Anal. Chem. 1990, 62,125-129
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Endothermic Ion-Molecule Reactions: Strategies for Tandem Mass Spectrometric Structural Analyses of Large Biomolecules Ron Orlando, Constance Murphy, and Catherine Fenselau Structural Biochemistry Center, Department of Chemistry, University of Maryland Baltimore County, Baltimore, Maryland 21228
Gordon Hansen Boehringer-Ingelheim Research and Development Center, Ridgefield, Connecticut 06876
Robert J. Cotter* Middle Atlantic Mass Spectrometry Facility, Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
Very low energy colllsions between the protonated peptide leucine-enkephalin and ammonla on a tandem mass spectrometer lead to the formation of a proton-bound collision complex, whlch dlssoclates to form fragment Ions of the peptide or by transferrlng a proton to ammonia (neutralizatlon). The endothermlcity of the proton transfer reaction suggests that the proton was inltlaiiy located on the amide bonds of the peptide. From these studies we conclude that endothermlc Ion-molecule reactions may be effectlve for the fragmentatlon of large peptldes and/or as the first step in a neutrailzation/chemical reionlzatlon scheme in which the reverse (exothermlc) reaction Is used for reprotonatlon.
INTRODUCTION Over the past several years there has been an increasing interest in the use of tandem mass spectrometry (or MS/MS) for the structural analysis of complex biomolecules (1-3). In the most common experiment the first mass analyzer (MS1) is used to select ions (generally molecular ions) of a particular mass. These are then fragmented in a field-free region between the analyzers and enter the second mass analyzer (MS2) which is scanned to produce a product ion spectrum. The advantages of MS/MS analysis for sequencing peptides (4) and carbohydrates (5) and for other structural problems have been described. The major advantage is, of course, the ability to select the molecular ion of a single analyte from a mixture and obtain its mass spectrum, for example a specific tryptic fragment in the presence of other peptides coeluting from a reversed-phase high-performance liquid chromatography separation. When fast atom bombardment, FAB (6), is used as the ionization technique, the normal mass spectra are characterized by an abundance of peaks arising from the liquid matrix, adduct ions, and a general peak-at-every-mass background (7).In many cases, several molecular ion species (e.g. MH+ and MNa+) are produced. Selection of a single molecular ion species in an MS/MS experiment produces a mass spectrum whose peaks are unambiguously attributable to the analyte and for which the signal/noise relative to background ions may be considerably improved. A second advantage is that the fragmentation induced in the field-free region between the analyzers may be more extensive than and/or different from the occurring during ionization. Reinhold et al. (5) have noted that FAB mass spectra of carbohydrates generally produce little sequence information. Although chemical derivatization has been proposed to improve fragmentation (8),the problem can also be alleviated
by collisionally induced dissociation (CID). Biemann et al. (9) have described a number of side-chain fragmentation pathways for peptides, which occur in addition to the normal N- and C-terminal sequence ions (10). Fragment ions arising from side-chain cleavages can be used to distinguish residues, i.e. leucine and isoleucine, whose normal sequence ions would fall a t the same mass. The relative abundances of such cleavages can be controlled somewhat by the beam energy in a collisionally induced dissociation analysis (9). Gross et al. (11) have reported an increase in charge site remote fragmentation in collisionally induced dissociation spectra and have demonstrated its usefulness in determining the location of double bonds in unsaturated fatty acyl moieties. While electrons (12), photons (13),and solid targets (14) have all been used to induce fragmentation, collision of molecular ions with neutral atoms or molecules has been the most common approach (15). The energy required for dissociation of the ions may be derived from the electronic, vibrational, and relative translational energies (in the center of mass frame) of the colliding particles (16). The latter is related to the ion kinetic energy in the laboratory frame (Elab)by
