Anal. Chem. 1998, 70, 213-217
Low-Voltage On-Resonance Ion Selection and Storage: An Alternative to Quadrupolar Axialization for FTMS Salvador J. Pastor and Charles L. Wilkins*
Department of Chemistry, University of California, Riverside, California 92521
A new alternative to quadrupolar axialization for ions trapped within a cubic Fourier transform mass spectrometer cell is presented. This method, low-voltage onresonance excitation, requires no electronic switching hardware, as is necessary for the quadrupolar axialization technique. However, it yields equivalent ion selection performance, both for single- and broad-band ion selection, as is demonstrated with spectra for a number of poly(ethylene glycol) samples, gramicidin S, and a bovine insulin b-chain sample.
Several years ago, Savard and co-workers1 introduced a new method for cooling ions in a Penning trap. Their paper demonstrated that it is possible to efficiently couple ion cyclotron, magnetron, and axial motions in the presence of a high background gas pressure, with the result that selected ions can be caused to relax to the center of the cell. Because both ion cyclotron and axial motions damp to the center of the cell, while relaxation of magnetron motion leads to increased orbital precession and eventual ejection of the ion, cyclotron and axial coupling with magnetron motion can eliminate ion loss and drive ions to the center of the cell as their kinetic energy is reduced. Subsequently, azimuthal quadrupolar excitation/ion cooling was adapted to Fourier transform ion cyclotron-resonance mass spectrometry (FT-ICR) by Marshall and co-workers2,3 and was quickly accepted as a technique for enhancing mass resolving power, sensitivity, and selectivity in FTMS. In fact, over the last few years, ion axialization has been demonstrated in many applications and was the subject of review4 just two years after its first demonstration for FTMS. Numerous publications have documented its theory5-8 and use in a variety of applications, including remeasurement,9,10 high resolution,11-13 sensitivity en(1) Savard, G.; Becker, S.; Bollen, G.; Kluge, H.-J.; Moore, R. B.; Otto, T.; Schweikhard, L.; Stolzenberg, H.; Wiess, U. Phys. Lett. A 1991, 158, 247252. (2) Schweikhard, L.; Guan, S.; Marshall, A. G. Int. J. Mass Spectrom. Ion Processes 1992, 120, 71-83. (3) Guan, S.; Wahl, M. C.; Wood, T. D.; Marshall, A. G. Anal. Chem. 1993, 65, 1753-1757. (4) Guan, S.; Kim, H. S.; Marshall, A. G.; Wahl, M. C.; Wood, T. D.; Xiang, X. Chem. Rev. 1994, 94, 2161-2182. (5) Xiang, X.; Guan, S.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1993, 5, 238-249. (6) Guan, S.; Xiang, X.; Marshall, A. G. Int. J. Mass Spectrom. Ion Processes 1993, 124, 53-67. (7) Guan, S.; Marshall, A. G. Rev. Sci. Instrum. 1995, 66, 63-66. S0003-2700(97)01126-8 CCC: $15.00 Published on Web 01/15/1998
© 1998 American Chemical Society
hancement,14 selectivity applications,15 selected-ion accumulation,16,17 and multiple MS.18,19 Ultimately, broad-band axialization was also shown to be feasible.20-23 Basically, any method that can couple the cyclotron/magnetron or axial/magnetron motions in the presence of a background pressure will produce the axialization effect. The original paper1 found that a quadrupolar rf field in the X-Y plane worked well. Since then, other combinations have been proposed. Guan et al.,6 in a theoretical study, proposed the use of a segmented cubic ion trap that could generate an rf excitation with X-Z symmetry. Here, magnetron motion would be periodically interconverted into axial motion, thus producing the axialization effect. Similarly, both Laude and co-workers24 and Ijames25 discovered that a quadrupolar field in the X-Y plane could be approximated by using two oppositely positioned electrodes with rf of the same phase applied. Here, an even simpler method that requires no cell modification at all is presented. Using the normal excitation plates of a cubic cell with rf applied on-resonance at millivolt levels, one can (8) Guan, S.; Huang, Y.; Marshall, A. G. J. Mass Spectrom. 