Anal. Chem. 1999, 71, 2945-2950
Narrow-Band Collisional Activation Technique for Ion Trap Mass Spectrometers Andrew G. Baker, Andrew Alexander, and Milos V. Novotny*
Department of Chemistry, Indiana University, Bloomington, Indiana 47405
Narrow-bandwidth signals were applied to the end caps of an ion trap mass spectrometer to excite ions during collisional activation. Excitation waveforms were created from a single-frequency component and a random noise component using a multiplier circuit. Tandem and higher order mass spectrometry experiments (MS3) can be performed without optimization of the supplemental frequency applied to the end cap electrodes. The usefulness of this method of ion excitation is demonstrated using singly and multiply protonated peptide ions as well as sodium-cationized carbohydrates. Tandem mass spectrometry using ion trap instruments is becoming increasingly popular for peptide and carbohydrate sequencing.1-5 The ion traps are now easily coupled to electrospray ionization or matrix-assisted laser desorption/ionization, two favored methods for creating gas-phase ions from high-mass, nonvolatile molecular species.6,7 Ion trap instruments offer the advantages of performing tandem-in-time experiments, namely, the efficient conversion of parent ions to fragment ions and the possibilities of MSn (n > 3).8,9 The addition of resonant ejection and slow scan rates have extended the mass range and maximum resolution obtainable during routine analyses.10,11 The methodologies for ion isolation as a precursor to ion activation have been well-developed.1,9,12 Ion activation is most usually accomplished by applying a low-amplitude supplemental * Corresponding author: (e-mail)
[email protected]; (phone) (812)-8554532; (fax) (812)-855-8300. (1) Cox, K. A.; Williams, J. D.; Cooks, R. G.; Kaiser, R. E. Biol. Mass Spectrom. 1992, 21, 226-241. (2) Korner, R.; Wilm, M.; Morand, K.; Schubert, M.; Mann, M. J. Am. Soc. Mass Spectrom. 1996, 7, 150-156. (3) Figeys, D.; Ning, Y. B.; Aebersold, R. Anal. Chem. 1997, 69, 3153-3160. (4) Bahr, U.; Pfenninger, A.; Karas, M.; Stahl, B. Anal. Chem. 1997, 69, 45304535. (5) Weiskopf, A. S.; Vouros, P.; Harvey, D. J. Rapid Commun. Mass Spectrom. 1997, 11, 1493-1504. (6) Van Berkel, G. J.; Glish, G. L.; McLuckey, S. A. Anal. Chem. 1990, 62, 1284-1295. (7) Doroshenko, V. M.; Cotter, R. J., Rapid Commun. Mass Spectrom. 1993, 7, 822-827. (8) Johnson, J. V.; Yost, R. A.; Kelley, P. E.; Bradford, D. C. Anal. Chem. 1990, 62, 2162-2172. (9) Louris, J. N.; Brodbelt-Lustig, J. S.; Cooks, R. G.; Glish, G. L.; Van Berkel, G. J.; McLuckey, S. A. Int. J. Mass Spectrom. Ion Processes 1990, 96, 117137. (10) Kaiser, R. E.; Cooks, R. G.; Stafford, G. C.; Syka, J. E. P.; Hemberger, P. H. Int. J. Mass Spectrom. Ion Processes 1991, 106, 79-115. (11) Schwartz, J. C.; Syka, J. E. P.; Jardine, I. J. Am. Soc. Mass Spectrom. 1991, 2, 198-204. (12) McLuckey, S. A.; Goeringer, D. E.; Glish, G. L. J. Am. Soc. Mass Spectrom. 1991, 2, 11-21. 10.1021/ac980930p CCC: $18.00 Published on Web 05/27/1999
© 1999 American Chemical Society
frequency across the end cap electrodes of the ion trap.13 Ions with a particular Mathieu stability parameter (qz) will come into resonance with the frequency applied to the end caps, resulting in large-amplitude ion motions in the z direction.14 As a result of this motion, analyte ions are activated by collisions with the buffer gas (present at ∼1 mTorr) and subsequently decomposed. Fragmentation efficiency is a function of qz, while collisional activation is usually performed in the range of qz ) 0.2-0.4. This range is a tradeoff between the fragmentation efficiency and the low-mass exclusion limit for smaller fragment ions.13,14 Several other approaches to ion activation have been documented. McLuckey et al.15 and Van Berkel and Goeringer16 demonstrated that broad-band activation using random noise is possible, either in conjunction with or without elevating the qz parameter of the ion. Other research groups17-19 have used broadband SWIFT pulses for ion isolation or excitation. Wang et al. described another method of nonresonant ion activation where a low-frequency, high-amplitude square wave is