(1) (EladMn) / (Mion + Mn) where Mn is the mass of the (target) neutral and Mionis the mass of the (projectile) ion, assuming that the target atoms or molecules are at rest. When translational energies are very low, ion-molecule (or ion-atom) reactions occur leading to charge transfer, dissociation, and other chemical reactions. For reactions that are endothermic, there is a threshold for the appearance of ionic products on the kinetic energy scale from which information on the heats of reaction, heats of formation, proton affinities, and electronic states of reactants and products may be determined (17, 18). Many of these reactions occur via a long-lived collision complex between the projectile ion and the target molecule (17, 18). As the translational energy of the projectile ion is increased, the internal energy of the collision complex is also increased, and fragment ions are formed as several dissociation channels are opened up. The important aspect of ion-molecule reactions in this energy regime is that the relative translational energy is almost completely utilized in the formation of the collision complex and subsequent dissociative products and is the reason that the relative energy scale can be used to determine endothermicity. While such reactions have been studied extensively by physical chemists for determining heats of formation and proton affinities, they have not been utilized for the dissociation of large biomolecules. At slightly higher translational energies, product ion formation follows more direct processes, where direct vibrational Ecm =
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 2, JANUARY 15, 1990
coupling leads to fragmentation with very high (10-100 A2) cross section (19). In the laboratory frame this generally corresponds to energies Elab= 10-100 eV, the low energy regime for collisionally induced dissociation reactions common on triple quadrupole (3) and hybrid (e.g. BEQQ) (20) instruments. Collisionally induced dissociation of high energy ions is generally carried out in the several kiloelectronvolt region (21). In this case, the interaction between the ion and the neutral target occurs in less than a vibrational period. The energy transferred to the ion is only a small fraction of the relative energy available and results in electronic excitation, energy equilibration according to the quasi-equilibrium theory (QET) and fragmentation patterns similar to those observed from (unimolecular) decomposition of ions formed by electron impact (22). High energy collisionally induced decomposition is most conveniently carried out on four sector instruments, i.e. two mass analyzers each composed of a double focusing electrostatic energy analyzer (E) and magnetic field (B) combination. For both high- and low-energy CID, eq 1 presents a distinct problem for heavy ions. For example, the molecular ion (MH+) of bovine insulin of mass 5775 daltons, when accelerated from the ion source to an energy of 8 keV, collides with a helium atom with a relative energy of only 5.5 eV. Only a portion of this energy is imparted to the molecular ion, and must be distributed over a very large number of degrees of freedom. The situation would appear to be improved by the use of heavier targets such as neon or xenon. However, scattering losses in these cases would greatly reduce the transmission of the product ions formed (23). Thus, while Biemann has reported the sequencing by MS/MS of a tryptic peptide of 3 kDa mass ( 4 ) , there are few, if any, examples of fragmentation of larger species. Laser induced photodissociation (13), and solid targets (14) for which M, would effectively be infinite and E,, = Elab, would seem to be solutions to the relative energy problem for high mass ions, and indeed they are currently under development. Alternatively, McLafferty et al. (24)have noted that structural information may also be obtained by reionizing the neutral products formed from unimolecular or collisionally induced dissociation. They have introduced a technique known as neutralizationlreionization mass spectrometry (NRMS), in which a mass selected high-energy ion beam from MS1 is neutralized by collision with a metal vapor and then reionized and mass analyzed by MS2 (25). The collision chamber consists of two regions (26). Vaporized metals in the first chamber favor change exchange over collisionally activated dissociation. Reionization by collision with O2is the most efficient (27,28) of the target gases tested and occurs in the second collision chamber, producing fragment ions as well for structural information. Both charge transfer processes (neutralization and reionization) take place at high kinetic energy and involve electron transfer. In McLafferty's experiments, mass analyses in MS2 were performed with an electric sector only. We suggest that ion-molecule reactions of low endothermicity between a protonated molecular ion and a suitable reactive target molecule would also offer a solution to the relative energy problem for high mass ions. The low relative energy required would place such reactions within range of the laboratory energy scale for even large molecular ions. The long-lived collision complexes between the projectile ion and the target molecule would lead to efficient fragmentation as well as neutralization, which could be utilized for reionization studies. We have noted that the neutralizationlreionization scheme of McLafferty et al. (25) involves electron transfer reactions