1995, 30, 15931598. (9) Speir, J. P.; Gorman, G. S.; Pitsenberger, C. C.; Turner, C. A.; Wang, P. P.; Amster, I. J. Anal. Chem. 1993, 65, 1746-1752. (10) Hendrickson, C. L.; Laude, D. A. Anal. Chem. 1995, 67, 1717-1721. (11) Guan, S.; Marshall, A. G. Rapid Commun. Mass Spectrom. 1993, 7, 857860. (12) Guan, S.; Wahl, M. C.; Marshall, A. G. Anal. Chem. 1993, 65, 3637-3653. (13) Pasa-Tolic, L.; Huang, Y.; Guan, S.; Kim, H. S.; Marshall, A. G. J. Mass Spectrom. 1995, 30, 825-833. (14) Wahl, M. C.; Kim, H. S.; Wood, T. D.; Guan, S.; Marshall, A. G. Anal. Chem. 1993, 65, 3669-3676. (15) Wood, T. D.; Ross, C. W.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1994, 5, 900-907. (16) Bruce, J. E.; Anderson, G. A.; Hofstadler, S. A.; Van Orden, S. L.; Sherman, M.S.; Rockwood, A. L.; Smith, R. D. Rapid Commun. Mass Spectrom. 1993, 7, 914-919. (17) Bruce, J. E.; Van Orden, S. L.; Anderson, G. A.; Hofstadler, S. A.; Sherman, M.G.; Rockwood, A. L.; Smith, R. D. J. Mass Spectrom. 1995, 30, 124133. (18) Guan, S.; Marshall, A. G.; Wahl, M. C. Anal. Chem. 1994, 66, 1363-1367. (19) Huang, Y.; Pasa-Tolic, L.; Guan, S.; Marshall, A. G. Anal. Chem. 1994, 66, 4385-4389. (20) Guan, S.; Wahl, M. C.; Marshall, A. G. J. Chem. Phys. 1994, 100, 61376140. (21) Guan, S.; Marshall, A. G. J. Chem. Phys. 1993, 98, 4486-4493. (22) Marto, J. A.; Guan, S.; Marshall, A. G. Rapid Commun. Mass Spectrom. 1994, 8, 615-620. (23) O’Conner, P. B.; Speir, J. P.; Wood, T. D.; Chorush, R. A.; Guan, Z.; McLafferty, F. W. J. Mass Spectrom. 1996, 31, 555-559. (24) Hendrickson, C. L.; Drader, J. J.; Laude, D. A. J. Am. Soc. Mass Spectrom. 1995, 6, 448-452. (25) Ijames, C. F. Proceedings of 43rd ASMS Conference on Mass Spectrometry and Allied Topics, Atlanta, GA, 1995; p 796.
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produce similar mass selection under the same high-pressure conditions employed for quadrupolar axialization. As will be shown here, there are no perceptible differences in ion mass selectivity or trapping lifetimes. EXPERIMENTAL SECTION Instrumentation. All spectral measurements were obtained with an FTMS-2000 Fourier transform mass spectrometer (Finnigan FT/MS, Madison, WI) equipped with a 2-in. dual cubic cell and a 7-T superconducting magnet (Oxford Instruments Inc., Oxford, U.K.). Internal MALDI ion generation was achieved by focusing optics both in the chamber and outside the system as described previously for a 3-T system.26 Matrix-assisted laser desorption/ionization employed a nitrogen laser operating at 337 nm (PTI Canada Inc., London, ON, Canada). The entire chamber is pumped by two Edwards diffusion pumps (Diffstak model 100, Crawley, West Sussex, England) achieving a working pressure of 8 × 10-8 Torr during experiments. The sample probe was inserted manually and positioned exactly 4 mm from the source trapping plate. Data acquisition and experiment control were carried out with an FT/MS Odyssey system (Finnigan FT/MS). The SWIFT cell controller allowed precise control of selected wave forms to implement broad-band selection of ions. Data analysis was done with a Sparc IPX Station running Odyssey software version 3.1. Polymer spectra were Hamming apodized. To provide constant-pressure intervals for the experiment, a combination pulsed valve/leak valve was situated on the source flange of the chamber and was triggered by the pulsed valve triggers of the FT/MS cell controller. This procedure has been described in detail elsewhere.27 All experiments took place in the source cell. Sample Preparation. Poly(ethylene glycol) 1000, gramicidin S, and insulin chain B (oxidized) were obtained from Sigma Chemical Co. (St. Louis, MO). Poly(ethylene glycol) 3000 and 6000 were obtained from Fluka Chemical Co. (Buchs, Switzerland). The samples were prepared in solutions of 5 mg/mL with methanol except for insulin B-chain, which was prepared at 3.7 mg/mL. 2,5-Dihydroxybenzoic acid (DHB, Fluka) was used for all MALDI polymer analyses and mixed in a 1:300 (analyte/matrix) ratio. For the gramicidin S and insulin B-chain experiments the analyte/matrix ratio was 1:200 and 1:750, respectively. Samples were sprayed onto the probe tip using a spray apparatus as described previously.31 The aerospray produces fine crystal surfaces. Approximately 200 µL of sample solution were deposited on the probe tip. No cations were added to enhance ionization. The spectra in Figures 1-5 resulted from samples prepared in this way. Experiment Control. Polymer Analysis. For polymer analysis, two different experimental scripts were used for ion selection. By definition, a script is the sum of all the sequences. Normally, several events (time slices) make up a single sequence, and should (26) (27) (28) (29)
