Anal. Chem. 1998, 70, 340-346
Boundary-Activated Dissociation of Peptide Ions in a Quadrupole Ion Trap Richard W. Vachet and Gary L. Glish*
Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599
Boundary-activated dissociation (BAD) of peptides has been investigated as an alternative to the use of resonant excitation to effect collision-induced dissociation in the quadrupole ion trap. BAD’s nonresonant excitation mechanism overcomes a major drawback in resonant excitation, namely, the variation of the resonant excitation frequency as a function of ion space charging. As with resonant excitation, the pulsed introduction of heavy gases (argon, xenon) extends the applicability of BAD when tandem mass spectrometry is performed on peptide ions. The presence of heavy gases during ion activation allows greater internal energy deposition and also enables BAD to be performed at much lower trapping field strengths (lower q values) than previously reported for this technique. This extends the mass range over which product ions can be collected. Primary sequence analysis of peptides by mass spectrometry (MS) has seen increased use because of the variety of advantages it has over more traditional sequencing methods.1-4 The speed and sensitivity with which MS can provide information leading to the structural elucidation of a wide variety of peptides are among its advantages. In addition, the selectivity afforded by tandem mass spectrometry (MS/MS) enables a single peptide to be analyzed from a complicated mixture of peptides. Many types of instruments have been used to obtain primary sequence information from peptides. Quadrupole1,5 and sector6-8 instruments have seen the most widespread use for structural studies of peptides, but trapping instruments such as Fourier transform ion cyclotron resonance (FTMS)9-13 and quadrupole
ion trap14-16 mass spectrometers have also been used to investigate peptide structure. The quadrupole ion trap has advantages, such as relative low cost, high sensitivity, high MS/MS efficiency, and the ability to perform multiple stages of MS/MS (MSn), that make it well suited for the routine structural analysis of peptides. The quadrupole ion trap, however, has some drawbacks with its typical experimental approach to providing structural information. In the quadrupole ion trap, MS/MS is usually carried out by the application of a supplementary ac signal to the end-cap electrodes at a frequency corresponding to the secular frequency of a chosen parent ion. If the amplitude of the supplementary ac signal is adequate, the parent ion will be resonantly excited and the amplitude of its motion will increase. The subsequent increase in kinetic energy will enable the ion to undergo energetic collisions with the background gas. These collisions can increase the internal energy of the ion and cause it to eventually dissociate. Two main problems exist with the most common means of implementing this ion activation technique. First, the internal energy deposited into the ions during collision-induced dissociation (CID) is relatively small. This low-energy deposition is due primarily to the low center-of-mass collision energies involved with CID in the presence of only the standard bath gas, helium. Second, the application of a resonance excitation signal to the end cap at the theoretically predicted frequency of the parent ion can fail to effect kinetic excitation, and hence internal excitation, of that ion. Space charge effects and higher order fields (present due to imperfections in the geometry of the ion trap) cause the secular frequencies of ions to be shifted from that theoretically predicted.17-20 Unless the number of ions present in the trap is carefully controlled, the optimum experimental resonant frequency must be empirically determined and this process can be time consuming. To overcome the drawbacks of low-energy deposition and optimization of the resonant frequency, a number of approaches have been proposed. A method has been described in which
* Author to whom correspondence should be addressed: phone, (919) 9622303; e-mail,
[email protected]. (1) Hunt, D. F.; Yates, J. R., III; Shabanowitz, J.; Winston, S.; Hauer, C. R. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 6233. (2) Biemann, K.; Martin, S. A. Mass Spectrom. Rev. 1987, 6, 1. (3) Carr, S. A; Hemling, M. E.; Bean, M. F.; Roberts, G. D. Anal. Chem. 1991, 63, 2802. (4) Papayannopolous, I. A. Mass Spectrom. Rev. 1995, 14, 49. (5) Tang, X.-J.; Thibault, P.; Boyd, R. K. Anal. Chem. 1993, 65, 2824. (6) Martin, S. A.; Biemann, K. Int. J. Mass Spectrom. Ion Processes 1987, 78, 213. (7) Johnson, R. S.; Martin, S. A.; Biemann, K.; Stults, J. T.; Watson, J. T. Anal. Chem. 1987, 59, 2621. (8) Johnson, R. S.; Martin, S. A.; Biemann, K. Int. J. Mass Spectrom. Ion Processes 1988, 86, 137. (9) Cody, R. B., Jr.; Amster, I. J.; McLafferty, F. W. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 6367. (10) Yang, L.-C.; Wilkins, C. L. Org. Mass Spectrom. 1989, 24, 409.