to neutralize and re-form radical ion species, while the molecular ions of complex biomolecules produced by desorption techniques are generally protonated, euen electron species. Thus, endothermic reactions, such as the proton transfer reactions H20+ + H2 H3+ OH (2) +
H 3 0 + + H2
-+
+ H3+ + H 2 0
(3)
which have been used previously by one of us to determine proton affinities (17,18), have been of interest to us for some time as a possible means for neutralizing protonated molecular ions, which would then be followed by reionization. Both reactions proceed via a collision complex near threshold and by direct (stripping) mechanism at higher energies. The abrupt decay in H3+intensity in reaction 3 indicated that the proton-bound complex between H20 and H2leads to a number of other fragmentation pathways. Thus we became interested in whether the formation of proton-bound complexes between protonated molecular ions and target molecules of slightly lower proton affinity would, in addition to neutralization, lead to an increase in ion fragmentation. The reaction
+ CH3CONH2
-
+ CH3CONH3+
(4) has been studied by Yamdagni and Kebarle (29) and is slightly exothermic PA(",) - PA(CH3CONH3+) = 207 kcal/mol - 210.4 kcal/mol = -3.4 kcal/mol NH,+
NH3
Assuming that protons are attached to the amide bonds in a peptide and that the proton affinity for attachment a t that site is similar to that of acetamide, the (reverse) reaction between a protonated peptide and ammonia should be barely endothermic. Thus, in this study, we followed the formation of the proton-bound collision complex, fragmentation, and neutralization that takes place in the reaction between protonated leucineenkephalin and ammonia near threshold and at higher translational energies where direct processes occur. We increased the target gas pressure to promote multiple collisions and compared the results with the reaction of stachyose with ammonia (for which proton transfer is exothermic). From these preliminary studies, we draw some conclusions on the value of endothermic and reactive collisions for inducing fragmentation and propose a scheme, known as neutralizationlchemical reionization mass spectrometry (NCRMS) which can be carried out in a single collision cell which is also a high-pressure chemical ionization (CI) source.
EXPERIMENTAL SECTION Product ion mass spectra were obtained for protonated leucine-enkephalinand stachyose using ammonia as the collision gas. Our initial experiments were carried out on a Kratos (Ramsey, NJ) MS8ORFQQ mass spectrometer, a hybrid instrument with an EBQQ configuration. Protonated molecular ions were formed by fast atom bombardment and mass selected by MS1 (EB). The quadrupole mass analyzer (Q2) was tuned to the molecular ion at a collision energy of 20 eV and the ammonia gas pressure in the collision chamber (61)was adjusted until the beam was attenuated by about 50%. Product ion spectra were then obtained at 20,40,60,20, and 10 eV, scanning a mass range which would also include adduct ions formed between the protonated molecular ion and ammonia. The instrument was then retuned to 20 eV and product ion spectra was obtained at 20,10,6, and 4 eV. The relative intensities in the spectra obtained at the overlapping points at 10 and 20 eV agreed to within 2%. Our most recent experiments employed a JEOL (Tokyo, Japan) Model HXllO/HX110 four-sector (EBEB configuration) tandem mass spectrometer. Protonated molecular ions were produced by fast atom bombardment, accelerated to 10 keV and mass selected by MS1. The collision cell voltage was placed at 10 kV - VIab, where VIab is the collision voltage which was varied from
ANALYTICAL CHEMISTRY, VOL. 62, NO. 2, JANUARY 15, 1990
127
Chart I 279
8 4
*
MNH~*
425
-t 397
* 1-
1-
656
573
279
J-co 397
0 to 80 V, measured as the potential difference between the collision cell and the ion source. Since the product ions of different masses are reaccelerated to (approximately) the same kinetic energy, the product ion spectra were obtained by scanning only the magnetic field, over a range which included the mass of the major fragment ions and the collision complex. (Strictly speaking, the electric field sector should be scanned as well. However, when the collision cell voltage is nearly equal to the ion source voltage, scanning the magnetic field alone has no appreciable adverse effech on the transmission of fragment ions of nearly equal energy and is operationally simpler.) A major advantage of this instrument is the much higher ion transmission at very low energies. This enabled the use of higher collision gas pressures to attenuate the protonated molecular ion beam by 80 and 99.5% in order to promote multiple collisions.
