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Anal. Chem. 1986, 58, 2898-2902
Fast Atom Bombardment Mass Spectrometry following Hydrogen-Deuterium Exchange Sunita Verma, Steven C. Pomerantz, Satinder K. Sethi, and James A. McCloskey*
Departments of Medicinal Chemistry and Biochemistry, University of Utah, Salt Lake City, Utah 84112
Exchange of labile hydrogen (protium) by deuterium can be carried out in the presence of O-perdeuterloglycerol and D,O, and the products can be directly analyzed by fast atom bombardment (FAB) mass spectrometry with mlnlmal back exchange. Exchange levels up to 97 % In Ions having 28 labile hydrogens are demonstrated. Methods of calculatlon and errors In measurement of lsotoplc patterns for ions having large numbers of deuterlum atoms have been studied. The use of computer-generated isotopic patterns for the comparison with experimentally measured isotoplc clusters for analysls of complex patterns Is described. The matrix exchange-FAB technique is useful to determine the exact active hydrogen content in molecules of unknown structure, in studies of condensed-phase reactlons Induced by particle bombardment, and In the Interpretation of mass spectra produced by desorption methods.
T h e exchange of deuterium (D) for protium in organic molecules has been used for many years in studies of mass spectrometric reactions and for structural studies by mass spectrometry ( I ) . A major limitation in the procedure has been the occurrence of reexchange reactions in the inlet system and ion source that result in an ambiguous isotopic pattern, and therefore, practical applications of the method have been limited to molecules containing four to six exchangeable hydrogens. Exchange in the gas phase under chemical ionization conditions may be useful in some cases (2-4), but serious uncertainties can result from exchange of nonlabile hydrogens (5, 6). A practical approach toward this problem is possible using fast atom bombardment (FAB) mass spectrometry in which 'H-D exchange reactions are carried out in deuteriated matrix on the sample probe tip. This approach results in very high levels of isotopic incorporation by providing a large excess of labile deuterium in intimate contact with solute molecules at the time of ionization, with minimal opportunity for reexchange. Further, the desorption-ionization process provides a means of obtaining mass spectra of polar molecules that contain larger numbers of active hydrogens than would be the case using thermal vaporization. A preliminary communication outlining the scope of the method has appeared (7). We presently report on methods of calculation and measurement of deuterium exchange levels and on potential sources of error in such calculations, and we demonstrate the use of computer-generated isotopic patterns for comparison with complex experimentally measured isotope clusters when large numbers of active hydrogens ( > - 2 O ) have been exchanged. As shown by recent studies from several laboratories, the matrix exchange technique is useful in three areas: (a) to determine the exact active hydrogen content in molecules of unknown structure, of particular value when unusual or unexpected structures are encountered (8),and to select between some isomeric alternatives (9); (b) studies of condensed-phase reactions induced by particle bombardment ( 10, 1 I ) ; (c) to assist in the interpretation of mass spectra produced
by desorption techniques (12). EXPERIMENTAL SECTION Materials. C-Perdeuterioglycerol,97+ atom % D (referred to as glycerol-d,), 0-perdeuterioglycerol, 98.8 atom % D (referred to as glycerol-d,), and DzO, 99.6 atom %D, were purchased from MSD Isotopes, St. Louis, MO. fl-Cyclodextrin and somatostatin were from Sigma Chemical Co., St. Louis, MO; leucine enkephalin (referred to as leu-enkephalin) was from U S . Biochemical Corp., Cleveland, OH. Glycerol-d, was found to contain small amounts of 0-deuterium and so was washed with H20 until pure glycerol-d, was obtained. Glycerol-d3was stored in a screw-capped vial in a desiccator after being opened. A new vial of DzOwas used for each experiment, when sample enrichment levels over 93% D were desired. Mass Spectrometry. Mass spectra were acquired with a MAT 731 instrument and an oscillographic recorder with magnetic or electric scanning a t 8 keV accelerating potential. In some cases ion abundance data were taken from spectra accumulated from 8 to 32 scans over a limited mass range using a Nicolet 1170 signal averager. FAB mass spectra were produced by using an Ion Tech FAB 11N ion source with a neutral Xe beam of 6 keV energy. For experiments requiring high deuterium incorporation levels (>95% D) the FAB probe tip was rinsed with ethanol and then cleaned by fast atom bombardment of the dry surface for 2-3 min, then rinsed with D20 immediately before loading the sample. Samples were prepared off-line by solution in D20 (3-5 p g / p L DzO); 1 pL was mixed on the probe tip with a 25% solution of glycerol-d3in DzOand then transferred to the probe vacuum lock in less than 10 s. Mass spectra acquired using glycerol or glycerol-d, matrix were determined by use of analogous protocols for cleaning the probe and using HzO in order to exclude labile D from previous runs. Calculation of Isotopic Distributions. Theoretical massabundance distribution patterns were calculated with a Digital Equipment Corp. PDP 11/24 computer, based on algorithms described in the following