[16]
DEUTERIUM EXCHANGE PROCEDURES
329
new opportunities to exploit derivatization. Likewise, the rapidly developing field of tandem mass spectrometry creates additional opportunities for the creative use of chemistry in conjunction with mass spectrometry to increase the ability to solve structural problems. In the area of protein structure, for example, mass spectrometry has already established itself as the "third leg of the stool" of protein primary structure methods (along with Edman and DNA sequencing). To date, however, mass spectrometry has been little exploited for other than determination of primary covalent structure. In conjuction with creative uses of chemical modifications (derivatizations), mass spectrometry offers the potential to yield three-dimensional information as well. Chemical linkage experiments can yield distance geometry measurements which, in conjunction with computer molecular graphics, can be used to refine three-dimensional structures. As a further extension, chemical trapping of intermediate states offers the potential to gain information on molecular dynamics. Thus, there appears to be a wide range of future applications for chemical derivatization in the broadest sense of the term. Just as with protein chemistry, whose demise was prematurely announced some years ago, 34 chemical derivatization in relation to mass spectrometry is far from dead. 34 A. D. B. Malcolm,
Nature (London) 275,
90 (1978).
[16] I n t r o d u c t i o n o f D e u t e r i u m b y E x c h a n g e for Measurement by Mass Spectrometry By
JAMES A. MCCLOSKEY
The chemical incorporation of deuterium followed by measurement of the intact product by mass spectrometry has for a number of years played a major role in three, often overlapping areas: the structural characterization of molecules of unknown structure; to gain information on the mechanisms of chemical or biological reactions; and as an aid in the interpretation of mass spectra. 1 In each of these areas mass spectrometry offers several general advantages: (1) relatively routine measurements can be made in the sample quantity range 10-11-10-6 g, sometimes in mixtures or without extensive sample purification; (2) the sites of labeling can often be deterI H. Budzikiewicz, C. Djerassi, and D. H. Williams, " S t r u c t u r e Elucidation of Natural Products by M a s s S p e c t r o m e t r y , " Vol. 1, Alkaloids, Chap. 2. Holden-Day, San Francisco, 1964.
METHODS IN ENZYMOLOGY, VOL. 193
Copyright © 1990by AcademicPress, Inc. All fights of reproduction in any form reserved.
330
GENERALTECHNIQUES
[16]
mined from the resulting mass spectrum; (3) in addition to the overall extent of labeling, the molar distribution of labeled species can be measured, which is not possible using methods in which the sample is cornbusted or similarly degraded prior to measurement. On the other hand, measurement of incorporation levels at several mole percent and below may be difficult (depending on molecular weight and method of sample introduction into the mass spectrometer) due to the necessity to correct for background ions and for naturally occurring heavy isotopes. The present chapter describes methods for the direct introduction of deuterium by simple exchange of heteroatom-bound protium, or "active" hydrogen [Eq. (1)]. excess
ROH
active D
~ ROD
excess
RCO2H
/
N--H
RSH
active D excess active D
excess active D
~ RCOzD
(1) ~
/
N--D
RSD
These reactions are generally quantitative in terms of incorporation level due to use of an excess of labeled solvent such as D20, and are essentially instantaneous without use of acid, base, or catalyst, thus generally excluding exchange of carbon-bound hydrogen. Although microscale deuterium exchange procedures are experimentally simple, they suffer from the principal disadvantage of ease of reexchange, which can occur readily due to adsorbed protium (water) on glassware or mass spectrometer surfaces, or in solvents or air. Compared with incorporation and measurement of nonactive deuterium or of other isotopes, sample handling and introduction into the mass spectrometer is usually a critical experimental issue, as described in later sections. Primarily due to reexchange, mass spectrometric methods employing thermal vaporization are generally limited to compounds having 4-6 exchangeable hydrogens, while with ion desorption methods which use deuterated matrix, the useful range may be extended to compounds having over 20 active hydrogen atoms. Some related methods not covered in detail in the present chapter include on-column exchange of active hydrogen with GC/MS 2 and ther2 j. A. McCloskey, this series, Vol. 14, p. 438.
