2232
Anal. Chem. 1988,60,2232-2236
in chloroaluminate and chlorogallate mass spectra. Interestingly, the presence is indicated, in the gas phase, of novel polynuclear aluminum and gallium chloro complexes that contain subvalent metal ions, e.g., A13Cl,, Ga2C15-,and Ga3C19-. Such species may have unusual structures with an electron located outside the molecule; for example, centrally within a six-membered ring of AI-C1 bridged atoms. Recent calculations have supported the premise that an electron can be localized within the cubic equilibrium configuration of Na4C14clusters a t low temperatures (19).
ACKNOWLEDGMENT Helpful discussions were held of Lewis structures of subvalent species with N. Kestner. LITERATURE CITED Berber, M.; Bordoli, R. S.;Sedgwick, R. D.; Tyler, A. N. J. Chem. Soc., Chem. Commun. 1981, 7, 325. Bojesen, G. Org. Mess Spectrom. 1985, 1 0 , 413. Ackermann, B. L.; Tsarbopoulos, A,; Allison, J. Anal. Chem. 1985, 57, 1766. Franzen, G.; Gilbert, B. P.; Peizer, G.; DePauw, E. Org. Mess Spectrom. 1988, 21, 443. Gale, R. J.; Gilbert, B. P.; Osteryoung. R. A. Inorg. Chem. 1978, 77, 2728.
(6) Frank J. Seiler Research Laboratory, Technical Memorandum FJSRL-
TM-83-00 16, USAF Academy, Colorado, 1983. (7) Berg, R. W.; Hjuler, H. A.; Bjerrum, N. J. Inorg. Chem. 1985, 2 4 , 4506. (8)Thewalt, U.; Stoilmaier, F. Angew. Chem., Int. Ed. Engl. 1982, 2 7 , 133. (9) Khvostenko, V. I.; Sultanov, A. Zh. Eksp. Teor. Fiz., 1964, 46, 1605. (10) Chai, B. J.; KO, H. C.; Greenbaum, M. A,; Farber, M. J . Phys. Chem. 1967, 77, 3331. (11) Heller, D. N.: Yergery, J.; Cotter, R. J. Anal. Chem. 1983, 55, 1310. (12) Kinsella, E.; Chadwick, J.; Coward, J. J. Chem. SOC.A 1968, 969. (13) Evans, C. A.; Hal-Tan, K.; Tapper, S. P.; Taylor, M. J. J . Chem. Soc., Dalton Trans. 1973, 988. (14) Beamish, J. C.: Small. R. W.; Worrall, I. J. Inorg. Chem. 1979, 18, 220. (15) Wicelinski, S. P.; Gale, R. J. Proceedings of the Jdnt International Symposium on Molten Salts, Honolulu; The Electrochemical Society: Pennington, NJ, 1987; Vol. 87-7, pp 591-601. (16) Baev, A. K.; Fadeev, V. M.; Ratkovskii, I. A. Obshch. Prikl. Khim. 1969, 7 , 90. (17) Wicelinski, S. P. Ph.D. Thesls, Louisiana State University, 1987. (18) Connolly, M. J.; Orth, R. G. Anal. Chem. 1987, 59,903. (19) Scharf, D.; Jortner, J.: Landman, U. J . Chem. Phys. 1987, 87(5), 2716.
RECEIVEDfor review October 14,1987. Accepted June 6,1988. This paper was presented orally at the 194th ACS National Meeting, New Orleans, LA, Sept 4, 1987.
Consecutive Reaction Monitoring in a Four-Sector Mass Spectrometer: MS4 and One Step Beyond Kenneth B. Tomer,* Christian R. Guenat, and Leesa J. Deterding
Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, North Carolina 27709
MS4 and MS' spectra In which decomposltlons occur In consecutlve field-free regions can be obtained on a four-sector tandem mass spectrometer of BEEB geometry. Appllcatlons to both Ion chemlstry and peptide structure elucldatlon are demonstrated. Examples of ion chemlstry Include the following: differentlation between the structure of benzoyl ions arlslng In the Ion source and vla consecutlve reactlons; monltorlng the decompodtlons of C,H7+ Ions formed via consecutive reactions from dlfferent precursors such as toluene, butylbenzene, and 2-bromoethylbenzene; and differentlatlon of CSH80+'Ions arlslng via consecutive McLafferty rearrangements and of acetone molecular Ions. I n the area of peptlde structure elucidations, leucine and isoleucine residues In trlpeptldes were dlstlngulshed by the decomposltlons of lmmonlum Ions ( m / z 86) found In MS4 analyses, and the amino acld sequence of a tetrapeptide was determined by followlng sequentlal amino acld residue loss.