RESULTS AND DISCUSSION Chart I shows the ions appearing in the product ion spectra of protonated leucine-enkephalin and ammonia gas that were followed in this study. These include the protonated molecular ion (556), the M + NH4+ion (573), the C-terminal sequence ions (425 and 397), and the N-terminal sequence ion (279). Collisions at 50% Attenuation. The results for the product ion spectra obtained on the hybrid instrument are plotted as a function of laboratory energy in Figure 1. At all collision energies, the molecular ion (MH+) was the most abundant species in the mass range examined. In Figure la, the fragment ions (279, 397, and 425) resulting from direct, low-energy collisionally induced decomposition are observed in the region from 10 to 60 eV. Below 10 eV, however, there is a large increase in the relative abundnces of these fragment ions, which reaches a maximum around 8 eV (0.24 eV in the CM frame). At the same time, the proton bound complex (M + NH4+) shows a similar maximum at 6 eV (0.18 eV in the CM frame). Thus, the results exhibit a behavior typical of endothermic ion-molecule reactions which proceed via a collision complex. In this case the collision complex itself can be recorded. It is stable at low energies but is not formed at energies where direct processes occur. The ion-molecule reaction products, in this case fragment ions of leucine enkephalin, reach their maximum intensities a t slightly higher energies than the collision complex as the internal energy of the complex is increased. Figure l b shows the strong attenuation of the MH+ ion in the region for which ion-molecule reactions occur. In contrast, the reaction between protonated stachyose and ammonia (Figure 2) is clearly exothermic. The M + NH4+ ion and an ammoniated fragment ion (505 + NH4+)are the most abundant species at the lowest beam energies studied. As they decrease at higher beam energies, the relative abundance of the MH+ ion increases, as do two fragment ions (505 and 487) formed by direct processes. Ion transmission and signal/noise is poor at the lowest beam energies, so that it was not possible to accurately determine thresholds for the reactions between leucine-enkephalin and ammonia. Alexander and Boyd (30) have noted that for reactions occurring in an rf-only quadrupole cell, the variations in intensity vs Ekb must be interpreted with some caution in view of the strong spatial focusing effecta on ion transmission,
0 1
0
= d E
0 0.0
r
0.0
.
0
2
0
3
0
4
0
5
0
6
0
4 1
10
30
20
60
50
40
BEAM ENERGY, lab ( r V ) .
,
0.3
.
I
,
I
.
l
.
12
0.9
06
t.3
I
-
,
!.E
E cm (ev) Figure 1. Reaction of protonated leucine-enkephalin with ammonia in the collision chamber of an EBQQ tandem hybrid mass spectrometer: (A) intensity of fragment ions of masses 279, 397, and 425 amu and the ammonium adduct ion relative to the molecular ion as a function of the ion kinetic energy, E;, (E) intensity of the molecular ion normalized to its intensity observed at 20 eV as a function of ion kinetic energy. For convenience, the relative energy scale, E, is also
included.
too -
80
-
n
EX
60-
* c
E
40-
-li 20-
B E A N EMERGY, lab. (eV)
0.00
0.10
020
0.30
0.40
0.30
E cm (eV) Figure 2. Intensities of the product ions from the reaction of protonated stachyose with ammonia in the collision chamber of an EBOQ tandem hybrid mass spectrometer as a function of the Ion kinetic energy, E,,, and relative energy, E, in the center of mass frame.
particularly for a configuration in which a sector mass analyzer (B, BE or EB) is coupled to a quadrupole collision chamber. In addition, ions entering a quadrupole collision chamber undergo an oscillatory motion induced by the rf-field, so that
128
ANALYTICAL CHEMISTRY, VOL. 62, NO. 2, JANUARY 15, 1990 15 r
x
L
:
g
5g I \
'
oa
I
Flgure 3. Relative intensities of the proton bound complex (0)and vs ion kinetic energy for the reaction of protonated ammonium ion (0) leucine-enkephalin with ammonia carried out at 80% attenuation of the molecular ion in the collision chamber of a four sector mass spectrometer.