Pastor, S. J.; Wilkins, C. L. J. Am. Soc. Mass Spectrom. 1997, 8, 225-233. Pastor, S. J.; Castoro, J. A.; Wilkins, C. L. Anal. Chem. 1995, 67, 379-384. Castoro, J. A.; Wilkins, C. L. Anal. Chem. 1993, 65, 2621-2627. Knobeler, M.; Wanczek, K. P. Int. J. Mass Spectrom. Ion Processes 1997, 163, 47-68. (30) Marshall, A. G.; Wang, T. L.; Ricca, T. L. J. Am. Chem. Soc. 1985, 107, 7893-7897. (31) Yao, J.; Dey, M.; Pastor, S. J.; Wilkins, C. L. Anal. Chem. 1995, 67, 36383642.
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one want to signal average, the sequence is looped. For singlefrequency selection experiments, ions were trapped using a gated trapping procedure28,29 and allowed to cool for 100 ms. To select a single isotopic cluster, the cyclotron frequency of the ion was applied at 0.5 Vp-p or less (optimized for each sample) in the presence of 5 × 10-5 Torr argon for 30-50 s. This combination of pressure and excitation time allows for high selectivity from the other m/z species, which are lost through high-pressure collisions that relax magnetron motion and remove them from the cell. Following selection, a 3-s delay is imposed to remove background pressure. For detection, a “chirp” excitation from 50 to 500 kHz at a sweep rate of 400 Hz/µs was applied. Detection was at a bandwidth of 1 MHz and used 32K data points. The trapping voltage was 1.0 V during the entire experiment. The script described is adequate for most purposes involving the selection of a single m/z or a narrow range of ions. However, for more complex ion selection, it becomes necessary to employ a broad-band selection approach. This parallels broad-band quadrupolar axialization20,22,23 in every way except that, for the present method, switching hardware is not required to change from a dipolar to a quadrupolar cell plate configuration. The hardware usually consists of a switching box made up of relays9,10,24,27 that re-direct excitation signals to (1) two opposite plates of the cell to form the normal dipolar configuration with out-of-phase signals producing a near-linear excitation or (2) two pairs of opposite plates of the cell to form a quadrupolar field with opposite plates sharing the same phase. A TTL pulse usually triggers the circuit experimentally. Alternatively, quadrupolar axialization has been applied in a hardwire configuration when a dual cell is available. In this hardware scenario, ions must be transferred to the analyzer cell for analysis because the source cell is no longer able to perform the normal differential detection.18,19 Because low-voltage on-resonance ion selection does not require a quadrupolar field, the experiment is greatly simplified. In the broad-band selection approach, a SWIFT30 wave form is generated with the excitation characteristics desired for the selection. The experiment then consists of three consecutive sequences. The first sequence is executed only once and is used for ionizing and trapping the ions. Because of the short time duration of the applied tailored SWIFT excitation (less than ∼4 ms), sequence 2 must be looped in conjunction with a time delay. For example, sequence 2 might consist of a 3-ms SWIFT slice and a 3-ms delay time slice, looped 5000 times to generate the requisite 30 s of ion selection. The final sequence allows for a 3-s delay in which the pulse gas is turned off, followed by the standard excitation and detection events. Thus, the experiment would consist of three sequences in the time ratio of 1:5000:1. Gramicidin S Analysis. The [M + Na]+ peak for gramicidin S was selected using a 0.4-Vp-p single-frequency dipolar excitation signal for 40 s at the cyclotron frequency of this ion, 92 980 Hz. Following a 3.0-s delay, a 1.0-s pulse was applied at 7.4 Vp-p and at a frequency that was 1000 Hz higher to initiate fragmentation using SORI-CID. Insulin B-Chain Analysis. The [M + H]+ peak for insulin B-chain was selected using a 0.2-Vp-p single-frequency dipolar excitation signal for up to 30 s at its cyclotron frequency of 30 960 Hz. Following the usual 3.0-s delay to allow for a return to baseline pressure, excitation was done from 50 Hz to 200 kHz at a sweep
rate of 350 Hz/µs. Detection was done at a bandwidth of 400 kHz and used 32K data points. RESULTS AND DISCUSSION During a study of SORI-CID of polymers,32 it was observed that low-voltage on-resonance excitation under high-pressure conditions could result in ion selection. This was seen in experiments in which, under high-pressure conditions, excitation was applied slightly off-resonance for m/z ions of interest for 1.0 s, with the excitation strength being varied systematically to determine combinations that produced the onset of fragmentation. It was found that SORI voltages of less than 0.5 Vp-p did little to promote fragmentation. However, as the mass range of the polymer increased and the cyclotron frequency spacing decreased, oligomer ions with frequencies nearest to the applied excitation began to show enhancement in their overall abundances. This surprising observation was unexpected because high background pressures normally promote ion loss through magnetron expansion, particularly in a cubic cell. There appeared to be an axialization process occurring, similar to that described in the numerous previous publications on quadrupolar excitation/ion cooling. Like quadrupolar excitation, it is found that, for optimum ion selection, the applied excitation is a low-voltage rf at the ion’s unperturbed cyclotron frequency. Comparison of Low Voltage On-Resonance and Quadrupolar Selection. Following discovery of the new ion selection procedure, a comparison was done between low-voltage onresonance and quadrupolar axialization. As illustrated in the accompanying figures, identical results could be obtained with both methods. For single-frequency excitation, ion selectivity in both selection methods is controlled by attenuation of the singlefrequency excitation amplitude. At excitation amplitudes of 0.4 Vp-p, complete isolation of an oligomer of PEG 3000, which served as the test polymer, is possible within 30 s at a pressure of 5 × 10-5 Torr argon, a time equal to that reported by Wood et al.15 using axialization. When the selected frequency is increased in amplitude to 3.7 Vp-p, there is selection of up to five oligomers of PEG 3000 for both methods. Further increase in the applied voltage causes ejection of ions from the cell. The control of the selection “width” by altering the applied voltage for quadrupolar excitation was demonstrated previously.15,27 The fundamental question remaining is whether low-voltage on-resonance selection, as reported here, is a form of ion axialization in which the magnetron motion is being interconverted to cyclotron motion faster than the magnetron motion is being damped. In other words, are ions being centered within the analysis cell as a result of their being selected? A simple experiment is to test the ion residence time in the cell following a 30-s selection event in which a single oligomer of PEG 3000 is isolated and allowed to remain trapped prior to observation, following reduction of background pressure to normal low levels. Again, for both quadrupolar excitation and the new selection method, ion abundances were high, following a 3-s delay to allow the trapping cell to return to baseline pressure of 8 × 10-8 Torr. For both techniques the ion abundance quickly decreased after 15 s, and all signal was gone by 30 s. Thus, if the quadrupolar method does result in “axialization” of selected ions, as seems to (32) Pastor, S. J.; Wilkins, C. L. Int. J. Mass Spectrom. Ion Processes, in press.
