Anal. Chem. 1995,67,319-384
High-Mass Analysis Using Quadrupolar Excitation/ Ion Cooling in a Fourier Transform Mass Spectrometer Salvador J. Pastor, John A. Castoro, and Charles L. Wilkins* Department of Chemistry, University of California, Riverside, California 92521
Quadrupolar axialization has been applied to matrixassisted laser desorptiodionization Fourier transform mass spectrometric analysis of singly charged polymer and protein ions. When a quadrupolar excitation is applied in the presence of argon collision gas, ions are driven to the center of the cell, where they can traverse the conductance limit and be detected in the lower pressure analyzer cell of a dual cell instrument. It is shown that single frequency quadrupolar excitation permits highly selective detection of single oligomers of poly(ethy1ene glycol) 6000. Resolution enhancement is demonstrated for bovine insulin and ubiquitin, where spectra with resolving powers of 92 000 and 25 000, respectively, are obtained. Finally, quadrupolar axialization is demonstrated to be effective for molecules with masses as large as 13 000 Da. Adaptation of matrix-assisted laser desorption/ionization (.MAL DI) to Fourier transform mass spectrometry (FTMS) is now well established. By using Fourier transform mass spectrometry in conjunction with MALDI, employing a gated trapping deceleration protocol,' high-resolution MALDI-FTMS spectra far superior to those available by MALDI-TOF for molecules with masses 10 000 is currently being investigated. One of the challenges in high-mass work is efficient trapping of ions generated by the MALDI process. Because desorbed ions have similar average velocities, their kinetic energies scale directly with increasing mass, making high-mass trapping somewhat more
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Figure 4. Selective isolation of the [M(n = 136) from PEG 6000; resolving power, 20 000.
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higher resolution measurements of individual oligomers, ion isolation becomes necessary. A number of ion isolation methods have been demonstrated, including selective sweep ejection and, more recently, the stored wave form inverse Fourier transform (SWIFT) t e c h n i q ~ e . ' ~However, -~~ these methods do not result in repositioning ions within the center of the cell along the z-axis and generally result in some loss in ion signal intensity. Quadrupolar excitation with collisional cooling shows enhanced S/N for the ions of interest. The ability to effectively isolate one or more oligomers from the PEG 6000 distribution allows experimental conditions to be further optimized. When quadrupolar axialization was used to center the selected oligomer ions in the source cell, the ions could be selectivelytransferred to the lower
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Flgure 5. Ultrahigh-resolutionUV-MALDI-FTMS mass spectrum of bovine insulin obtained using a 1:4000:2000analyte/DHB/o-fructose molar ratio and quadrupolar axialization. Inset shows expanded scale for molecular ion, [M 382 Analytical Chemistry, Vol. 67, No. 2, January 15, 7995
+ HI+; average resolving power, 92 000.
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Figure 7. Effects of quadrupolar axialization upon transfer of MALDI-derived cytochrome c ions to the analyzer cell (a) in the presence of 5 x 10-6 Torr argon, no quadrupolar axialization, and Torr argon, with quadrupolar (b) in the presence of 5 x axialization. 8530.00
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Figure 6. High-resolution UV-MALDI-FTMS mass spectrum of ubiquitin obtained using a 1:3000:3000analyte/DHB/D-fructose molar ratio and analyzer cell detection following axialization; resolving power, 25 000.