their transverse velocities become significant as one approaches zero beam energy and ion-molecule reactions can be observed below the expected threshold. Errors in threshold may also be brought about by Doppler broadening, i.e. an increase in the low energy tail near the reaction threshold due to the thermal velocity distributions of reactant ions and neutrals (31). This effect, in fact, becomes more pronounced (in the laboratory frame) in cases in which a heavy projectile collides with a relatively light target (18). Product ions may also be observed below the expected threshold for reactions in which the projectile ion contains appreciable precollision internal energy (30). In reaction 3, for example, a distribution of H30+ ions formed in the ground and first excited (triplet) states resulted in product ion intensities vs beam energy which reflected both endothermic and exothermic processes (18). Thus, in this study, it is encouraging that endothermic behavior is observed for the reaction between Leu-enkephalin and ammonia, that it occurs at relative energies consistent with the 0.15 eV endothermicity predicted from the results of Kebarle (29), and that there is a dramatic qualitative difference between that reaction and that of stachyose with ammonia. Collisions at Higher Attenuations. The four-sector tandem mass spectrometer enables us to overcome some of the problems encountered in studying these reactions at very low beam energies. The high ion transmission permitted measurements with good signal/noise down to beam energies of 1 eV, which could be maintained at collision chamber pressures for which the precursor ion was attenuated up to 99.5% Figure 3 shows the results for the proton transfer ionmolecule reaction MH+ NH, [M-H+-NHJ M NH4+ (5)
+
-+
+
I
I
40
30
Flgure 4. Relative intensities of fragment product ions vs ion kinetic energy for the reaction of protonated leucine-enkephalin with ammonia carried out at 80% attenuation of the molecular ion in the collision chamber of a four sector mass spectrometer (A,425; 0, 397; 0, 279
mlz). 150
A
t .-
2
loo-
t
C
u
.I
7
3
5
10
15
EL,b
20
-25
(eV)
Figure 5. Relative intensities of the proton bound complex (0)and vs ion kinetic energy for the reaction of protonated ammonium ion (0) leucine-enkephalin with ammonia carried out at 99.5% attenuation of the molecular ion in the collision chamber of a four sector mass spectrometer.
B
: I
c .-
1501
-P i
+
when the collision gas pressure has been adjusted to attenuate the MH+ ion by 80% at 20 eV. The collision complex and the NH4+product ion show very similar behavior, suggesting that redissociation of the complex with the proton attached to ammonia occurs with little excess energy. The threshold for the proton transfer reaction is observed at 6 eV, or about 0.18 eV in the CM frame. At higher energies, other dissociation channels are available as shown in Figure 4. The picture is more detailed than that obtained on the hybrid instrument and suggests that the fragment ion at 279 is formed with less excess internal energy than the ions at 425 and 391 (which differ by the loss of a neutral CO). At higher energies, direct, collisionally induced decomposition is observed. When the pressure of the collision gas is increased so as to attenuate the protonated molecular ion by 99.5%, several
I
20
10
0
ELab (ev) Flgure 0 . Relative intensities of fragment product ions vs ion kinetic energy for the reaction of protonated leucine-enkephalin with ammonia carried out at 99.5 % attenuation of the molecular ion in the collision chamber of a four sector mass spectrometer (0,425; 0, 397; A,279
mlz).
interesting things happen, which can most likely be attributed to an increase in multiple collisions. Figure 5 shows that the M + NH4+ ion continues to increase in relative intensity reaching a value at 10 eV which exceeds that of the MH+ ion. This may result from collisions of the protonated molelcular ion, which reduce its energy prior to the formation of the collision complex, or collisions occurring after formation of the complex, which stabilize it and reduce the possibilities for fragmentation. The proton transfer to ammonia is also con-
ANALYTICAL CHEMISTRY, VOL. 62, NO. 2, JANUARY 15, 1990
siderably increased, as reflected in the relative intensity of the NH4+ion. For both ions, the energy range for which they can be observed has been broadened. In Figure 6, the formation of fragments from the collision complex has been eliminated, and only those formed by direct processes at higher energies are observed. Thus, it would appear that muliple collisions at low energies favor neutralization (proton transfer) and stable complex formation over fragmentation.