One advantage of the development of multiple analyzer mass spectrometers (e.g. EBE and EBQQ) has been the capability of investigating sequential reactions in MS" experiments. In theory, the number of sequential reactions that can be studied in a magnetic sector instrument is ( n + 1)where n is equal to the number of analyzers and (n + 1)is equal to the number of field-free regions present in the instrument. Defining the initial reaction monitored as an MS2 experiment,
* Author to whom correspondence should be addressed.
we then define the results of monitoring n sequential reactions as an MSn+' experiment where the ion accelerating region serves as an analyzer. Practical considerations and design constraints typically limit investigations to n sequential reactions. Maquestiau (1)and Gross (2)have demonstrated the ability of acquiring MS3 data for sequential reaction sequences on three-sector instruments. In what is probably the first report of the use of tandem mass spectrometry to confirm the sequence of an unknown peptide (the cyclic peptide HCToxin), MS3 was employed to verify the key reaction sequence ( 3 ) . MS3 experiments have also been reported for BEQ and multiple quadrupole instruments (4-6). Multiple reaction sequences have also been investigated by using FT instruments (7). For example, Nibbering has obtained an MS4 spectrum from acetophenone (8),and Jacobson and Freiser have followed a nine-step (MS'O) reaction sequence (9). With the advent of four-sector instruments ( I O ) , multiple reaction sequence monitoring with n = 4 (i.e. MS4 and MS5) should be feasible. We have investigated and demonstrate here the MS4 and MS5 capabilities of a four-sector instrument of BEEB configuration in monitoring consecutive reactions. Applications to ion chemistry and peptide structural elucidation are shown.
EXPERIMENTAL SECTION Materials. Ethyl benzoate, 4-heptanone, acetone, n-butylbenzene, 2-pentanone, butyrophenone, acetophenone, (2bromoethyl)benzene, and toluene were purchased from Aldrich Chemical Co. (Milwaukee, WI). 2-Bromoethyl benzoate was purchased from American Tokyo Kasei, Inc. (Portland, OR). Phe-Gly-Leu-Met-NH, was purchased from Peninsula Laboratories (Belmont, CA). Leu-Gly-Gly,Gly-Gly-Leu, and Gly-Gly-ne
This article not subject to US. Copyright. Published 1988 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 20, OCTOBER
15, 1988 2233
Table I. Consecutive Reactions Studied reaction region precursor butyrophenone
ethyl benzoate acetophenone toluene butylbenzene (2-bromoethyl)benzene 4-heptanone
ion examined 148 148 148 148 148 120 105 150 120
Leu-Gly-Gly + H Gly-Gly-Ile + H Gly-Gly-Leu + H
4
120 120
92 134 92 91 105 91 58 58 58 466 466 466 246 246 246
105 105
77
+
105 105
---*
122
4
92
105 105 910 91 91 91 91
+
4
+ 4 4
86
318 171 132 132
4
fragments
fragments fragments fragments fragments fragments fragments fragments fragments fragments fragments fragments fragments fragments fragments fragments fragments fragments fragments fragments fragments fragments fragments fragments fragments fragments fragments
120
91 91
114
2-pentanone acetone Phe-Gly-Leu-Met-NH2+ H
4
eq no.
