/z 300) showed two CAD daughter ions at m/z 99 and 228 (Fig. 1). A similar spectrum was obtained from the pseudo-molecular ion at m/z 300 of citrulline extracted from cells incubated in the presence of (14NG)~-arginineand under an atmosphere of air Received 25 June 1991 Accepted (Revised) 26 August I991
A.
760
M. LEONE ET AL.
228
1
80
100
I
120 140
I
160
I
I
180 200
I
I
I
I
220
240
260
280
1
300
320
m/z Figure 1. The daughter ion spectrum of the dimethylaminomethylene methyl ester of authentic citrulline.
loo
3 I0
II
99
80
100
120
140
160
180 200
220
240
260
280
c,
300 320
mtz Figure 2. The daughter ion spectrum of derivatized citrulline extracted from 5774 cells incubated with ('4NG)~-argininein air.
(Fig. 2; n = 3), where n = the number of replicate incubates. When the cells were incubated with (14NG)-~arginine but under an atmosphere of "0, , the pseudomolecular ion shifted to m/z 302 and gave rise to ions at m/z 230 and 101 (Fig. 3; n = 3). When the cells were incubated with (' 'NG)~-arginineunder an atmosphere of "O,, the pseudo-molecular ion shifted to m/z 303 and gave rise to ions at m/z 230 and 102 (Fig. 4; n = 2).
DISCUSSION We have recently shown15 that N O synthase is a dioxygenase. In that study we monitored the pseudomolecular ion of intact derivatized citrulline to minimize background interference. Although this conferred a high degree of specificity on the analysis, allowing us to demonstrate that oxygen was incorporated from dioxygen and not water, it did not allow us to determine the site of incorporation. To do this it is necessary to analyze fragment ions diagnostic of the possible sites of incorporation for a mass shift due to the presence of
"0. As the derivatized extracts give rise to a complex low-mass background these mass shifts could not be easily determined from the full mass spectra. Consequently, to reduce this chemical noise, we used tandem mass spectrometry, in which the pseudo-molecular ion was selected by MS1, fragmented in the collision cell and the daugher ions analysed by MS2. The CAD spectrum of the pseudo-molecular ion of citrulline showed fragments at m/z 99 and 228 which have been assigned the structures shown in Scheme 1. If "0 were to be incorporated into the ureido position,
L228
Scheme 1.
N
II
MS/MS ANALYSIS OF OXYGEN INCORPORATION INTO CITRULLINE
80
100
120
140
160
180 200
220
240
260
280 300
761
320
mlz Figure 3. The daughter ion spectrum of derivatized citrulline extracted from J774 cells incubated in ''0,and arginine.
in the presence of ( ' * N C ) ~ -
3 3
7 230
80
100 120
140
160
180 200 220 m/z
240
260
280
30
1 320
Figure 4. The daughter ion spectrum of derivatized citrulline extracted from J774 cells incubated in ''0,and arginine.
then both the pseudo-molecular ion and the m/z 99 fragment would increase by two mass units. However, if the oxygen were to be incorporated into the a-carboxyl then the m/z 99 fragment would remain unchanged. The m/z 228 fragment would increase by two mass units in either case. When the cells were incubated in " O , , the m/z 99 fragment increased by two mass units, indicating that the ureido position is the site of oxygen incorporation. Furthermore, when (15NG)~-arginine was used as substrate in an incubation under the m/z 99 fragment increased by 3 u; i.e. an additional one mass unit,
in the presence of
(15NG)~-
due to the presence of a guanido "N atom. This observed mass shift clearly shows that the " 0 and 15N atoms are present in the same fragment (m/z 99) and this confirms both the assigned structure and the ureido position as the site of oxygen incorporation into citrulline by NO synthase. In conclusion, we have confirmed our previous finding that di-oxygen is the source of the oxygen incorporated into citrulline by inducible N O synthase and have further shown unambiguously that it is incorporated into the ureido position.
REFERENCES 1. R. M. J. Palmer, D. S. Ashton & S. Moncada, Nature (Lond.) 317, 524-526 (1988). 2. R. M. J. Palmer & S. Moncada, Biochem. Biophys. Res. Commun. 159,348 (1989). 3. R. G. Knowles, M. Palacios, R. M. J. Palmer & S. Moncada, Proc. Natl. Acad. Sci. USA 86, 5159 (1989). 4. M. W. Radomski, R. M. J. Palmer & S. Moncada, Proc. Nati. Acad. Sci. USA 87,5193 (1990). 5. M. Palacios, R. G. Knowles. R. M. J. Palmer & S. Moncada, Biochem. Biophys. Res. Commun. 165,802 (1989). 6. J. €3. Hibbs. R. R. Taintor and 2. Vazrin, Science 253, 473 (1987).
7. M. A. Marletta, P. S. Yoon, R. lyengar, C. D. Leaf and J. S. Wishnok, Biochemistry 27,8706 (1988). 8. M. W. Radomski, R. M. J. Palmer and S. Moncada, Proc. Natl. Acad. Sci. USA 87, 10043 (1990). 9. D. D. Rees, S. Cellek, R. M. J. Palmer and S. Moncada, Biochem. Biophys. Res. Commun. 173, 541 (1990). 10. T. R. Billiar, R. 0.Curran, 0.J. Stuehr, K. Hofmann and R. L. Simons, J. Exp. Med 169, 1467 (1989). 11. R. G. Knowles, M. Merrett, M. Salter and S. Moncada. Biochem.J. 270. 833 (1990). 12. T. 6. McCall, N. K. Boughton-Smith. R. M. J. Palmer, 6. J. R. Whittle and S. Moncada, Biochem.J. 262, 293 (1989).
162
A.
M. LEONE ET AL.
13. J. B. Hibbs, R. R. Taintor, Z. Vavrin. D. L. Granger, J.-C. Drapier, 1. J. Amber & J. R. Lancaster, in Nitric Oxide from L-Aginine: A Bioregulatory Mechanism, ed by s. Moncada and E. A. Higgs, p. 189. Excerpta Medica 897 (1 990). 14. E. G. De Master, L. Raij. S. L. Archer and E. K. Weir, Biochem. Biophys. Res. Commun. 163, 527 (1989).
15. A. M. Leone, R. M. J. Palmer, R. G. Knowles, P. L. Francis, D. S. Ashton and S. Moncada, J . Biol. Chem. (in press). 16. N. S. Kwon, C. F. Nathan, C. Gilker, 0. W. Griffith, D. E. Matthews and D. J. Stuehr, J. Biol. Chem. 265, 13442 (1 990).