[16]
DEUTERIUM EXCHANGE PROCEDURES
331
mospray LC/MS, 3 direct liquid introduction of deuterated solutions by thermospray,4 gas-phase exchange of active hydrogen by chemical ionization reagentss,6 such as D20,7 and the exchange of active hydrogen8and the hydrogen alpha to carbonyl groups 9 by preparative gas chromatography. Fetizon and Gramain] ° and, in particular Thomas] 1 have cataloged numerous methods and reactions for the incorporation of deuterium into organic compounds. Reagents
1)20. 98-99 atom%. D20 is widely available commercially with D concentrations above 98 atom % at reasonable cost due to its extensive use as an NMR solvent. Suppliers include: Cambridge Isotope Laboratories, Woburn, MA; Isotec, Inc., Miamisburg, OH; Merck Sharp & Dohme Isotopes, St. Louis, MO; and Icon Services, Summit, NJ. Although very highly labeled water (e.g., >99.998 atom %D) is available at higher cost, there is rarely any justification for use of solvents over 98 atom %D for exchange reactions to be monitored by mass spectrometry. A number of other deuterated solvents and reagents, such as CD3OD (or CH3OD at higher cost) and ND3, are also available from the above suppliers. Glycerol-(OD) 3. 98.8 atom %D, MSD Isotopes, St. Louis, MO. 3-Nitrobenzy[ Alcohol-OD. 12 3-Nitrobenzyl alcohol (NBA) is widely used as a matrix for desorption mass spectrometry. NBA (I ml) is vigorously shaken by hand in a I : l (v/v) mixture with D20 (I ml 99 atom %D) for I rain in a 3-ml Reacti-Vial (Pierce Chemical Co., Rockford, IL). The resulting two-phase system is allowed to separate for 5 min and the top D20 layer removed by aspiration. The procedure is repeated twice, for a total of three times. The resulting level of deuterium incorporation is reported as approximately 84%. 12 (No special precautions were taken
3 C. G. Edmonds, S. C. Pomerantz, F. F. Hsu, and J. A. McCloskey, Anal. Chem. 60, 2314 (1988). 4 M. M. Siegel, Anal. Chem. 60, 2090 (1988). 5 D. F. Hunt, C. N. McEwen, and R. A. Upham, Tetrahedron Lett. p. 4539 (1971). 6 W. Blum, E. Sclflumpf, J. G. Liehr, and W. J. Richter, Tetrahedron Lett. p. 565 (1976). 7 D. F. Hunt, C. N. McEwen, and R. A. Upham, Anal. Chem. 44, 1292 (1972). s G. J. Kallos and L. B. Westover, Tetrahedron Lett. p. 1223 (1967). 9 M. Senn, W. J. Richter, and A. L. Burlingame, J. Am. Chem. Soc. 87, 680 (1965). l0 M. Fetizon and J.-C. Gramain, Bull. Soc. Chim. Fr. p. 651 (1969). II A. F. Thomas, "Deuterium Labeling in Organic Chemistry." Appleton Century Crofts, New York, 1971. 12 A. M. Reddy, V. V. Mykytyn, and K. H. Schram, Biomed. Environ. Mass Spectrom. 18, 1087 (1989).
332
GENERALTECHNIQUES
[16]
to exclude traces of H20, so that higher incorporation levels should be attainable.)
Storage of Reagents Active deuterated reagents are subject to significant back-exchange in a period of minutes or less when reagents are in use or storage vessels are uncapped, and may undergo slow reexchange when Stored for long periods unless precautions are taken. Ampules or containers should be very tightly sealed and stored in a desiccator. In areas of high humidity, subdivision into smaller storage containers, or other manipulations, should be carried out in a dry glove-box if very high levels of enrichment are to be maintained. After preparation by exchange of active deuterated compounds, storage or transfer of the labeled product in organic solvents should be made with caution. Traces of H20 or other protic compounds in solvents can provide sufficient protium on a molar ratio basis to effect significant backexchange. Thomas has discussed methods for drying of glassware and maintenance of high levels of active deuterium. ~3 Procedures
Direct Exchange for Measurement by Electron Ionization (EI) or Chemical Ionization (CI) Mass Spectrometry Sample (2/zl), these bubbles may displace the solution from the capillary, therefore the pumping should proceed uniformly and slowly over several minutes in order to reduce bubble formation. Because this occasional problem is dependent in part on the sample holder and pumping geometry of the inlet system, it is advisable 13A. F. Thomas, "Deuterium Labeling in Organic Chemistry," Chap. 1. Appleton Century Crofts, New York, 1971.