section. The pattern matching programs outlined in a later section and Figure 2 are written in FORTRAN IV for interactive graphics display on a Wicat Systems MG 8000 terminal. RESULTS AND DISCUSSION The number of active hydrogens ( n )in a molecule or ion is determined from the mass shift that occurs following hydrogen-deuterium exchange. In the case of molecules that contain fewer than 10 exchangeable hydrogens and have molecular weights below about 600, the number of exchangeable hydrogens can usually be established by visual inspection of the FAB mass spectrum (7). As the value of n increases, or as contributions to the observed pattern from natural heavy isotopes increase (higher molecular weight), or as the level of deuterium incorporation descreases, it becomes increasingly difficult to establish the value of n from the observed isotopic cluster. In those cases, interpretation of the isotopic pattern can be aided by measurement of the level of deuteriation using glycerol cluster ions produced in the same recording. The level of deuterium enrichment thus determined is then used to calculate the expected isotopic cluster patterns from the sample molecule, for incremental values of n as described in later sections. Calculation of Deuterium Enrichment and Distribution. Deuterium enrichment, % D, is expressed as a per-
0003-2700/86/0358-2898$01,50/0 C 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
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the modest incorporation level of 85% D, which is readily achieved experimentally. On the other hand when n = 20, the lower isotopic species predominates even at the relatively high enrichment level of 95% D. Experience has shown that levels of 91-93% D can be routinely achieved with modest effort, but the region 93-97% D requires particular care (see Experimental Section) to ensure high initial incorporation levels and minimal back exchange from atmospheric water. As an alternative method of calculation, % D can be derived from abundance data taken from the entire isotopic cluster, as shown in eq 3, where r is the number of D atoms in each
3
2
M D' M D+- I I
0 05
95
90
r=O
IO0
%D
Figure 1. Relative abundances of MD' and (MD - 1)' ions calculated from eq 2 for various numbers of exchangeable hydrogens (n).
centage of the maximum possible incorporation level (Le., three deuteriums for each glycerol moiety in glycerol-d3, or five deuteriums for glycerol-d,). Percent D can be determined from glycerol cluster peaks in the FAB mass spectrum by two methods: measurement of intensities I, and I,+, following correction for natural heavy isotopes, for any two successive peaks p and p + 1,or measurement of the intensities of peaks from the entire isotopic cluster. With either method, the values of n for peaks chosen for measurement are readily apparent in the glycerol-d, mass spectrum. For the two-peak method, the ratio R of intensities for peaks p and p + 1 can be defined as
where D is the mole fraction corresponding to % D (Le., 95% D = 0.950), and n!
(;)
When peak p rangement to
+ 1 = n in
= (n-p)!n!
MD+, eq 1 reduces upon rear-
D=- nR
1 + nR
The ratio R from eq 1 for the case in which I,,, corresponds to MD+ and I, to (MD - 1)+ is plotted in Figure 1for several values of n over a range of deuterium enrichment. The relationships shown predict that (exclusive of contributions from natural heavy isotopes) when n = 5, the MD+ ion will be more abundant than the lower isotopic species (MD - l)+, even at
I '
ESTIMATE ELEMENTAL COMPOSITION
r=O
ion, I , is the relative intensity for any peak containing r D atoms, after correction for natural heavy isotopes, and Wris the ratio r / n and represents the fraction of D present in each ion relative to the maximum number of D atoms possible. The value of % D measured from glycerol ions can be used to determine the isotopic distribution due to deuterium, using terms from expansion of the binomial function [D (1- D)]", Le., for 0 Si%, the probability of the (i + 1)th peak is given by
+
where i is any integer between 0 and n. The isotopic pattern for any deuterium-exchanged molecule can then be calculated by combining the deuterium distribution values with those calculated from elemental composition by standard methods (13,14) or taken from the isotopic pattern produced by the FAB mass spectrum of the unlabeled compound. A critique of these alternative methods of calculation is given in a later section. As predicted from Figure 1,the unambiguous determination of n from the spectra of large molecules containing many exchangeable hydrogens may be difficult by simple visual inspection. The problem may be further compounded in cases involving certain polyisotopic elements (e.g., C1, Br) or large numbers of natural-abundance elements. In all such cases, the analysis of the isotopic pattern can be assisted by comparison of experimental data with calculated patterns, as outlined in Figure 2. The calculated pattern can be based on either known or estimated elemental composition (Figure 2, point 1) or on the isotopic pattern observed for the unlabeled material (point 2). The experimental and calculated patterns for the deuteriated molecular clusters are graphically displayed, with provision for incrementing the value of n, or for changing % D. The latter option is less commonly used, but permits the observer to readily assess the effect of small errors in measurement of % D from glycerol cluster ions.