(11) Castoro, J. A.; Wilkins, C. L.; Cotter, R. J. J. Mass Spectrom. 1995, 30, 94. (12) Speir, J. P.; Amster, I. J. J. Am. Soc. Mass Spectrom. 1995, 6, 1069. (13) Price, W. D.; Schnier, P. D.; Williams, E. R. Anal. Chem. 1996, 68, 859. (14) Qin, J.; Chait, B. T. J. Am. Chem. Soc. 1995, 117, 5411. (15) Doroshenko, V. M.; Cotter, R. J. Anal. Chem. 1995, 67, 2180. (16) Vachet, R. W.; Asam, M. R.; Glish, G. L. J. Am. Chem. Soc. 1996, 118, 6252. (17) Todd, J. F. J.; Waldren, R. M.; Mather, R. E. Int. J. Mass Spectrom. Ion Phys. 1980, 34, 325. (18) Todd, J. F. J.; Waldren, R. M.; Freer, D. A; Turner, R. B. Int. J. Mass Spectrom. Ion Phys. 1980, 35, 107. (19) Franzen, J. Int. J. Mass Spectrom. Ion Processes 1991, 106, 63. (20) Vedel, F.; Vedel, M. Phys. Rev. A 1990, 41, 2348.
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mass-selected ions undergo collisions with the ring electrode via the application of a rapid (ms) dc pulse to the end cap.21 This experiment removes the possible need for empirical optimization of a resonant frequency and results in greater energy deposition but at the cost of MS/MS efficiency. Another method that does not require a resonant signal involves the application of a lowfrequency (50-500 Hz) dipolar square wave to the end-cap electrodes.22 The dipolar square wave causes the electrical center of the trapping field and the center of ions’ oscillation to change instantly with no change in the spatial location of the ions. In response to the change in their oscillation center, ions accelerate and can undergo energetic collisions with the background gas. Simulations suggest that ions can be accelerated to kinetic energies in excess of 40 eV,22 thus enabling ion/neutral collisions to be more energetic than in normal resonance excitation. An off-resonance excitation method recently described23 also overcomes some of the drawbacks of traditional resonant excitation. This technique, termed red-shifted off-resonance large-amplitude excitation (RSORLAE), involves the application of a largeamplitude (21 Vp-p) ac signal to the end cap that is red-shifted ∼5% from the secular frequency of the ion and is preceded by a rapid “jump” in the rf voltage applied to the ring electrode. RSORLAE has been shown to be effective in dissociating MALDIgenerated ions. Another approach used to increase the energy deposition is the addition of heavy gases (argon, xenon, etc.) to the helium bath gas.24-29 Experiments in which heavy gases are pulsed in just prior to the MS/MS stage of the analysis have been shown to be much better than static pressures of the same heavy gas with regard to spectral resolution, ion detection, and energy deposition.28,29 This means of heavy gas introduction has also addressed the problem of empirical optimization of the resonant excitation frequency, because efficient MS/MS can be obtained at the theoretically predicted frequency in the presence of heavy gases.29 Another excitation method that does not require the application of a resonant signal is one referred to as boundary activation.30-34 In boundary activation, a dc pulse (ms) is applied to the ring electrode to bring ions’ a and q parameters into the proximity of one of the stability boundaries in the Mathieu stability diagram for the quadrupole ion trap. This method, however, has resulted (21) Lammert, S. A.; Cooks, R. G. J. Am. Soc. Mass Spectrom. 1991, 2, 487. (22) Wang, M.; Schachterle, S.; Wells, G. J. Am. Soc. Mass Spectrom. 1996, 7, 668. (23) Qin, J.; Chait, B. T. Anal. Chem. 1996, 68, 2108. (24) Gronowska, J.; Paradisi, C.; Traldi, P.; Vettori, U. Rapid Commun. Mass Spectrom. 1990, 4, 307. (25) Nourse, B. D.; Cox, K. A.; Morand, K. L.; Cooks, R. G. J. Am. Chem. Soc. 1992, 114, 2010. (26) Morand, K. L.; Cox, K. A.; Cooks, R. G. Rapid Commun. Mass Spectrom. 1992, 6, 520. (27) Charles, M. J.; McLuckey, S. A.; Glish, G. L. J. Am. Soc. Mass Spectrom. 1994, 5, 1031. (28) Doroshenko, V. M.; Cotter, R. J. Anal. Chem. 1996, 68, 463. (29) Vachet, R. W.; Glish, G. L. J. Am. Soc. Mass Spectrom. 1996, 7, 1194. (30) Paradisi, C.; Todd, J. F. J.; Vettori, U. Org. Mass Spectrom. 1992, 27, 251. (31) Paradisi, C.; Todd, J. F. J.; Vettori, U. Org. Mass Spectrom. 1992, 27, 1210. (32) Curcuruto, O.; Fontana, S.; Traldi, P.; Celon, E. Rapid Commun. Mass Spectrom. 1992, 6, 322. (33) Creaser, C. S.; O’Neill, K. E. Org. Mass Spectrom. 1993, 28, 564. (34) March, R. E.; Weir, M. R.; Londry, F. A.; Catinella, S.; Traldi, P.; Stone, J. A.; Jacobs, W. B. Can. J. Chem. 1994, 72, 966.