[16]
DEUTERIUM EXCHANGE PROCEDURES
10() A
333
-25.6
74 91T74
)-
I~ZO] - 9 5 % ) , the sample probe tip is rinsed with ethanol and then cleaned by fast atom (or ion) bombardment of the dry surface for 2 to 3 14 B. S. Freiser, R. L. Woodin, and J. L. Beauchamp, J. Am. Chem. Soc. 97, 6895 (1975). t5 D. F. Hunt and S. K. Sethi, J. Am. Chem. Soc. 102, 6953 (1980). 16 A. G. Harrison, "Chemical Ionization Mass Spectrometry," p. 129. CRC Press, Boca Raton, FL, 1983. 17 S. Verma, S. C. Pomerantz, S. K. Sethi, and J. A. McCloskey, Anal. Chem. 58, 2898 (1986).
[16]
DEUTERIUM EXCHANGE PROCEDURES
335
M + 2H)+ 517 I-Z I--
_z C-uJ -
(517 -2 H20)+ 497 (G5 +2H) II
III
(Gs +2H)+ 558
__IX.I
572
'
m/z
FIG. 2. Fast atom bombardment mass spectrum of melezitose following deuterium exchange. M is defined as the molecule in which all l I hydroxyl hydrogens have been replaced by deuterium, and similarly for the glycerol (G) cluster ions. (Adapted with permission from Sethi et al.lS)
min, then rinsed with D20 immediately before loading the sample. Samples are prepared off-line in D20 solutions, at concentrations dependent on the ionization efficiency of the sample, but generally 0.5-3/.~g//~l). One microliter of the D20 solution is mixed on the probe tip with a 25% solution of glycerol-(OD)3 in D20 and then transferred to the probe vacuum lock in less than l0 sec. Syringes, glass capillaries, or other devices used for sample or solvent transfer should be prerinsed with D20 immediately before use. If lower deuterium incorporation levels are satisfactory, for example, when the sample molecule contains few ( < - 6 ) exchangeable hydrogens or a solvent of lower deuterium content is used, some of the above precautions can be relaxed, in particular, the procedure for cleaning the probe by ion or atom bombardment. The FAB mass spectrum of melezitose following deuterium exchange in glycerol-(OD)3 and D20 is shown in Fig. 2, and is representative of cases in which the number of hydrogens exchanged can be ascertained by visual inspection. The isotopic pattern in the cluster m/z 514-518 is asymmetric because m/z 517 represents the maximum exchange possible (DIIin melezitose, plus the ionizing deuteron), with m/z 518 corresponding very closely to the required 13C isotope peak for 12 carbon atoms. The decrease in abundances of m/z 516-514 corresponds to statistical decrease in deuterium content dictated by the H/D ratio in the sample at the time of ionization.
336
GENERALTECHNIQUES
[16]
Caution in interpretation of the data should be exercised when the resulting isotope pattern is sufficiently symmetrical that the mass of the fully exchanged ion cannot be determined by visual inspection. This circumstance results when the number of exchangd hydrogens becomes large (> ~ 18), or the extent of deuteration falls below -85-90%. In those cases, the correct number of deuterium atoms introduced can often be determined by comparison of the experimentally observed isotope pattern with theoretical patterns, calculated using test values of n, the number of deuterium atoms present. This method 17 requires knowledge of the approximate elemental composition of the unlabeled molecule, and the level of deuterium exchange. This latter value can be determined simply and accurately from matrix cluster ions in the spectrum, 17for example, the m/z 477 region in Fig. 2.18 Calculation of Deuterium Content Both the molar distribution of deuterium and the overall percentage of deuterium incorporated can be readily calculated from the resulting mass spectrum. 19The method is illustrated below using the data from phenylalanine in Fig. lb. The same principles can be extended to other stable isotopes, such as 180,2° and to more complex patterns as might result from presence of multiisotopic elements (Br, Cl). For extended treatment of isotopic abundance calculations, the reader is referred to earlier literature. 17,21 The tabulation below, with data taken from Fig. lb, illustrates how deuterium content can be calculated. Ion
m/z m/z m/z m/z m/z
165 166 167 168 169
Relative intensity (%) M M M M M
+ + + +
1 2 3 4
0.70 1.92 2.60 1.63 0.16
1. Estimate the abundance of naturally occurring heavy isotopes in the molecule (see also Appendixes 1 and 4 at the end of this volume). Is S. K. Sethi, D. L. Smith, and J. A. McCloskey, Biochem. Biophys. Res. Commun. 112, 126 (1983). 19 K. Biemann, "Mass Spectrometry: Organic Chemical Applications," p. 233. McGrawHill, New York, 1962. 2o R. C. Murphy and K. L. Clay, this volume [17]. ~: 21 H. Yamamoto and J. A. McCloskey, Anal. Chem. 49, 281 (1977).