applied to the end cap electrodes, resulting in increased ion kinetic energy that can be internalized during CID.20 Yet another method for generating broad-band noise sources, the filtered noise field (FNF), has also been utilized to selectively isolate or excite ions of defined m/z ratios.21 Other approaches utilizing narrow-bandwidth waveforms for collisional activation have also been reported. Yates et al. applied a series of signals over a range of frequencies bracketing the ion secular frequency.22 These signals are generated by the on-board frequency synthesizer under software control. Alternatively, a narrow-band SWIFT pulse encompassing these frequencies was reported.23 A multifrequency approach using a series of signals centered around the ion secular frequency was also described.24 (13) Louris, J. N.; Cooks, R. G.; Syka, J. E. P.; Kelley, P. E.; Stafford, G. C.; Todd, J. F. J. Anal. Chem. 1987, 59, 1677-1685. (14) Johnson, J. V.; Basic, C.; Pedder, R. E.; Kleintop, B. L.; Yost, R. A. In Practical Aspects of Ion Trap Mass Spectrometry; March, R. E., Todd, J. F. J., Eds.; CRC Press: Boca Raton, 1995; Vol. III, p 257. (15) McLuckey, S. A.; Goeringer, D. E.; Glish, G. L. Anal. Chem. 1992, 64, 1455-1460. (16) Van Berkel G. J.; Goeringer, D. E. Anal. Chim. Acta 1993, 277, 41-54. (17) Julian, R. G.; Cooks, R. G. Anal. Chem. 1993, 65, 1827-1833. (18) Doroshenko, V. M.; Cotter, R. J. Rapid Commun. Mass Spectrom. 1996, 10, 65-73. (19) Asam, M. R.; Ray, K. L.; Glish, G. L. Anal. Chem. 1998, 70, 1831-1837. (20) Wang, M.; Schachterle, S.; Wells, G. J. Am. Soc. Mass Spectrom. 1996, 7, 668-676. (21) Goeringer, D. E.; Asano, K. G.; McLuckey, S. A.; Hoekman, D.; Stiller, S. W. Anal. Chem. 1994, 66, 313-318. (22) Yates, N. A.; Yost, R. A.; Bradshaw, S. C.; Tucker, D. B. Proceedings of the 39th ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, 1991; p 132.
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Figure 1. Schematic of noise-modulation circuit used for narrow-band CA.
The purpose of this report is to describe a novel method for applying narrow-bandwidth waveforms to the end cap electrodes of an ion trap to induce collisional activation. The use of narrowbandwidth signals centered near the resonant frequency for activation reduces the tuning requirements necessary for collisional activation, while not exciting the product ions during tandem mass spectrometry experiments. EXPERIMENTAL SECTION A modified ion trap described by Van Berkel et al. was used for all experiments.6 Peptide ions from bradykinin and angiotensin I (2.0 × 10-4 M concentration in 50:50:1 water/acetonitrile/acetic acid) and sodium-cationized oligosaccharides were generated using a nanospray emitter as described by Ji et al.25 Maltotetraose and stachyose were dissolved at 5.0 × 10-4 M in 50:50 water/ acetonitrile, with aqueous sodium acetate added to form 1 × 10-3 M Na+ solutions prior to analysis. Both the peptides and oligosaccharides were obtained from Sigma (St. Louis, MO). Software control for the modified ion trap was accomplished using the ICMS software package developed by Yates and Yost.26 Parent ion isolation for the tandem mass spectrometry experiments was accomplished using either single-ramp resonant ejection12 or reverse-then-forward scan1 techniques. Collisional activation (CA) of ions during the MS2 or MSn experiments was performed at qz ) 0.2. During single-frequency CA, the frequency and amplitude of the excitation waveform applied to the end cap electrodes were iteratively optimized for the minimum amplitude. Narrow-band CA was controlled from the ICMS software by a TTL signal applied during the resonant excitation step(s) of the (23) Yates, N. A.; Griffin, T. P.; Yost, R. A.; Borum, P. R. Proceedings of the 41th ASMS Conference on Mass Spectrometry and Allied Topics, San Francisco, 1993; p 444a. (24) Plomley, J. B.; March, R. E.; Mercer, R. S. Anal. Chem. 1996, 68, 23452352. (25) Ji, Q.; Tomlinson, A. J.; Johnson, K. J.; Kieper, W. C.; Jameson, S. C.; Naylor, S. Proceedings of the 45th ASMS Conference on Mass Spectrometry and Allied Topics, Palm Springs, 1997; p 1289. (26) Yates, N. A.; Yost, R. A. ICMS Software; University of Florida, 1992.