CONCLUSIONS MS/MS experiments in which ammonia was used as the target gas have been reported by a number of other laboratories (32, 33). White et al. (32) investigated the products formed from the reaction of benzoyl cation with NH3. They observed products for which the reaction is exothermic, e.g.
C6H6COf
+ 3"
-
CGHb+ + CO + NH3
(6)
and may proceed via a collision complex; but they did not observe a reaction threshold or a dramatic decrease in molecular ion abundance indicative of extensive neutralization. (In this case, charge rather than proton transfer would be involved.) In contrast, Schmit et al. (33) observed neutralization of protonated molecular ions of several compounds by proton transfer to ammonia. Formation of this proton-bound collision complex was barely endothermic with a maximum around 0.02 eV (relative energy), while NH4+ ion formation showed a steep maximum at and decreased at 'lightly higher beam energies. In some cases the NH4+ ion current rose again at still higher beam energies reflecting proton transfer (neutralization) reactions by more direct processes. These experiments, as well as our own suggest at least two strategies for the MS/MS analysis Of large for which the low relative energy in direct (both low- and high-energy CID) processes is a problem. The first strategy exploits the efficient fragmentation resulting from endothermic ion-molecule reactions between a protonated peptide and ammonia* In addition to the molecular ion in MS1, one would also select a collision voltage. For leucine enkephalin, we have noted that the fragment ion signal is maximized at 10 eV. For insulin, it would occur at approximately 100 eV. Interestingly, the latter is an energy though not a mass range for quadrupole instruments. Thus, it would seem that there is a need for more studies of low-energy (and reactive) collisions of large molecules on sector tandem instruments. Our studies of low-energy collisions are based upon an assumption that the is attached to the amide bond and' in addition, that the endothermicity can be estimated from the exothermicity of the reverse reaction studied by Kebarle (29). It is likely that peptides containing more basic residues will exhibit greater endothermicity toward proton transfer, and our future studies will focus on peptides containing one or more arginine and/or lysine residues. At the same time, our threshold results appear to support these assumptions and suggest that similar studies could be used to locate the sites for proton attachment. The second strategy involves development of conditions that promote neutralization by proton transfer. As a first step in a neutralization/reionizutionscheme, endothermic proton transfer reactions could provide several advantages over the scheme proposed by McLafferty (25). First, proton transfer reactions would be a more logical approach than charge (electron) transfer for the protonated, even electron molecular ions produced by desorption techniques. Secondly, an en-
129
dothermic reaction would permit neutralization and reionization to take place in the same collision cell. An ion entering the cell with high kinetic energy would undergo multiple collisions until its energy was reduced to permit transfer of a proton in an endothermic reaction. If the cell was also a CI source, it would then reionize after further collisions, by the reverse, exothermic reaction. We propose the name neutralization/chemical reionization mass spectrometry (NCRMS) for this technique. A suitable collision cell, known as C12, has been constructed and is currently being tested in our laboratory,