[16]
DEUTERIUM EXCHANGE PROCEDURES
337
Depending on the a c c u r a c y desired, the second isotope peak (due to 13C2, etc.) can usually be ignored for molecules under Mr - 4 0 0 if only C, H, N, and O are present. Phenylalanine = C9HIIN First isotope peak = (9 x 1.1) + (1 × 0.4) = 10.3% 2. Correct peak M + 1 for natural heavy isotopes. 0.70 × 0.103 = 0.072 1.92 - 0.07 = 1.85% = (M + 1)¢o~ 3. Using the value of (M + 1)corr, calculate the natural heavy isotope contribution to M + 2, and correct the abundance of M + 2. 1.85 x 0.103 = 0.19 2.60 = 0.19 = 2.41% = (M + 2)tour 4. Make similar corrections to M + 3 and M + 4. 2.41 x 0.103 = 0.25 1.63 - 0.25 = 1.38% = (M + 3)tour 1.38 × 0.103 = 0.14 0.16 - 0.14 = 0.02% = (M + 4)corr 5. The sum of corrected abundance values from steps 2-4 is 6.36. Each ion can be expressed as a percentage of all molecular species.
M M M M M
+ + + +
I 2 3 4
0.70% 1.85% 2.41% 1.38% 0.02%
I1% 29% 38% 22% 0%
D0 D~ D, D3 D4
100%
The calculated distribution, therefore, confirms that phenylalanine has three exchangeable hydrogen atoms, even though the extent o f labeling is incomplete due to re-exchange. A similar calculation can be applied to the data in Fig. 2, showing that a maximum o f 12 deuterium atoms have been incorporated, or 11 in the neutral molecule. The deuterium content can be calculated as a percentage of the maximum possible, as follows: 6. Calculate the percentage deuterium in each molecular species, relative to the maximum value (three in phenylalanine).
338
GENERALTECHNIQUES
[17]
M M + 1 M + 2 M + 3
0% 33% 66% 100%
7. The percentage of deuterium is determined by multiplying the above values by the appropriate mole fraction (step 5 above).
DO D1 0 2
D3
0 33 66 100
x × x x
0.11 0.29 0.38 0.22
= 0.0 = 9.6 = 25.0 = 22.0
The sum of these values, 57%, represents the total extent of deuteration. When applied to the data for MD ÷ from melezitose in Fig. 2, a similar calculation shows 93% deuteration, indicating a much lower level of re-exchange, reflecting presence of an excess of active deuterium in intimate contact with solute molecules at the time of ionization, and on ion source surfaces.
[17] P r e p a r a t i o n of L a b e l e d M o l e c u l e s b y E x c h a n g e with O x y g e n - 18 W a t e r
By ROBERT C. MURPHY and KEITH L. CLAY The combined use of stable isotope-labeled molecules and mass spectrometry has been a powerful means to address numerous problems in chemistry and biochemistry which had been difficult or impossible to probe by other techniques. Molecules labeled with 180 have been used in numerous mass spectrometric studies from the classic determination of the fate of the carboxyl oxygen atoms in ester hydrolysis,1 mechanistic studies of gas-phase ion decompositions, internal standards for quantitative mass spectrometric analysis, and detailed studies of chemical and biochemical reaction mechanisms. This chapter will emphasize the synthesis of 1SO-labeled molecules to equilibrium exchange reactions with H 2 1 8 0 . Several functional groups containing oxygen have the property of undergoing exchange of their I M. L. Bender, J. Am. Chem. Soc. 73, 1626 (1951).