I
VARY n AND/OR 0
NO
GLYCEROL
-
DETERMINE FAB/MS OF LPBELED COMPOUND IN GLYCEROL-d3
-
CALCULATE %D FROM GLYCEROL CLUSTER IONS
CALCULPTE ISOTOPIC PATTERN FOR n ACTIVE HYDROGENS AT %O CALCULATED FROM GLYCEROL CLUSTER IONS
-
DISPLAY AND COMPARE
__c CALCULATE0 AND OBSERVED PATTERNS
SO TOPIC
Figure 2. Procedures for determination of n in complex isotopic clusters by comparison of calculated and observed isotopic patterns.
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
Table I. Calculated Effect of Presence of Skeletal Protium in G,D+ Cluster Ions from Glycerol-d3
apparent 70D 70D true value
GI
G*
G1
92 94 96
3.9" 91.112 93.092 95.073
4.3" 91.438 93.426 95.414
5.6" 91.488 93.477 95.465
Assumed level of protium orginating from glycerol carbon skeleton.
Figure 3. Variations in deuterium content of cluster ions from glycerol-d, during fast atom bombardment based on (a) fuikluster method of calculation and (b) two-peak method of calculation.
Measurement of Deuterium Incorporation. The interpretation of isotopic cluster patterns for large values of n requires that deuterium enrichment levels be accurately known. As a corollary, the question arises as to whether 70 D in the substance being examined is the same as that in the matrix. A related concern is whether H+ or D+ that is transferred from matrix to analyte in the ionization step originates solely from glycerol oxygen as opposed to carbon. In response to these issues, % D in glycerol (G) cluster ions from glycerol-d3was determined as a function of time using the two-peak and full-cluster methods of calculation, shown in Figure 3. These data demonstrate that (i) stable values of % D over a period of time (until evaporation of glycerol is nearly complete) are obtained by the two-peak method, but a significant decrease of % D with time is observed when calculations are made using the full-cluster method (Figure 3a). The effect is larger when G3D+is used for measurement as compared to when G2D+is used. (ii) Percent D values from G2D+and G3D+are experimentally indistinguishable (Figure 3b) but are approximately 0.5% higher than for GD'. The difference in 70 D values resulting from different methods of calculation shown in Figure 3a is attributed to the presence of (G,H+ - 2H)-type ion species ( x = 1,2,3, ...), which in unlabeled glycerol occur a t m / z 91, 183, 275, ..., which are unresolved from G,H+-type ions in clusters from glycerol-d3. The interfering ions increase in relative abundance with time (15) and are relatively more abundant in larger cluster ions (16), thus producing the effect shown in Figure 3a. This problem is avoided by use of the two-peak method of calculation because the G,H+ - 2H ions principally contain three exchangeable hydrogens per glycerol unit, in support of an earlier proposal for their structures by Field (15),and therefore do not interfere with measurement of MD+/(MD - 1)+ratios. The labeling patterns in cluster ions from glycerol-d5were examined to determine whether carbon-bound hydrogen might contribute significantly to protonation of analyte and thus influence measured values of 70D. Excess ion currents above natural 13C isotope levels during the first 3 min of bombardment were determined to be 3.9% in GH+, 4.3% in G2H+,and 5.6% in G3H+,resulting from deuterium originating from the glycerol carbon skeleton. These results qualitatively support a 3% incorporation value earlier reported by Ligon (17) and imply that a small error is introduced in the determination of % D when determined from the labeling pattern in glycerol ions. The potential error from this source from each of the three principal glycerol cluster ions can be readily calculated for assumed true values of 70D. As shown in Table I, the errors from measurement of GD+ are about 0.9% D and approximately 0.5% D for GzD+ and G3D+,exclusive of deuterium isotope effects. Similarly, transfer of hydrogen from the carbon skeleton