challenging.22Another consequence of laser desorption is that ions are not ideally positioned within the cell, making transfer of direct laser-desorbedhigh-mass species to the analyzer cell nearly impossible.n However, because quadrupolar excitation combined with collisional cooling effectively eliminates magnetron motion and allows ions to relax to the center of the trap, it provides a means to make possible the desired t r a n ~ f e r . ~This ~ - ~ paper ~ presents the first demonstration of axialization of singly charged ions with masses greater than 3496 Da (the molecular ion from the b-chain of bovine insulin), which is the highest mass ion previously reported to have been axialized.27 Figure 7 illustrates the successfultransfer of cytochrome c to the analyzer cell. Figure 7a is the spectrum obtained when MALDI-FTMS is carried out in the presence of a collision gas in the source cell, followed by a delay to allow transfer to the analyzer. Clearly this is an inefficient process, and very little signal is seen. Figure 7b shows the spectrum obtained by tranferring the molecular ion species following quadrupolar excitation during the cooling period, resulting in much better transfer across the 2 mm conductance limit. The two peaks observed at masses greater than that of the molecular ion multiplet correspond to matrix adducts. In this case, (20) Chen, L.; Wang, T. L.; Ricca, T. L.; Marshall, A G. Anal. Chem. 1987,59, 449-454. (21) Ijames, C. F.; Wilkins, C. L]. Am. Chem. SOC.1988, 110,2687-2688. (22) Beavis, R C.; Chait, B. T. Chem. Phys. Lett. 1991, 181,479-484. (23) Honovich, J. P.; Markey, S. P. Int. J. Mass Spectrom. Ion Processes 1990, 101, 21 -34. (24) Guan,S.; Marshall, A G.J. Chem. Phys. 1993, 98,4486-4493. (25) &an, S.; Xiang, X;Marshall, A G. Int. J. Mass Spectrom. Ion Processes 1993, 124, 53-67. (26) Xiang, X.; Guan, S.; Marshall, A. G. J. Am. SOC.Mass Spectrom. 1994, 5, 238-249. (27) Huang, Y.; Pasa-Tolic, L;Kim, H. S.; Guan, S.; Marshall, A G. Proceedings of the 42nd ASMS Conference on Mass Spectrometry and Allied Topics, Chicago, IL, May 29-June 4, 1994; p 687.
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Figure 8. UV-MALDI-FTMS mass spectra for PEG 10000: (a) full distribution using source cell detection and a single laser shot and (b) selection of five oligomers using axialization.
resolution is sufficient to observe M - 17 fragment ions for each of the three main peaks. Unit mass resolution could be obtained, albeit with greatly reduced S/N. Figure 8 shows the mass spectrum obtained following the successful axialization of oligomers of PEG 10000 with masses in the range of 12 000-14 000 Da. The low-resolution spectrum at the top shows the full polymer distribution obtained using source cell detection following a single laser pulse. The bottom spectrum is that obtained using a single frequency axialization procedure to select five oligomers for subsequent analysis. Resolving power, in this case, was improved immediately and averaged 1100 for these five oligomer species. Analytical Chemistry, Vol. 67, No. 2, January 15, 1995
383
Clearly, axialization shows much potential for Fourier transform mass spectrometry. As is evident from the present results, it is generally useful for ion selection, relaxation, and storage. In the present study, it was found that the molecular ions of cytochrome c can be stored for several minutes in the analyzer cell before the excitation/detection events when axialization is applied. CONCLUSION The present results demonstrate that quadrupolar excitation with collisional cooling is practical for compounds with masses up to 13 000 Da. These observations suggest that the technique has promise for improving resolving power for even higher mass singly charged MALDI-derived species which, to date, have been detected only with poor resolution (generally with resolving power of 100 or less). Thus, high-resolution MALDI-FTMS spectra have been limited to compounds with masses not greater than that of cyctochrome c. For lower mass species, axialization shows great promise for facilitating ultrahigh resolution applicable to accurate
384 Analytical Chemistry, Vol. 67, No. 2, January 15, 1995
mass measurements and sequence studies. In addition, application of broadband quadrupolar excitation for relaxation of ions over broad mass ranges should prove invaluable for applications to product ions generated from collision-induced,surface-induced, or photodissociation experiments. ACKNOWLEDGMENT We would l i e to thank Dan Bui for his early work in the project, Jerry Sorrels for his help in building the switching circuit, and Dr. Michael Kahr for constructing the gate valve/leak valve assembly. This research was supported by the National Institutes of Health (GM-44606) and the National Science Foundation (CHE 92-01277). Received for review July 26, 1994. Accepted October 27, 1994.a AC940745L @Abstractpublished in Advance ACS Abstracts, December 1, 1994.