METHODS IN ENZYMOLOGY, VOL. 193
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
DEUTERIUM EXCHANGE PROCEDURES
329
new opportunities to exploit derivatization. Likewise, the rapidly developing field of tandem mass spectrometry creates additional opportunities for the creative use of chemistry in conjunction with mass spectrometry to increase the ability to solve structural problems. In the area of protein structure, for example, mass spectrometry has already established itself as the "third leg of the stool" of protein primary structure methods (along with Edman and DNA sequencing). To date, however, mass spectrometry has been little exploited for other than determination of primary covalent structure. In conjuction with creative uses of chemical modifications (derivatizations), mass spectrometry offers the potential to yield three-dimensional information as well. Chemical linkage experiments can yield distance geometry measurements which, in conjunction with computer molecular graphics, can be used to refine three-dimensional structures. As a further extension, chemical trapping of intermediate states offers the potential to gain information on molecular dynamics. Thus, there appears to be a wide range of future applications for chemical derivatization in the broadest sense of the term. Just as with protein chemistry, whose demise was prematurely announced some years ago, 34 chemical derivatization in relation to mass spectrometry is far from dead. 34 A. D. B. Malcolm,
Nature (London) 275,
90 (1978).
[16] I n t r o d u c t i o n o f D e u t e r i u m b y E x c h a n g e for Measurement by Mass Spectrometry By
JAMES A. MCCLOSKEY
The chemical incorporation of deuterium followed by measurement of the intact product by mass spectrometry has for a number of years played a major role in three, often overlapping areas: the structural characterization of molecules of unknown structure; to gain information on the mechanisms of chemical or biological reactions; and as an aid in the interpretation of mass spectra. 1 In each of these areas mass spectrometry offers several general advantages: (1) relatively routine measurements can be made in the sample quantity range 10-11-10-6 g, sometimes in mixtures or without extensive sample purification; (2) the sites of labeling can often be deterI H. Budzikiewicz, C. Djerassi, and D. H. Williams, " S t r u c t u r e Elucidation of Natural Products by M a s s S p e c t r o m e t r y , " Vol. 1, Alkaloids, Chap. 2. Holden-Day, San Francisco, 1964.
METHODS IN ENZYMOLOGY, VOL. 193
Copyright © 1990by AcademicPress, Inc. All fights of reproduction in any form reserved.
330
GENERALTECHNIQUES
[16]
mined from the resulting mass spectrum; (3) in addition to the overall extent of labeling, the molar distribution of labeled species can be measured, which is not possible using methods in which the sample is cornbusted or similarly degraded prior to measurement. On the other hand, measurement of incorporation levels at several mole percent and below may be difficult (depending on molecular weight and method of sample introduction into the mass spectrometer) due to the necessity to correct for background ions and for naturally occurring heavy isotopes. The present chapter describes methods for the direct introduction of deuterium by simple exchange of heteroatom-bound protium, or "active" hydrogen [Eq. (1)]. excess
ROH
active D
~ ROD
excess
RCO2H
/
N--H
RSH
active D excess active D
excess active D
~ RCOzD
(1) ~
/
N--D
RSD
These reactions are generally quantitative in terms of incorporation level due to use of an excess of labeled solvent such as D20, and are essentially instantaneous without use of acid, base, or catalyst, thus generally excluding exchange of carbon-bound hydrogen. Although microscale deuterium exchange procedures are experimentally simple, they suffer from the principal disadvantage of ease of reexchange, which can occur readily due to adsorbed protium (water) on glassware or mass spectrometer surfaces, or in solvents or air. Compared with incorporation and measurement of nonactive deuterium or of other isotopes, sample handling and introduction into the mass spectrometer is usually a critical experimental issue, as described in later sections. Primarily due to reexchange, mass spectrometric methods employing thermal vaporization are generally limited to compounds having 4-6 exchangeable hydrogens, while with ion desorption methods which use deuterated matrix, the useful range may be extended to compounds having over 20 active hydrogen atoms. Some related methods not covered in detail in the present chapter include on-column exchange of active hydrogen with GC/MS 2 and ther2 j. A. McCloskey, this series, Vol. 14, p. 438.