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experiment. A narrow-band excitation waveform was created by multiplying the output of the ITMS frequency synthesizer with the output signal from a random-noise generator. A low-frequency noise signal (5 Vpp ) from a Wavetek model 132 noise source (San Diego, CA) was multiplied with a 78 800-Hz sine wave generated by the ITMS frequency synthesizer as described in Figure 1 (full details of the circuit are available on request). The resulting excitation waveform had a bandwidth of ∼400 Hz, centered on the frequency generated by the ITMS frequency synthesizer. This waveform is then amplified by the ITMS power amplifier on the frequency synthesizer board and then passed through the ITMS “Balun Box” before being applied to the end cap electrodes. The circuit described in Figure 1 also includes an analog switch after the multiplier. This switch allows either the narrow-band excitation signal or the output signal from the frequency synthesizer to be applied to the end cap electrodes during collisional activation. During the ion isolation and resonant ejection segments of the scan function, the unmodulated signal from the frequency synthesizer is applied to the end caps. RESULTS AND DISCUSSION Peptides. Tandem mass spectra obtained from the (M + H)+ and (M + 2H)2+ charge states of angiotensin I are shown in Figure 2. The spectrum in the top panel was obtained after singlefrequency CA, while the bottom spectrum was obtained after narrow-bandwidth CA. As expected, the singly charged precursor ions were much more difficult to fragment, resulting in few fragment ions.27 Fewer fragmentation channels were accessed during the narrow-band CA, although the parent ion was attenuated more than in the single-frequency CA. Tandem spectra from the (M + 2H)2+ charge state were more informative, yielding a large number of structurally diagnostic ions. The two spectra have a comparable number of fragment ions, while the ion intensities are slightly different. The parent ion ((M + 2H)2+) in the narrow(27) Cox, K. A.; Gaskell, S. J.; Morris, M.; and Whiting, A. J. Am. Soc. Mass Spectrom. 1996, 7, 522-531.
Figure 2. Ion trap MS/MS spectra obtained for (M + H)+ (a) and (M + 2H)2+ (b) charge states of angiotensin I (DRVYIHPFHL). Fragment ions are labeled using the system of nomenclature described in ref 38. The spectra in the top panels were obtained using an optimized singlefrequency CA, while the bottom spectra were recorded using the narrow-band CA. Single-frequency excitation signal amplitudes of 100 and 45 mV0-p were used for the (M + H)+ and (M + 2H)2+ ions, while the narrow-band CA experiments used waveform amplitudes of 1500 and 390 mV0-p for the (M + H)+ and (M + 2H)2+ ions, respectively. For all experiments, a 10-ms ion activation period was used.
band CA spectra appears as a shoulder on the b5 ion. This is not due to a loss in resolution, but rather due to a more complete fragmentation of the precursor ion. Similar experiments were performed using the (M + H)+ and (M + 2H)2+ charge states of bradykinin, with qualitatively similar results (data not shown). Several figures of merit used to describe tandem MS experiments were proposed by Yost and Enke.28 Given that ΣFi is the sum of fragment ion currents, P is the precursor ion current observed in the MS/MS experiment, and P0 is the precursor ion current obtained without ion activation, the fragmentation efficiency and collection efficiency for the CID process are defined as EF ) ΣFi/(P + ΣFi) and EC ) (P + ΣFi)/P0, respectively. Figure 3 shows the dependence of EF on signal amplitude and ion activation time observed for collisional activation of the (M + 2H)2+ charge state from angiotensin I. As previously observed, there is an inverse relationship between the activation waveform amplitude and time needed for efficient fragmentation.12 Using (28) Yost, R. A.; Enke, C. G. J. Am. Chem. Soc. 1978, 100, 2274-2275.
the narrow-band CA at 400 mV0-p for 20 ms or longer, the ratios of parent ion to total fragment ion peak height were similar to those obtained using the optimized single-frequency CA. As previously observed, large activation signal amplitudes result in substantial ejection of parent ions before dissociation can occur.13,29 The narrow-band CA spectra obtained using the highest signal amplitudes had somewhat lower S/N ratios, consistent with the ejection of the precursor ion. Intermediate signal amplitudes had much better S/N ratios, with very little precursor ion. Tandem MS experiments performed on the angiotensin I (M + 2H)2+ charge state using the narrow-band CA were compared with the results from single-frequency CA experiments. For both sets of experiments, excitation amplitudes were varied, using either a 5or 40-ms ion activation period. The fragmentation efficiency as a function of activation waveform amplitude is shown in Figure 4A. As expected, there is little (29) Charles, M. J.; McLuckey, S. A.; Glish, G. L. J. Am. Soc. Mass Spectrom. 1994, 5, 1031-1041.