of glycerol-d, to produce protonated analyte molecules (MH+) would lead to deviations of % D in analyte ions compared with % D in the bulk matrix in which the analyte is dissolved. That such is the case is suggested by the observation that first isotope peaks of MH+ ions from a variety of materials using glycerol-d5 as matrix are usually higher than expected from either abundance values based on elemental composition or from FAB mass spectra taken using unlabeled glycerol. As typical examples, melezitose and leu-enkephalin exhibit 5-7 % enhancement of MH' first isotope peaks in mass spectra acquired using glycerol-d,. As a consequence, errors of 0 . 8 4 9 % are introduced in the measurement of % D in these compounds. Fortunately, the direction of the error is the same as that estimated in glycerol cluster ions (Table I), and so the overall effect of hydrogen transfer from glycerol carbon is small, with regard to establishing the value of n. Similar errors are expected in the case of FAB mass spectra that yield significant peaks one or two mass units above MH+ (in unlabeled matrix), designated as MHz'+ or MH3+ (18). Accurate assessment of errors in such cases requires knowledge of whether the extra hydrogen atoms are active (thus changing the value of n for those components), as illustrated for the peptide somatostatin in the following section. Taking a general case as an example, in which MH2'+ ions are present at 5% the abundance of MH+, for n = 25, the error in calculating % D resulting from lower isotope peaks of MH;+-type ions is only about 0.2% in the worst case, namely that in which the value of n is the same for both species. As shown in a later section, this type of error will be minimized if the mass spectrum of the unlabeled compound (point 2 in Figure 2) rather than elemental composition is used as a basis for calculation of the isotopic pattern following deuterium enrichment. To test the overall hypothesis that the extent of deuterium labeling in the molecule of interest is accurately represented by 70D in glycerol, as determined by glycerol ions, the pentapeptide leu-enkephalin (molecular weight (M,) = 555, n = 8 in the neutral molecule) was examined. Percent D levels were determined as a function of time for both peptide and glycerol, as shown in Figure 4,based on the two-peak method of calculation. After initial instability of the ion beam, % D levels were found to be the same within experimental error ( < 0 . 5 % ) and are thus indistinguishable when comparing peptide and G2D+cluster ions. The lower value observed for GD+ ions reflects the same difference obtained using neat glycerol-d, (Figure 3b) and indicates that GD+ should not be used to establish 70D unless a correction factor of about 0.5% is introduced. Similar results and conclusions were reached from separate experiments using melezitose, M , = 504, n = 16, angiotensin 11, M , = 1045, n = 16, and P-cyclodextrin, M , = 1134, n = 21 (values of n for neutral molecules). Analogous studies of glycerol-d,, glycerol-d,, and model substrates were carried out with negative ion measurements, based on glycerol ions of the type (G, - H)-. The results were essentially the same as for positive ion mass spectra, so the method can be applied to saccharides, nucleotides, and other
ANALYTICAL CHEMISTRY, VOL. 58,
NO. 14, DECEMBER 1986 2901
MD+ 1157
1 0
0
4 Minutes
Flgure 4. Variations in deuterium content of ions from glycerol (individual data points not shown) and leu-enkephalln (open circles) during fast atom bombardment, following deuterium exchange. Deuterium content was calculated by use of the two-peak method.