[16]
DEUTERIUM EXCHANGE PROCEDURES
331
mospray LC/MS, 3 direct liquid introduction of deuterated solutions by thermospray,4 gas-phase exchange of active hydrogen by chemical ionization reagentss,6 such as D20,7 and the exchange of active hydrogen8and the hydrogen alpha to carbonyl groups 9 by preparative gas chromatography. Fetizon and Gramain] ° and, in particular Thomas] 1 have cataloged numerous methods and reactions for the incorporation of deuterium into organic compounds. Reagents
1)20. 98-99 atom%. D20 is widely available commercially with D concentrations above 98 atom % at reasonable cost due to its extensive use as an NMR solvent. Suppliers include: Cambridge Isotope Laboratories, Woburn, MA; Isotec, Inc., Miamisburg, OH; Merck Sharp & Dohme Isotopes, St. Louis, MO; and Icon Services, Summit, NJ. Although very highly labeled water (e.g., >99.998 atom %D) is available at higher cost, there is rarely any justification for use of solvents over 98 atom %D for exchange reactions to be monitored by mass spectrometry. A number of other deuterated solvents and reagents, such as CD3OD (or CH3OD at higher cost) and ND3, are also available from the above suppliers. Glycerol-(OD) 3. 98.8 atom %D, MSD Isotopes, St. Louis, MO. 3-Nitrobenzy[ Alcohol-OD. 12 3-Nitrobenzyl alcohol (NBA) is widely used as a matrix for desorption mass spectrometry. NBA (I ml) is vigorously shaken by hand in a I : l (v/v) mixture with D20 (I ml 99 atom %D) for I rain in a 3-ml Reacti-Vial (Pierce Chemical Co., Rockford, IL). The resulting two-phase system is allowed to separate for 5 min and the top D20 layer removed by aspiration. The procedure is repeated twice, for a total of three times. The resulting level of deuterium incorporation is reported as approximately 84%. 12 (No special precautions were taken
3 C. G. Edmonds, S. C. Pomerantz, F. F. Hsu, and J. A. McCloskey, Anal. Chem. 60, 2314 (1988). 4 M. M. Siegel, Anal. Chem. 60, 2090 (1988). 5 D. F. Hunt, C. N. McEwen, and R. A. Upham, Tetrahedron Lett. p. 4539 (1971). 6 W. Blum, E. Sclflumpf, J. G. Liehr, and W. J. Richter, Tetrahedron Lett. p. 565 (1976). 7 D. F. Hunt, C. N. McEwen, and R. A. Upham, Anal. Chem. 44, 1292 (1972). s G. J. Kallos and L. B. Westover, Tetrahedron Lett. p. 1223 (1967). 9 M. Senn, W. J. Richter, and A. L. Burlingame, J. Am. Chem. Soc. 87, 680 (1965). l0 M. Fetizon and J.-C. Gramain, Bull. Soc. Chim. Fr. p. 651 (1969). II A. F. Thomas, "Deuterium Labeling in Organic Chemistry." Appleton Century Crofts, New York, 1971. 12 A. M. Reddy, V. V. Mykytyn, and K. H. Schram, Biomed. Environ. Mass Spectrom. 18, 1087 (1989).
332
GENERALTECHNIQUES
[16]
to exclude traces of H20, so that higher incorporation levels should be attainable.)
Storage of Reagents Active deuterated reagents are subject to significant back-exchange in a period of minutes or less when reagents are in use or storage vessels are uncapped, and may undergo slow reexchange when Stored for long periods unless precautions are taken. Ampules or containers should be very tightly sealed and stored in a desiccator. In areas of high humidity, subdivision into smaller storage containers, or other manipulations, should be carried out in a dry glove-box if very high levels of enrichment are to be maintained. After preparation by exchange of active deuterated compounds, storage or transfer of the labeled product in organic solvents should be made with caution. Traces of H20 or other protic compounds in solvents can provide sufficient protium on a molar ratio basis to effect significant backexchange. Thomas has discussed methods for drying of glassware and maintenance of high levels of active deuterium. ~3 Procedures
Direct Exchange for Measurement by Electron Ionization (EI) or Chemical Ionization (CI) Mass Spectrometry Sample (2/zl), these bubbles may displace the solution from the capillary, therefore the pumping should proceed uniformly and slowly over several minutes in order to reduce bubble formation. Because this occasional problem is dependent in part on the sample holder and pumping geometry of the inlet system, it is advisable 13A. F. Thomas, "Deuterium Labeling in Organic Chemistry," Chap. 1. Appleton Century Crofts, New York, 1971.