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Figure 3. Fragmentation efficiency (EF) as a function of the excitation signal amplitude (b) 190, (9) 290, (4) 390, (3) 490, ([) 590, (]) 790, and (O) 990 mV0-p. For reference, the ratio obtained under an optimized single-frequency CA is also plotted (0).
difference in the single-frequency CA as a function of activation time. At a given excitation waveform amplitude using the narrowband CA, longer times are needed for efficient fragmentation. Immediately after acquisition of an MS/MS spectrum, the precursor ion current (P0) was monitored by acquiring a spectrum without the activation waveform (“tickle voltage” turned off). The collection efficiency is a measure of the number of precursor ions retained in the trap (and at the higher CA signal amplitudes, transformed into product ions). Figure 4B shows that, during the narrow-band CA, more ions are retained in the trap, even at much higher signal amplitudes. Results from the complementary experiment, where ion current is monitored during the CA segment of the experiment (ion current is due to the ejection of precursor ions22), are plotted in Figure 4C, showing much the same results and indicating that the narrow-band CA results in greater fragmentation efficiency and a reduced ion loss during activation. Ion activation times of 40 ms and waveform amplitudes of 600 mV were used to maximize the production of useful fragment ions for the remaining studies. A process of nonresonant ion excitation has recently been applied to tandem MS studies of MALDI-generated peptide ions. Using red-shifted off-resonance large-amplitude excitation (RSORLAE), Chait and co-workers demonstrated that parent ions can be efficiently fragmented using a single-frequency activation waveform.30-32 This activation method deposits higher amounts of internal energy into the parent ion but at the same time results in less ejection of the parent ion during the activation period.32 Doroshenko and Cotter also reported an efficient parent ion activation method using “stretched-in-time” SWIFT pulses, which also result in reduced ejection of the precursor ion.18 The efficient precursor ion fragmentation of these two techniques, along with the narrow-band CA technique described in this report, results from the combined advantages of using higher-amplitude excitation signals than normally used in single-frequency CA, but with lower levels of precursor ion ejection during the ion activation period. 2948 Analytical Chemistry, Vol. 71, No. 14, July 15, 1999
Figure 4. (A) Fragmentation efficiency (EF ) for single-frequency and narrow-band CA as a function of the excitation signal amplitude (O, single-frequency CA, 5-ms duration; 0, single-frequency CA, 40ms duration; b, narrow-band CA, 5-ms duration; 9, narrow-band CA, 40-ms duration). (B) Collection efficiency (EC) during single-frequency and narrow-band CA. The symbols used correspond to those described in (A). (C) Integrated area for the ion current ejected during a precursor ion activation step as a function of the excitation waveform amplitude. The symbols used correspond to those described in (A). All experiments were performed using the (M + 2H)2+ ion from angiotensin I.
Carbohydrates. Figure 5 shows the tandem mass spectra obtained for maltotetraose. The top spectrum was obtained after a single-frequency CA, while the bottom spectrum was obtained after narrow-band CA. Conversion efficiency is somewhat better for the narrow-band CA spectrum, where a number of lower intensity ions are observed. These lower-intensity ions result from cross-ring cleavages, which could be especially useful in determining carbohydrate linkages.33 One of the main disadvantages of the ion trap is that fragmentation during MSn studies with this instrument often occurs through the lowest-energy channels available.34,35 However, the longer activation time available with the ion trap CA results in increased fragment yield for some processes, especially with sugars, compared to the MS/MS studies using triple-quadrupole systems.36 The elevated signal amplitudes that can be used in the (30) Qin, J.; Steenvoorden, R. J. J. M.; Chait, B. T. Anal. Chem. 1996, 68, 17841791. (31) Qin, J.; Chait, B. T. Anal. Chem. 1996, 68, 2102-2107. (32) Qin, J.; Chait, B. T. Anal. Chem. 1996, 68, 2108-2112. (33) Domon, B.; Costello, C. E. Glycoconjugate J. 1988, 5, 397-409. (34) Vachet, R. W.; Asam, M. R.; Glish, G. L. J. Am. Chem. Soc. 1996, 118, 6252-6256. (35) McLuckey, S. A.; Goeringer, D. E. J. Mass Spectrom. 1997, 32, 461-474. (36) Sheely, D. M.; Reinhold, V. R. Anal. Chem. 1998, 70, 3053-3059.