molecules that exhibit suitable negative ion FAB mass spectra. Comparison of Experimental and Calculated Isotopic Abundance Patterns. As the value of n increases and the value of % D decreases, isotopic patterns of increasing complexity are produced, so unambiguous assignment of the value of n may be difficult. Under such circumstances the generation of expected isotopic patterns for various value of n (Figure 2) can be used to clarify or substantiate interpretation of the data. The approach is illustrated by the FAB mass spectrum of P-cyclodextrin ( M , = 1134, n = 21 in the neutral molecule) following deuterium exchange, shown in Figure 5. The patterns shown in Figure 6a-c were produced by an interactive computer program in which experimental data were compared with patterns calculated from the elemental comand % D was deterposition of b-cyclodextrin (C42H70035) mined from the G2Df ion isotopic distribution (96.6%), as represented by point 1 in Figure 2. For three test values of n, the value n = 22 in the ion (21 in the neutral molecule) clearly represents the best fit (Figure 6b). No correction was made for ions of the type (MH - 2)' from the spectrum of unlabeled P-cyclodextrin,resulting in signals in the lower mass section of Figure 6b for which there are no calculated counterparts. It is noted that the calculated abundance patterns in Figure 6a-c appear very similar, but with the major difference of being shifted in mass. The program used to calculate abundance patterns also permits variation of % D for any value of n, to determine what effect errors in measurement of % D might have. Patterns in Figure 6d-f were generated by use of a hypothetical value of 94.6% D, 2% D lower than for Figure 6a-c, well outside the range of errors discussed in the preceding section. The correct value of n = 22 (Figure 6b) is readily apparent, and it was not possible to find an incorrect combination of n and 70 D to form a better match than by using the correct set, represented in Figure 6b. The accuracy of assignment of n ultimately depends on the quality of the recorded mass spectrum in the molecular ion region and the extent to which other processes or contaminants may contribute to the observed spectrum. An example of this is given in application of the exchange technique to peptides that contain disulfide bridges. Reductive opening of the disulfide bridge
c)S-S
FAB
SH HS
m/ z Figure 5. Molecular ion region of the FAB mass spectrum of @-cyclodextrin following deuterium exchange. The m / z 1157 ion contains 22 deuterium atoms.
1157
1152
1160
I152
9465bD
(e) n :22
1152
1160
1152
1160
Figure 6. Observed isotopic patterns from P-cyclodextrin following compared with calculated deuterium exchange at 96.6% D (-) patterns (---) at 96.6% D (a-c) and 94.6% D (d-f).
during FAB has been proposed to produce the measurable amounts of the bis-thiol derivative (eq 4) (19-21) for which the value of n will be 2 greater (for each reduced disulfide
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
5j:
rC'
r i
Figure 7. Molecular ion region from the FAB mass spectrum of somatostatin (-) compared wilh the theoretical isotope distribution for MH' calculated from elemental composition (- - -). Contributions from m l r 1635 and 1636 are omitted from the calculated distribution.
ented (22). Their results, and rationale in design of their experiment in which deuterium exchange was not expected, remains unclear. After deuterium exchange of somatostatin at a measured incorporation level of 97.6%, the resulting experimental pattern can be compared with patterns based either on elemental composition (Figure 8a) or on the pattern from the unlabeled peptide (Figure 8b). By either method, a value of n = 27 in the MD+ ion ( n = 26 in the neutral molecule) can be clearly established by incrementing the value of n as outlined in Figure 2. A better match of patterns is obtained in Figure 8b compared with Figure 8a because peaks representing other processes such as reduction are in part taken into account. In Figure 8a,b the greater experimental signals at (MD + 4)+ ( m / z 1668) and (MD + 5 ) + reflect reduced species considered to be an estimate because ions from the reduced species increase in relative abundance somewhat during bombardment (21). It is interesting to note the excess experimental peak contributions at m / z 1666 and 1667,which remain in Figure 8c. The peak height comparisons at these mass values are consistent with the presence of a reduced component having the same number of active hydrogens as somatostatin, but the present data do not further address this point. Independent of corrections that may be applied to improve the fit for somatostatin, it is noted that the correct value of n was readily established without corrections. ACKNOWLEDGMENT We are indebted to Andrew Iverson and William R. Gray for helpful discussions. LITERATURE CITED
0-
Figure 8. Molecular ion region from the FAB mass spectrum of deuterium-exchanged somatostatin (-) at 97.6% D cornpared with isotopic distributions calculated (- --) at 97.6% D from (a) elemental composition (n = 27), (b) isotope distribution exhibited in the mass spectrum of unlabeled somatostatin (n = 27), and (c) elemental composition (n = 27) plus 2 5 % reduced somatostatin (n = 29). Contributions from (MH - 1)'- and (MH - 2)+-type ions are omitted from calculated distributions.