MS"
1
58
318 205 86 86
86
5
1 2 3
4 3 2
4 5 6
3 3 2 4
7
8 9 10
3 2 2
11 12
3 4 3 2
13
3
16 17 18
14 15
2 4 2 2 2 2 3 4 4
19
20 21 22
23 24
25 26
4 4
27
O 4 kV.
were purchased from Sigma Chemical Co. (St. Louis, MO). Mass Spectrometry. All experiments were performed on a VG ZAB-4F, which has been previously described ( I I , 1 2 ) . This instrument has been designed such that each sector can be set and/or scanned independently of the others. In a typical set of experiments (Scheme I), the MS/MS spectrum of the parent ion Scheme I ml+ m2+ m3+ m4+ m5+
- - - -
species, ml+, under unimolecular or collisional activation conditions, is obtained by scanning MS-I1 (a linked scan of E2B2). The first daughter ion, m2+,of the reaction sequence to be studied is identified, and its fragmentations are determined in an MS3 experiment. In the MS3 experiment, ml+ is focused through B1 and collisionally activated in the second field-free region. E l is set such that the daughter ion, m2+,is focused into the third field-free region. m2+ is then collisionally activated, and the spectrum of ita daughter ions is obtained by an E2B2 linked scan. The appropiate granddaughter ion, m3+,is determined from the MS3spectrum. In theory, the MS4experiment can be set up either by starting with the first transition and working toward the last transition or by focusing the granddaughter ion, ma+,first. In practice, the latter approach is easier and is described here. The instrument is set to transmit the parent ion through all four sectors to the final detector. Collision gas is introduced into the third field-free region so that the beam is reduced by about 50%. E2 and B2 are set in a linked mode to transmit the m3+ion. (For MS5 experiments, E2 and B2 are set to transmit m4+. Then the potentials applied to E2 are adjusted to transmit (m4/mJEo where Eo is the kinetic energy of the main beam.) E l is then set to transmit (m3lml)Eo. Collision gas is admitted into the second field-free region so that the intensity of m3+at the final detector is optimized. B1 is then tuned to transmit m$/ml. Collision gas pressures are then reoptimized. The MS4 spectrum is then acquired by scanning E2B2 in a linked mode. For the acquisition of MS6 spectra, only B2 is scanned. Due to the absence of a collision cell between E2 and B2, only unimolecular fragmentations are observed in the MS5 experiment. The reduction in intensity on going from ml+ being focused to m4+ being focused (Le. from an MS experiment to an MS5 experiment) is approximately 5 orders of magnitude. The major loss in signal intensity is typically observed in the first step. To
Scheme I1
M I 2 148
M I 2 120
M I Z 105
M I Z 77
M I Z 51
enhance sensitivity, MS-I1 was typically operated at low resolution (R= 250). Scan rates of 30-300 s/decade were used and multiple scans were acquired and signal averaged. Total data acquisition times varied from 10 min to 3 h (for MS5) and were dependent on the abundance of the ion whose fragmentation was monitored. For example, an MS4spectrum of butyrophenone or 4-heptanone could be acquired within 0.5 h while an MS4spectnun from a small peptide required from 1.5 to 3 h (including sample loading as well as data acquisition). Mass calibration used computerized mass assignments based on a linear scan function of B2. These assignments were verified by comparison with the MS/MS spectra of standards and by manual mass marking when necessary.
RESULTS AND DISCUSSION To characterize the capabilities of the instrument, several sets of reaction sequences were examined (Table I). Nibbering had previously studied the acetophenone benzoyl cation -,phenyl cation C4H3+sequence (8)in demonstrating MS4 capabilities of an FT instrument. Since the ZAB-4F is theoretically capable of doing MS5 experiments, we chose to investigate the butyrophenone sequence, as this sequence is similar to that of acetophenone and also contains an additional step in its fragmentation pathway (Scheme 11). C7H50+.The complete reaction series from MS to MS5for butyrophenone was obtained (Figure 1). These data successfully demonstrate that it is possible to employ each sector separately so as to realize the instrument's full potential for reaction sequence monitoring. In addition to butyrophenone, the C7H50+ion from several other origins was also investigated (Table I and Figure 2). When the C7H50+ion is formed as part of a reaction sequence, losses of 16 and 18 daltons are observed, while source-formed m / z 105 ions do not exhibit these losses (