in low efficiencies, and conflicting results have been reported with regard to energy deposition.32,33 Boundary-activated dissociation (BAD), however, may be a promising technique for the dissociation of peptide ions due to its nonresonant excitation mechanism. In particular, when used in conjunction with the pulsed introduction of a heavy gas, such as xenon or argon, some of the disadvantages to its present use may be overcome. The current application of BAD as a massselective method to dissociate ions is limited for a couple of reasons. Due to the nature of the activation mechanism, BAD has not been effectively performed at q values much below 0.3 when helium is used as the collision gas (which is similar to the lower limit for resonance excitation). This relatively high qz value limits the product ion mass range. At lower q values, parent ion trajectories are less stable when their a and q parameters are in the proximity of the boundaries of the stability diagram, and as a result, ion ejection predominates over ion dissociation. This limitation would be detrimental to the study of peptides because the dissociation of a parent ion at m/z 900, for example, would preclude the detection of product ions below m/z 300. Also, MS/ MS efficiencies with BAD are lower than efficiencies obtained by resonant excitation at any q value. Previous reports show that MS/MS efficiencies from BAD range from 10 to 90% with a rapid drop at lower q values.30-34 These values are compared to 75100% efficiencies obtained during resonant excitation over a larger range of q values. This report focuses on the details of BAD in the presence of heavy gases and its merits as an activation method to provide primary sequence information for peptides. It is observed that in the presence of heavy gases the limitations of this activation method can be reasonably overcome. EXPERIMENTAL SECTION Experiments were performed on a Finnigan MAT ITMS (San Jose, CA) modified with a custom-built electrospray source of a design similar to one previously reported.35 A Model 22 Harvard Apparatus (South Natick, MA) syringe pump was used to infuse solutions at 0.25-0.50 µL/min into a capillary that was connected to a needle via a Teflon union. The needle voltage was held at 3-4 kV. A TTL pulse from the ITMS electronics was used to trigger an HP 8013B pulse generator that supplied a dc pulse of variable length and amplitude to a high-speed amplifier that was connected to the rf coil. The HP pulse generator allowed the manual adjustment of the length, amplitude, and delay of the dc pulse that was eventually supplied to the ring electrode to effect BAD. The same TTL pulse that triggered the HP pulse generator was also used to trigger a General Valve Corp. Iota One (Fairfield, NJ) pulse driver that actuated a series 9 high-speed solenoid valve. The solenoid valve was mounted on the vacuum manifold to which a xenon or argon gas line was connected with a gas backing pressure of ∼600 Torr. When BAD was performed in the presence of xenon or argon, a delay (usually 15 ms) was manually entered into the HP pulse generator to allow the heavy gas to diffuse into the volume of the electrodes before the dc pulse was applied to the ring electrode. In all cases in which argon or xenon (35) Van Berkel, G. J.; Glish, G. L.; McLuckey, S. A. Anal. Chem. 1990, 62, 1284.