bridge) than the native peptide and will contribute to the observed isotopic abundance pattern. Figure 7 shows the FAB mass spectrum in the molecular region of the tetradecapeptide somatostatin ( M , = 1636, n = 26 in the neutral molecule) compared with the isotope pattern required by its elemental The presence of approxicomposition (Ci6H104N18019S2). mately 26% of a reduced species is suggested by the excess peak height in the experimental pattern a t (MH + 2)' ( m / z 1639) and the corresponding first isotope peak ( m / z 1640). Following deuterium exchange, the MH+ ion shifts in mass to m / z 1664, shown by the solid lines in Figure 8, demonstrating that nearly complete exchange of all active hydrogens in somatostatin has taken place prior to ionization. These results are in conflict with the report by Buko and co-workers (22) who studied the FAB mass spectrum of somatostatin produced using glycerol-d, as the matrix. Their results were interpreted in terms of deuterium incorporation into the dithiol moiety (eq 4) but without exchange of the remaining 26 active hydrogens. The fragment ions from their deuteriated matrix experiment have no clear correlation with those resulting from use of unlabeled matrix, and no discussion of deuterium incorporation in various fragment ions was pres-
(1) Budzikiewicz, H.; Djerassi, C.; Williams, D. H. Structure Elucidation of Natural Products by Mass Spectrometry, Vol . I . A/kaloMs ; HoldenDay: San Francisco, CA, 1964; Chapter 2. (2) Hunt, D. F.; McEwen, C. N.; Upham, R. A. Anal. Cbem. 1972, 44, 1292-1 294. (3) Blum, W.; Schlumpf, E.; Liehr, J. G.; Richter, W. J. Tetrabedron Lett 1976, 565-568. (4) Lin, Y. Y.; Smith, L. L. Biomed. Mass Spectrom. 1974, 6 , 15-18. (5) Hunt, D. F.; Sethi, S. K. J . A m . Cbem. SOC. 1980, 102, 6953-6963. (6) Martinsen, D. P.; Buttrill, S. E., Jr. Org. Mass Spectrom. 1976, 1 1 , 762-772. (7) Sethi, S. K.; Smith. D. L.; McCloskey, J. A. Biocbem. Biopbys. Res Commun. 1983, 112, 126-131. (8) Isono, K.; Uramoto, M.; Kusakabe, H.; Miyata, N.; Koyama, T.; Ubukata, M.; Sethi, S. K.; McCloskey, J. A. J . Antibiot. 1984, 3 7 , 670-672. (9) Carroll, S. F.; McCloskey, J. A.; Crain, P. F.; Oppenheimer, N. J.; Marschner, T. M.; Collier, R. J. Proc. Natl. Acad. Sci. U . S . A . 1985, 82, 7237-724 1. (10) Ligon, W . V., Jr. I n t . J . Mass Spectrom. Ion Pbys. 1983, 5 2 , 183- 187. (11) Sethi, S, K.; Nelson, C. C.; McCloskey, J. A. Anal. Cbem. 1984, 56, 1975-1977. (12) Sindona, G.; Uccella, N.; Weciawek, K. J . Cbem. Res. 1982, 104-185. (13) Beynon, J. H. Mass Spectrometry and Its Applications to Organic Chemistry; Elsevier: New York, 1960; pp 294-302. (14) Yamamoto, H.; McCioskey, J. A. Anal. Cbem. 1977, 4 9 , 281-283. (15) Field, F. H. J . Phys. Cbem. 1982, 86, 5115-5123. (16) Martin, S. A,; Costeiio, C. E.; Biemann, K . Anal. Cbem. 1982, 54, 2362-2368. (17) Ligon, W. V., Jr. I n r . J . Mass Spectrom. Ion Pbys. 1983, 5 2 , 189-. 193. (18) Cerny, R. L.; Gross, M. L. Anal. Cbem. 1985, 57, 1160-1163. (19) Fuiita, Y.; Matsuo, T.; Sakurai, T.; Matsuda, H. Inf. J . Mass Spectrom, Ion. Processes 1985, 6 3 , 231-240. (20) Buko, A. M.; Fraser, B. A. Biomed. Mass Spectrom. 1985, 12, 577-585. (21) Yazdanparast, R.; Andrews, P.; Smith, D. L.; Dixon, J. E. Anal. Biocbem. 1986, 153, 340-353. (22) Buko, A, M.; Phillips, L. R.; Fraser. B. A. Biomed. Mass Spectrom 1983. 10, 408-409.
RECEIVED for review June 10,1986. Accepted July 30, 1986. This work was supported by the National Institute of General Medical Sciences through Grant GM 21584.