Figure 1. Spectra of poly(ethylene glycol) 3000 sample. Each spectrum results from a single laser shot except for (b) which is the sum of four scans: (a) full distribution; (b) isolation of [M(n)71) + Na]+ oligomer ion; (c) broad-band selection of five oligomer ions; (d) selection of a single type of oligomer and a set of other oligomer ions cover a larger mass range.
be the case, then the low-voltage ion selection procedure described here might be doing the same thing. Poly(ethylene glycol) 3000. PEG 3000 was the first sample for which the enhancement effect produced under a high background pressure and a low-voltage applied frequency was observed. Figure 1 shows a series of spectra with different ion isolation strategies. Figure 1a is spectrum obtained from MALDI FTMS of this particular PEG 3000 sample, showing the full sodium-attached oligomer ion distribution. Upon application of low-voltage on-resonance selection, a single oligomer ion, [M(n)71) + Na]+, is selectable from the rest of the distribution (Figure 1b). By generating a SWIFT wave form that selects specific peaks, one can choose which m/z ions are being stored, as shown in Figure 1c. Figure 1d demonstrates the versatility of the method by illustrating selection of a single oligomer ion, in addition to another range of ions, for retention. Thus, as with broad-band axialization, any combination of ions within a mass spectrum is selectable for further study. The main difference between the present method and other selection techniques is that most others specifically eject unwanted ions. Here, one stores the selected ion while all others are lost (presumably by magnetron relaxation during the high-pressure phase of the experiment). It has been suggested that axialization is especially efficient for high-mass ions.3 Indeed, successful axialization has been demonstrated for singly-charged masses up to 13 000 Da.27 For Analytical Chemistry, Vol. 70, No. 2, January 15, 1998
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Figure 2. High-resolution spectra of low-mass PEG 1000 sample. Each spectrum results from a single laser shot: (a) full spectrum of sodium-attached species; (b) isolation of [M(n)28) + Na]+ oligomer ion; (c) broad-band selection of three oligomer ions; (d) broad-band selection of seven oligomer ions.
the experiments presented here, low-voltage on-resonance has been confirmed for selection of ions with masses up to 6000 Da from a PEG 6000 sample (vide infra). Poly(ethylene glycol) 1000. The ability to obtain low-mass selection spectra, while retaining high resolving power, was examined using a low molecular weight PEG. Figure 2 shows spectra that retain their nominal 5-6K resolving power, despite the variety of selections employed. Figure 2a shows the MALDIFTMS spectrum of a PEG 1000 sample, with no ion selection applied. Figure 2b is the spectrum obtained after selection of the relatively low abundance [M(n)28) + Na]+ oligomer. Figure 2c shows an interesting spectrum containing ions from three oligomers that were subjected to low-voltage broad-band SWIFT excitation to select them. The ions detected represent the selection of ions representative of every fifth oligomer. Finally, Figure 2d is the spectrum obtained after broad-band selection of ions from seven different oligomers (n ) 28, 30, 32, 34, 36, 38, 40) where every other alternate oligomer is selected. Clearly, there is much versatility, as the ability to select is strictly governed by the generated SWIFT wave form. Poly(ethylene glycol) 6000. As a graphic illustration of the parallelism between low-voltage on-resonance selection and quadrupolar axialization, Figure 3 presents spectral results from a series of experiments where only the method of selection was changed, while the electronics necessary for quadrupolar axialization remained in-line with the cell, but were either activated or not, depending on which method was being used. Figure 3a shows the full MALDI mass spectrum of the PEG 6000 sample, with its sequence of sodium-attached oligomer ions. Panels b and 216 Analytical Chemistry, Vol. 70, No. 2, January 15, 1998
Figure 3. MALDI FTMS spectra of PEG 6000 sample. Each spectrum results from four laser shots: (a) full spectrum of sodiumattached species; (b) low-voltage on-resonance selection of a single oligomer ion species; (c) low-voltage on-resonance selection of two ranges of oligomer ion species; (d) quadrupolar axialization selection of a single oligomer ion species; (e) quadrupolar axialization selection of two ranges of oligomer ion species.