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was pulsed in, a constant pressure of helium was maintained (8.6 × 10-4 Torr). The pulse lengths for gas introduction varied from 250 µs to 9 ms. The actual pressure maximum of gas entering the chamber at these pulse lengths ranged from ∼0.1 to 2 mTorr. The dc pulse lengths could be varied over a large range but were usually 50 ms. The sign and amplitude of the dc pulse were dependent upon the voltage required to bring the chosen ion to either the βz ) 0 boundary (+dc) or the βr ) 0 boundary (-dc) of the stability diagram. The magnitude of the voltage was dependent upon the m/z of the ion of interest and the qz value at which it was stored and could be easily calculated. Peptides were either obtained from Sigma Chemical Co. (St. Louis, MO) or synthesized in-house by standard Fmoc chemistry. The peptides were dissolved in either a 20:75:5 mixture of water/ methanol/acetic acid or a 25:75 mixture of water/methanol to a concentration of 250 µM. Argon (99.997% purity) and helium (99.995% purity) were purchased from International Welders, Inc. (Charlotte, NC), and xenon (99.997% purity) was obtained from Airco Gases (Murray Hill, NJ). RESULTS AND DISCUSSION In BAD, an ion can be activated when its Mathieu parameters, a and q, are chosen such that its working point is located close to the boundary of the stability diagram. The application of a dc pulse moves ions into regions of the trap where the rf restoring field is greater, causing them to accelerate to greater kinetic energies and undergo energetic collisions with the background gas. If enough internal energy can be deposited into an ion to cause it to dissociate before its trajectory becomes unstable, product ions can be stored and later detected during a normal analytical scan. A means of envisioning this competition between ion ejection and internal energy deposition (and eventual dissociation) can be done by considering motion of an ion in a pseudopotential well. When an ion has a stable trajectory in the quadrupole ion trap, it can be considered to be oscillating in a pseudopotential well.36 The depth (Dz) of this well is related to the ion’s qz parameter and is shown in eq 1 where m is the mass of the ion in kilograms,
Dz ) qz2mr2Ω2/32e
(1)
r is the radius of the ring electrode in meters, Ω is the frequency of the rf drive, and e is the fundamental unit of charge. When an ion’s stability parameters (a and q) are chosen to bring it to the boundary of the stability diagram, its oscillation in the pseudopotential well begins to increase in amplitude. In the absence of any damping force, the oscillation of the ion will eventually lead to a kinetic energy that results in an unstable trajectory, and the ion will be ejected. In the presence of a damping force, such as collisions with the background gas, ion ejection will be slowed. During these damping collisions, some of an ion’s kinetic energy can be converted into internal energy, which when sufficiently accumulated can cause the ion to dissociate. If enough internal energy accumulates before the ion’s trajectory becomes unstable, then the ion will dissociate and the product ions may be stored (36) Major, F. G.; Dehmelt, H. G. Phys. Rev. 1968, 179, 91.