[16]
DEUTERIUM EXCHANGE PROCEDURES
10() A
333
-25.6
74 91T74
)-
I~ZO] - 9 5 % ) , the sample probe tip is rinsed with ethanol and then cleaned by fast atom (or ion) bombardment of the dry surface for 2 to 3 14 B. S. Freiser, R. L. Woodin, and J. L. Beauchamp, J. Am. Chem. Soc. 97, 6895 (1975). t5 D. F. Hunt and S. K. Sethi, J. Am. Chem. Soc. 102, 6953 (1980). 16 A. G. Harrison, "Chemical Ionization Mass Spectrometry," p. 129. CRC Press, Boca Raton, FL, 1983. 17 S. Verma, S. C. Pomerantz, S. K. Sethi, and J. A. McCloskey, Anal. Chem. 58, 2898 (1986).
[16]
DEUTERIUM EXCHANGE PROCEDURES
335
M + 2H)+ 517 I-Z I--
_z C-uJ -
(517 -2 H20)+ 497 (G5 +2H) II
III
(Gs +2H)+ 558
__IX.I
572
'
m/z
FIG. 2. Fast atom bombardment mass spectrum of melezitose following deuterium exchange. M is defined as the molecule in which all l I hydroxyl hydrogens have been replaced by deuterium, and similarly for the glycerol (G) cluster ions. (Adapted with permission from Sethi et al.lS)
min, then rinsed with D20 immediately before loading the sample. Samples are prepared off-line in D20 solutions, at concentrations dependent on the ionization efficiency of the sample, but generally 0.5-3/.~g//~l). One microliter of the D20 solution is mixed on the probe tip with a 25% solution of glycerol-(OD)3 in D20 and then transferred to the probe vacuum lock in less than l0 sec. Syringes, glass capillaries, or other devices used for sample or solvent transfer should be prerinsed with D20 immediately before use. If lower deuterium incorporation levels are satisfactory, for example, when the sample molecule contains few ( < - 6 ) exchangeable hydrogens or a solvent of lower deuterium content is used, some of the above precautions can be relaxed, in particular, the procedure for cleaning the probe by ion or atom bombardment. The FAB mass spectrum of melezitose following deuterium exchange in glycerol-(OD)3 and D20 is shown in Fig. 2, and is representative of cases in which the number of hydrogens exchanged can be ascertained by visual inspection. The isotopic pattern in the cluster m/z 514-518 is asymmetric because m/z 517 represents the maximum exchange possible (DIIin melezitose, plus the ionizing deuteron), with m/z 518 corresponding very closely to the required 13C isotope peak for 12 carbon atoms. The decrease in abundances of m/z 516-514 corresponds to statistical decrease in deuterium content dictated by the H/D ratio in the sample at the time of ionization.
336
GENERALTECHNIQUES
[16]
Caution in interpretation of the data should be exercised when the resulting isotope pattern is sufficiently symmetrical that the mass of the fully exchanged ion cannot be determined by visual inspection. This circumstance results when the number of exchangd hydrogens becomes large (> ~ 18), or the extent of deuteration falls below -85-90%. In those cases, the correct number of deuterium atoms introduced can often be determined by comparison of the experimentally observed isotope pattern with theoretical patterns, calculated using test values of n, the number of deuterium atoms present. This method 17 requires knowledge of the approximate elemental composition of the unlabeled molecule, and the level of deuterium exchange. This latter value can be determined simply and accurately from matrix cluster ions in the spectrum, 17for example, the m/z 477 region in Fig. 2.18 Calculation of Deuterium Content Both the molar distribution of deuterium and the overall percentage of deuterium incorporated can be readily calculated from the resulting mass spectrum. 19The method is illustrated below using the data from phenylalanine in Fig. lb. The same principles can be extended to other stable isotopes, such as 180,2° and to more complex patterns as might result from presence of multiisotopic elements (Br, Cl). For extended treatment of isotopic abundance calculations, the reader is referred to earlier literature. 17,21 The tabulation below, with data taken from Fig. lb, illustrates how deuterium content can be calculated. Ion
m/z m/z m/z m/z m/z
165 166 167 168 169
Relative intensity (%) M M M M M
+ + + +
1 2 3 4
0.70 1.92 2.60 1.63 0.16
1. Estimate the abundance of naturally occurring heavy isotopes in the molecule (see also Appendixes 1 and 4 at the end of this volume). Is S. K. Sethi, D. L. Smith, and J. A. McCloskey, Biochem. Biophys. Res. Commun. 112, 126 (1983). 19 K. Biemann, "Mass Spectrometry: Organic Chemical Applications," p. 233. McGrawHill, New York, 1962. 2o R. C. Murphy and K. L. Clay, this volume [17]. ~: 21 H. Yamamoto and J. A. McCloskey, Anal. Chem. 49, 281 (1977).