LITERATURE CITED (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33)
Busch, K. L.; Cooks, R. G. Anal. Chem. 1983, 55, 38A. McLafferty, F. W. Science 1981, 241, 280-287. Yost, R. A.; Enke, C. G. J. Am. Chem. Soc.1978, 100, 2274. Biemann, K. Anal. Chem. 1986, 58, 1289A-1300A. Carr, S. A.; Reinhold, V. N.; Green, B. N.; Hass, J. R. Biomed. Environ. Mass Spectrom. 1985, 12, 288. Barber, M.; Bordoli. R. S.; Sedgwick. R. D.; Tyler, A. N., J. Chem. Soc., Chem. Commun. 1981, 325-326. Fenselau, C.; Cotter, R. J. Chem. Rev. 1987, 87, 501-512. Dell, A.; Thomas-Oates, J. E. I n Ana&& of Carbohydrates by GLC and MS; Biermann, C. J., McGinnis, G. D., Eds.; CRC Press: Boca Raton, FL, 1988; pp 217-236. Johnson, R. S.; Martin, S. A.; Biemann, K. Int. J. Mass. Spectrom. Ion Processes 1988q86, 137-154. Roepstorff, P.; Fohlman, J. Biomed. Mass Spectrom. 1984, 1 1 , 601. Adams, J.; Gross, M. L. J. Am. Chem. SOC. 1986, 108, 6915-6921. cOdy, R. B.; Freiser, B. S. Anal, Chem. 1979, 5 1 , 547, Baykut, G.; Watson, C. H.; Weller, R. R.; Eyler, J. R. J. Am. Chem. SOC. 1985. 107, 8036-8042. Mabud, Md. A,; Dekrey, M. J.; Cooks, R. G. Int. J . Mass Spectrom. Ion Processes 1985, 6 7 , 285-294. Collision Spectroscopy; Cooks, R. G., Ed.; Plenum: New York, 1978. Futrell, J. H.; Tiernan, T. 0. I n Ion-Mdecule Reactions; Franklin, J. L., Ed.; Plenum: New York, 1972; Vol. 2, pp 520-534. Cotter, R. J.; Rozett, R. W.; Koski, W. S. J. Chem. Phys. 1972, 57, 4 100-4 103. Cotter, R. J.; Koski, W. S. J . Chem. phys. 1973, 59, 784-787. Dawson, P. H.; Douglas, D. J. I n Tandem Mass Spectrometry; McLafferty, F. W., Ed.; John Wiley: New York, 1983; pp 125-148. Glish, G. L.; McLuckey, S. A.; Ridley, Et. Y.; Cooks, R. G. Int. J . Mass Spectrom. Ion phys. 1982, 4 1 , 157-177. Todd, P. J.; McLafferty, F. W. I n Tandem Mass Spectrometry; McLafferty, F. W., Ed.; John Wiley: New York, 1983; pp 149-174. Levsen, K.; Schwartz, H, Mass specporn. Rev. 1983, 83, 77-148, Laramee, J. A.; Cameron, D.; Cooks, R. G. J . Am. Chem. Soc. 1981, 103, 12-17. McLafferty, F. W.; Todd, P. J.; McGilvery, D. C.; Baidwin, M. A. J . Am. Chem. SOC. 1980, 102, 3360-3363. Danis, P. 0.; Wesdembtis, C.; McLafferty, F. W. J. Am. Chem. SOC. 1983, 105, 7454-7456. Danis, P. 0.;Feng, R.; McLafferty, F. W. Anal. Chem. 1986, 5 8 , 348-354. Gellene, G. I.; Porter, R. F. Int. J. Mass Spectrom. Ion Processes 1985, 6 4 , 55-66. Danis, P. 0.;Feng, R.; McLafferty, F. W. Anal. Chem. 1986, 5 8 , 355-358. Yamdagni, R.; Kebarle, P. J. Am. Chem. SOC.1973, 95, 3504-3510. Alexander, A. J.; Boyd, R. K. Int. J . Mass Spectrom. Ion Processes 1989, 90, 211-240. Chantry, P. J. J . Chem. Phys. 1971, 55, 2746. White, E. L.; Tabet, J.-C.; Bursey, M. M. Org. Mass Spectrom. 1987, 22, 132-139. Schmit, J.-P.; Beaudet, S.; Brisson, A. Org. Mass Spectrom. 1988, 21, 493-498.
RECEIVED for review July 24,1989. Accepted October 19,1989. Mass spectral measurements were carried out at Boehringer-Ingelheim (MS80RFQQ) and the Structural Biochemistry Center at the University of Maryland Baltimore County (HXllO/HX110), an NSF supported Biological Center. Research was supported in part by Grant DIR 8714238 from the National Science Foundation. The authors gratefully acknowledge the support of Dr. Frank Hatch for research carried out at Boehringer-Ingelheim. This work was presented in part at the Annual Meeting of the Federation of Analytical Chemists and Spectroscopy Societies (FACSS), October 1986.