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and subsequently detected. Simulations done by March et al. show that an ion’s kinetic energy can also be increased when the ion undergoes a collision with helium and its working point is close to the boundary.37 This collision could lead to either an unstable trajectory or another more energetic collision that deposits internal energy. As was already mentioned, the ability to dissociate an ion by this method will depend on the competition between ion activation and ion ejection. This competition is dependent upon several parameters. The first is the depth of the pseudopotential well in which the parent ion is trapped; the well depth determines the maximum kinetic energy the ion can achieve before ejection and will be a major factor determining the length of time before an ion is ejected. The second parameter is the length of time the dc is applied to move the ion to the boundary of the stability diagram; this factor will influence the length of time the ion has to achieve a potential energy that allows it to escape the pseudopotential well or undergo a collision that leads to increased kinetic energy and subsequent ion ejection. As the dc pulse is increased, the percentage of the parent ions that are ejected increases faster than the percentage of parent ions that are dissociated (i.e., at shorter dc pulse lengths ion activation competes more favorably with ion ejection than at longer pulse lengths). Another important parameter in the competition between ion ejection and dissociation is the energetics of the ion dissociation; the greater the amount of internal energy needed to dissociate an ion, the less likely it will be dissociated and the more likely it will be ejected. Because the depth of the pseudopotential well in which an ion resides affects the competition between ion dissociation and ejection, it is not surprising that the q value at which an ion is stored affects the MS/MS efficiency in BAD. MS/MS efficiency (EffMS/MS) is defined as the sum of the intensities of all product ions (∑[fi+]) detected divided by the intensity of the parent ion (∑[p+]) before dissociation (eq 2).38,39 It has been observed
EffMS/MS )
∑[f ]/∑[p +
i
+
]
(2)
previously that EffMS/MS decreases in BAD as the q value decreases.31 This effect was also observed for peptides in this study, and an example with the tetrapeptide GGGG is shown in Figure 1. The reduction in EffMS/MS coincides with the reduction in the pseudopotential well depth (eq 1) when the q value is decreased. The MS/MS efficiency and the extent of dissociation can be increased by the introduction of heavy gases during the activation stage of the experiment. Figure 2 shows a comparison of the BAD MS/MS spectra obtained for the protonated species of leucine enkephalin (YGGFL, m/z 556) in the presence of helium (a) and after a 1.5 ms pulse of argon (b), at a q value of 0.180. A positive dc pulse (67.1 V) was applied to the ring electrode to bring the ions’ working point into the proximity of the βz ) 0 (37) March, R. E; Tkacyzk, M.; Londry, F. A.; Alfred, R. L. Int. J. Mass Spectrom. Ion Processes 1993, 125, 9. (38) Yost, R. A.; Enke, C. G.; McGilvery, D. C.; Smith, D.; Morrison, J. D. Int. J. Mass Spectrom. Ion Phys. 1979, 30, 127. (39) McLuckey, S. A.; Glish, G. L.; Van Berkel, G. J. Int. J. Mass Spectrom. Ion Processes 1991, 106, 213.
Figure 1. MS/MS efficiency for the boundary-activated dissociation of the protonated species of GGGG over a range of q values.
a
Figure 3. MS/MS efficiency for the boundary-activated dissociation of the protonated species of YGGFL at varying a values in the presence of only helium (2), helium and a 1500 µs pulse of argon (b), and helium and a 1500 µs pulse of xenon (9).
than a collision between the ion and helium (eq 3). The center-
(
Ecom ) Elab
b
Figure 2. Boundary-activated dissociation MS/MS spectrum of the protonated species of YGGFL (a) with only helium and (b) the same conditions as (a) plus a 1500 µs pulse of argon.