[16]
DEUTERIUM EXCHANGE PROCEDURES
337
Depending on the a c c u r a c y desired, the second isotope peak (due to 13C2, etc.) can usually be ignored for molecules under Mr - 4 0 0 if only C, H, N, and O are present. Phenylalanine = C9HIIN First isotope peak = (9 x 1.1) + (1 × 0.4) = 10.3% 2. Correct peak M + 1 for natural heavy isotopes. 0.70 × 0.103 = 0.072 1.92 - 0.07 = 1.85% = (M + 1)¢o~ 3. Using the value of (M + 1)corr, calculate the natural heavy isotope contribution to M + 2, and correct the abundance of M + 2. 1.85 x 0.103 = 0.19 2.60 = 0.19 = 2.41% = (M + 2)tour 4. Make similar corrections to M + 3 and M + 4. 2.41 x 0.103 = 0.25 1.63 - 0.25 = 1.38% = (M + 3)tour 1.38 × 0.103 = 0.14 0.16 - 0.14 = 0.02% = (M + 4)corr 5. The sum of corrected abundance values from steps 2-4 is 6.36. Each ion can be expressed as a percentage of all molecular species.
M M M M M
+ + + +
I 2 3 4
0.70% 1.85% 2.41% 1.38% 0.02%
I1% 29% 38% 22% 0%
D0 D~ D, D3 D4
100%
The calculated distribution, therefore, confirms that phenylalanine has three exchangeable hydrogen atoms, even though the extent o f labeling is incomplete due to re-exchange. A similar calculation can be applied to the data in Fig. 2, showing that a maximum o f 12 deuterium atoms have been incorporated, or 11 in the neutral molecule. The deuterium content can be calculated as a percentage of the maximum possible, as follows: 6. Calculate the percentage deuterium in each molecular species, relative to the maximum value (three in phenylalanine).
338
GENERALTECHNIQUES
[17]
M M + 1 M + 2 M + 3
0% 33% 66% 100%
7. The percentage of deuterium is determined by multiplying the above values by the appropriate mole fraction (step 5 above).
DO D1 0 2
D3
0 33 66 100
x × x x
0.11 0.29 0.38 0.22
= 0.0 = 9.6 = 25.0 = 22.0
The sum of these values, 57%, represents the total extent of deuteration. When applied to the data for MD ÷ from melezitose in Fig. 2, a similar calculation shows 93% deuteration, indicating a much lower level of re-exchange, reflecting presence of an excess of active deuterium in intimate contact with solute molecules at the time of ionization, and on ion source surfaces.
[17] P r e p a r a t i o n of L a b e l e d M o l e c u l e s b y E x c h a n g e with O x y g e n - 18 W a t e r
By ROBERT C. MURPHY and KEITH L. CLAY The combined use of stable isotope-labeled molecules and mass spectrometry has been a powerful means to address numerous problems in chemistry and biochemistry which had been difficult or impossible to probe by other techniques. Molecules labeled with 180 have been used in numerous mass spectrometric studies from the classic determination of the fate of the carboxyl oxygen atoms in ester hydrolysis,1 mechanistic studies of gas-phase ion decompositions, internal standards for quantitative mass spectrometric analysis, and detailed studies of chemical and biochemical reaction mechanisms. This chapter will emphasize the synthesis of 1SO-labeled molecules to equilibrium exchange reactions with H 2 1 8 0 . Several functional groups containing oxygen have the property of undergoing exchange of their I M. L. Bender, J. Am. Chem. Soc. 73, 1626 (1951).
METHODS IN ENZYMOLOGY, VOL. 193
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