Figure 5. Ion trap MS/MS spectra obtained for the (M+Na)+ adducts of maltotetraose ((R-D-Glc-[1f4]-)4). Fragment ions are labeled using the Domon-Costello system of nomenclature.34 The spectrum in the top panel was obtained using an optimized single-frequency CA, while the bottom spectrum was obtained using the narrow-band CA.
Figure 6. Ion trap MS/MS spectra obtained for the (M + Na)+ adduct of stachyose (R-D-Gal-[1f6]-R-D-Gal-[1f6]-R-D-Glc-[1f2]-R-D-Fru). Fragment ions are labeled as described for Figure 5. The spectrum in the top panel was obtained using an optimized single-frequency CA, while the bottom spectrum was obtained using the narrow-band CA.
narrow-band CA experiments, while minimizing parent ion ejection, result in increased collision energy that can be internalized. This increased internal energy allows the decomposition processes requiring higher internal energy to occur, such as in the sugar cross-ring fragmentation.
Figure 6 shows tandem mass spectra for stachyose, an isomeric tetrasaccharide. Either single-frequency or narrow-band CA was effective in converting the parent ion to the isobaric C3 and Y3 ions. The narrow-band CA spectrum shown in Figure 6 was obtained immediately after activating the multiplying circuit, before Analytical Chemistry, Vol. 71, No. 14, July 15, 1999
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Figure 7. Pseudo-MS3 spectra of stachyose obtained through the application of two narrow-band CA pulses. After isolation of the parent ion with m/z 689 and excitation at qz ) 0.2 with a narrow-band CA pulse, the ring electrode rf level was lowered to bring the m/z 527 ion to qz ) 0.2, while another narrow-band CA pulse was applied.
any adjustment of the signal amplitude occurred. The efficiency of conversion was not as great for the narrow-band CA, however, while an increase in amplitude of the CA signal did result in higher conversion efficiency (data not shown). This indicates that highquality tandem mass spectra can be obtained with little optimization. In contrast to the data obtained with maltotetraose, CA of stachyose resulted in dissociation through only one channel. To study further dissociations of this sugar, a pseudo-MS3 experiment using two sequential narrow-band CA steps was performed (Figure 7). After fragmentation of the parent ion at qz ) 0.2, the ring electrode rf level was lowered to bring the m/z 527 ion to qz ) 0.2, and another narrow-band CA signal was applied. The resulting spectrum contains the undissociated component of the parent ion as well as the undissociated m/z 527 ion and its fragment ions. The same narrow-band CA signal was used for both activation steps, indicating that this method is effective with different numbers of ions in the trap. In the single-frequency CA, it is necessary to optimize the ac signal applied to the end cap electrodes for each segment of a tandem MS experiment, owing to the space charge-induced changes in the ion secular frequency.14 Fragmentation of the parent sugar formed an ion of m/z 527. From this information, it would be impossible to determine the structure, as the C3 and Y3 ions are isomeric. The fragmentation pattern observed during the pseudo-MS3 experiment was most consistent with the cross-ring fragmentation that would be predicted from 1,6 cleavages.37 This indicates that the fragment (37) Garozzo, D.; Giuffrida, M.; Impallomenti, G.; Ballistreri, A.; Montaudo, G. Anal. Chem. 1990, 62, 279-286. (38) Biemann, K.; Papayannopoulos, I. A. Acc. Chem. Res. 1994, 27, 370-378.
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ion observed at m/z 527 was a C3-type ion formed by a loss of the reducing end of the tetrasaccharide. CONCLUSIONS The use of a simple circuit for modulating the signal applied to the end caps of an ITMS during tandem MS experiments has been demonstrated. The narrow-band CA waveforms efficiently dissociated multiply charged peptide ions as well as sodiumcationized oligosaccharides. Narrow-band CA was also applied to pseudo-MS3 experiments, alleviating the CA frequency-tuning requirements necessary for an efficient ion fragmentation. Higheramplitude activation waveforms could be applied using narrowband CA without ejection of parent ions, allowing access to higher energy decomposition channels. The narrow-band CA should be amenable to tandem MS experiments coupled with highperformance separation techniques. ACKNOWLEDGMENT This work was supported by Grant GM 24349 from the National Institute of General Medical Sciences, U.S. Department of Health and Human Services. The authors acknowledge Dr. David Clemmer for many useful discussions and John Poehlman of the Indiana University Electronic Instrument Services for valuable technical assistance designing the multiplier circuit.
Received for review August 18, 1998. Accepted April 13, 1999. AC980930P