boundary. Clearly, the introduction of argon during BAD increases the extent of dissociation and thus the amount of information available, including the formation of the immonium ions of tyrosine (Y) and phenylalanine (F). What is not obvious from the spectra in Figure 2 is that EffMS/MS has increased from ∼10% in (a) to ∼20% in (b). A collision between an ion and argon can convert more of the ion’s kinetic energy into internal energy
)
mn mn + mp
(3)
of-mass collision energy (Ecom) is the maximum amount of internal energy that can be deposited into an ion, with a mass (mp) and kinetic energy (Elab), upon collision with a neutral target gas, with a mass (mn). The greater mass of argon relative to helium (factor of 10) allows more of the ion’s kinetic energy to be transferred into internal energy, and this affects not only the extent of dissociation but also the MS/MS efficiency of the BAD process. Increased conversion of kinetic energy into internal energy in the presence of argon allows a greater number of parent ions to accumulate enough internal energy to dissociate before they achieve a kinetic energy sufficient for ejection. The increase in MS/MS efficiency upon the pulsed introduction of argon (Figure 2b) is expected, but the somewhat modest increase is surprising. To further understand the effect of heavy gases on the BAD process, the MS/MS efficiency for BAD of leucine enkephalin was monitored at various distances from the βz ) 0 boundary (at q ) 0.180) in the presence of different gases. The amplitude of the dc pulse was changed to vary the a value at which the parent ion was stored, and the MS/MS efficiency was measured with a 1500 µs pulse of xenon, a 1500 µs pulse of argon, and helium alone (Figure 3). From Figure 3 it is clear that the optimum MS/MS efficiency for the BAD process occurs at different a values when the presence and the nature of the heavy gas is changed. The EffMS/MS reaches nearly 60% in the presence of a pulse of xenon or argon but at different a values. Thus, pulsed introduction of argon or xenon results in a 6 times increase in EffMS/MS at the optimum a value for each gas when compared to the results obtained in the presence of helium alone. Figure 3 suggests that an ion’s kinetic energy begins to increase farther away from the edge of the stability diagram than might be expected. A slight increase in an ion’s kinetic energy in the presence of xenon can lead to efficient dissociation, but in Analytical Chemistry, Vol. 70, No. 2, January 15, 1998
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the presence of argon or helium alone, no dissociation is observed (e.g., dc is 62.3 V, a ) -0.0181). If the ions’ kinetic energies are presumed to increase as their Mathieu parameters approach the edge of the stability diagram, it is not surprising to observe the trend that higher dc voltages (increased magnitude of a parameter) are needed to bring about ion dissociation in the presence of xenon, argon, and helium respectively (eq 3). Another issue that Figure 3 raises is what m/z range of ions is activated when the desired parent ion is brought to the stability boundary in the presence of various gases. Figure 3 shows that a range of dc voltages (and a values) can bring about the dissociation of a particular parent ion. If a dc voltage is applied to bring the protonated species of YGGFL [M + H]+ to the point of maximum efficiency in the presence of only helium (a ) -0.0196; Vdc ) 67.1 V), ions at m/z 552-560 should be activated as well. This determination is based upon the fact that because YGGFL can be effectively dissociated at dc voltages above and below the optimum (a ) -0.0196; Vdc ) 67.1 V), then ions that are above or below their optimum a value could dissociate as well. If this same rationale is applied to the cases in which argon is pulsed in during activation, the range of m/z that can be activated is m/z 540-560 when the protonated species is given an a value of -0.0196 (i.e., value for maximum MS/MS efficiency in presence of helium). The range changes to m/z 547-567 if the protonated species of YGGFL is brought to the point of maximum MS/MS efficiency in the presence of argon (a ) -0.0191; Vdc ) 66.2 V). For xenon the range of m/z that can be activated at the optimum az value for helium is m/z 518-560, while the range changes to m/z 536-578 at the optimum a value for the introduction of xenon. In the case of xenon, that means product ions of YGGFL could possibly be activated as well. For example, a product ion (m/z 538) corresponding to a loss of 18 Da (-H2O) would be expected to be activated as well as the parent ion [M + H]+, and indeed, the BAD MS/MS spectrum of the protonated species of YGGFL in the presence of xenon is missing the peak corresponding to a loss of H2O (spectrum not shown). Another point of interest from Figure 3 is the fact that 100% MS/MS efficiency is not observed with the pulsed introduction of xenon. If helium alone is present and a dc pulse of 62.3 V is applied (point of maximum MS/MS efficiency when xenon is present), there is no dissociation of the parent ion, and there is no detectable parent ion loss. This observation indicates that there is some ion loss (either product ions or parent ions) when xenon is present. The ion loss is presumably due to the scattering of ions from collisions with xenon into phase space areas that lead to unstable ion trajectories or possibly the formation of low-mass product ions that have m/z values below the low-mass cutoff (i.e., q > 0.908). The low-mass cutoff in the case described above is 110 Da, and based upon resonance excitation experiments at a reduced low-mass cutoff (