c are the spectra obtained with two low-voltage on-resonance selection experiments, and panels d and e are the corresponding quadrupolar axialization results. Clearly, the two techniques can produce equivalent results for ion selection. A small difference is the applied excitation voltage where the two-plate on-resonance selection requires a slightly higher (g2× axialization) applied voltage. Some slight difference in experimental conditions can be expected, changing the applied voltage or altering the selection time to compensate for the differing number of plates used. Gramicidin S. Application of low-voltage on-resonance selection to biomolecules was tested with the cyclic peptide gramicidin S. A normal MALDI experiment produces the spectrum in Figure 4a. Because gramicidin S is cyclic, requiring the breaking of two bonds to produce lower mass product ions, spectra are usually free of large abundances of fragment ions. Low-voltage onresonance excitation at 0.4 Vp-p was used to select the [M + Na]+ peak at high pressure for 40 s (Figure 4b). Upon applying a sustained off-resonant excitation (1000 Hz higher) at a higher voltage of 7.4 Vp-p, SORI-CID produced the spectrum shown in Figure 4c. In common with all experiments that probe for structural information, this experiment shows the benefit of using a trapped ion cell. Because FTMS experiments can grow quite complex in terms of hardware and software, the ability to select ions without the need for a set of modified hardware is advantageous. In order
Figure 5. Fourier transform mass spectra for bovine insulin B-chain. Each spectrum results from a single laser shot: (a) MALDI spectrum; (b) MALDI spectrum with added 100-ms delay prior to excitation/ detection; (c) on-resonance selection for 10 s; (d) on-resonance selection for 20 s; (e) on-resonance selection for 30 s. Figure 4. Fourier transform mass spectra for gramicidin S. Each spectrum results from a single laser shot: (a) MALDI spectrum; (b) isolation of [M + Na]+ peak; (c) SORI-CID mass spectrum showing fragmentation of parent ion.
to obtain the gramicidin S spectra in Figure 4, the only additional hardware requirement is a pulsed valve system that can maintain a constant pressure over a reasonable length of time. Insulin B-Chain. It should be noted that low-voltage onresonance ion selection appears to work equally well for all samples tested to date. Of particular interest is the selection of fragile peptides and proteins that exhibit instability in the gas phase following MALD ionization. Metastable decay can quickly reduce the amount of material trapped within the FTMS analysis cell. By using cooling techniques such as quadrupolar excitation/ ion cooling (axialization) and on-resonance ion selection, the amount of material lost can be minimized. Figure 5 demonstrates the superior results capable with the on-resonance selection method. Figure 5a is indicative of a MALDI-generated mass spectrum showing the absence of any time delays following the ionization event and immediately performing the excitation/detection events. Figure 5b shows the application of a short 100-ms delay following the MALDI event demonstrating the reduction of molecular ion signal intensity and the gain in fragmentation at lower m/z values. Panels c-e of Figure 5 show the enhancement in the molecular ion signal by application of on-resonance ion selection for increasing selection times of 10, 20, and 30 s. Not only are the fragment ions removed from the cell at high background pressure but the parent ions are sufficiently relaxed to promote an increased molecular ion
intensity and a better ion coherence during ion excitation/ detection. CONCLUSION A new ion selection method, low-voltage on-resonance excitation, which produces results equivalent to the popular quadrupolar axialization technique, is presented. The spectra presented here do not indicate severe limitations for the new selection technique, which appears to work equally well for both high- and low-mass applications. Based upon direct comparisons between this method and the quadrupolar axialization technique, there seems to be no advantage to using the hardware switching required by the latter in terms of ion selection. Quadrupolar axialization, however, is very well understood and will remain the model technique. Future work will address the already successful application of onresonance selection to ion remeasurement and MSn applications. It will also be interesting to see to what extent on-resonance ion selection can cool ions and if there is comparable performance between the two techniques. In view of the simplicity of lowvoltage on-resonance excitation, as demonstrated here, it may become the method of choice for single-, multiple-, or broad-band ion selection MS applications. ACKNOWLEDGMENT This work was partially supported by NSF Grant CHE-92 0 1277. Received for review November 19, 1997.X
October
10,
1997.
Accepted
AC971126Q X
Abstract published in Advance ACS Abstracts, December 15, 1997.
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