Thermally Assisted Infrared Multiphoton Photodissociation in a

Anal. Chem. 2001, 73, 3542-3548

Thermally Assisted Infrared Multiphoton Photodissociation in a Quadrupole Ion Trap Anne H. Payne and Gary L. Glish*

CB#3290 Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

Thermally assisted infrared multiphoton photodissociation (TA-IRMPD) provides an effective means to dissociate ions in the quadrupole ion trap mass spectrometer (QITMS) without detrimentally affecting the performance of the instrument. IRMPD can offer advantages over collision-induced dissociation (CID). However, collisions with the QITMS bath gas at the standard pressure and ambient temperature cause IR-irradiated ions to lose energy faster than photons can be absorbed to induce dissociation. The low pressure required for IRMPD (e10-5 Torr) is not that required for optimal performance of the QITMS (10-3 Torr), and sensitivity and resolution suffer. TA-IRMPD is performed with the bath gas at an elevated temperature. The higher temperature of the bath gas results in less energy lost in collisions of the IR-excited ions with the bath gas. Thermal assistance allows IRMPD to be used at or near optimal pressures, which results in an ∼1 order of magnitude increase in signal intensity. Unlike CID, IRMPD allows small product ions, those less than about one-third the m/z of the parent ion, to be observed. IRMPD should also be more easily paired with fluctuating ion sources, as the corresponding fluctuations in resonant frequencies do not affect IRMPD. Finally, while IR irradiation nonselectively causes dissociation of all ions, TA-IRMPD can be made selective by using axial expansion to move ions away from the path of the laser beam. Tandem mass spectrometry (MS/MS) has become an invaluable tool for investigating the identity and structure of an analyte.1-6 For example, MS/MS is increasingly used for sequencing peptides rather than the traditional solution-phase Edman degradation.7,8 The quadrupole ion trap mass spectrometer (QITMS) offers particular advantages including multiple stages (1) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 7, 994-999. (2) Lattimer, R. P.; Mu: nster, H.; Budzikiewicz, H. Int. J. Mass Spectrom. Ion Processes 1989, 90, 119-129. (3) Loo, J. A.; Edmonds, C. G.; Smith, R. D. Anal. Chem. 1991, 63, 24882499. (4) McLuckey, S. A.; VanBerkel, G. J.; Glish, G. L. J. Am. Soc. Mass Spectrom. 1992, 3, 60-70. (5) Papayannopoulos, I. A. Mass Spectrom. Rev. 1995, 14, 49-73. (6) March, R. E. J. Mass Spectrom. 1997, 32, 351-369. (7) Masselon, C.; Anderson, G. A.; Harkewicz, R.; Bruce, J. E.; Pasˇa-Tolic´, L.; Smith, R. D. Anal. Chem. 2000, 72, 1918-1924. (8) McCormack, A. L.; Schieltz, D. M.; Goode, B.; Yang, S.; Barnes, G.; Drubin, D.; Yates, J. R. Anal. Chem. 1997, 69, 767-776.

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of MS/MS (MSn), high sensitivity and duty cycle, relatively simple vacuum requirements, a small footprint, fast analysis, and ruggedness and is relatively inexpensive. The versatility and availability of this instrument makes the QITMS an increasingly popular choice for a mass spectrometer. The QITMS traps ions in the volume encompassed by three electrodes: two end caps and one ring electrode. The volume is defined in the radial dimension, r, by the ring electrode and in the axial direction, z, by the end cap electrodes. Ion stability within the QITMS depends on several factors: the m/z of the ion, the dimensions of the electrodes, and the applied ac and dc voltages. Ion motion in a QITMS can be described by a second-order differential equation. A general solution to this type of equation was developed by Mathieu over a century ago. From Mathieu’s solution, the combinations of ac and dc voltages that result in a stable ion trajectory in a QITMS can be determined.9 The QITMS, as it is typically operated, traps ions by means of an applied ac voltage and no dc voltage.10 In this case, the Mathieu parameter of interest, qz, is given by following equation:

qz ) 8eV/m(r20 + 2z20)Ω2

(1)

The relevant values included in this equation are the mass (m) and charge (e) of the ion, the trap dimensions both radial (ro) and axial (zo), the ac frequency (Ω), and the ac amplitude (V). Because the ac frequency is in the radio frequency range, it is commonly referred to as rf. For any combination where qz < 0.908, the ion trajectory is stable and ions are trapped. In practical terms, because qz is inversely proportional to the mass-to-charge ratio (m/z), this means that all ions above some low-mass cutoff are trapped. The m/z of this cutoff is varied by changing the amplitude of the rf, and the rf level is commonly referred to by the resulting m/z cutoff level rather than by the amplitude of the voltage. The qz value for a selected m/z can also be controlled by changing the m/z cutoff. A MS/MS analysis in the QITMS typically involves four steps: accumulation of ions, isolation of the parent ion, dissociation of that ion, and mass analysis of the product ions. Trapped ions have a periodic motion induced by the trapping rf voltage, so the manipulation of the trapped ions can be achieved using reso(9) March, R. E.; Londry, F. A. In Practical Aspects of Ion Trap Mass Spectrometry; March, R. E., Todd, J. F. J., Eds.; CRC Press: Boca Raton, FL, 1995; Vol. I, pp 25-48. (10) Kelley, P. E.; Stafford, G. C.; Stephens, D. R. U.S. patent 4,540,884, 1985. 10.1021/ac010245+ CCC: $20.00

© 2001 American Chemical Society Published on Web 06/22/2001

nance.11 Each m/z has a different frequency of motion, called the secular frequency. By applying to the end caps a supplemental ac voltage matching an ion’s secular frequency, that ion’s kinetic energy can be increased.12 With sufficient kinetic energy, the ion motions exceed the bounds of the trap, and the ion is ejected. Resonance ejection can be used in both the isolation and analysis steps. If the kinetic energy gained by an ion is lower than that required for ejection, the amplitude of its motion is increased while trapping is maintained. The ion undergoes collisions with the bath gas, which converts some of the kinetic energy into internal energy.13 If the internal energy level reaches the critical energy, the parent ion dissociates and product ions are formed. However, because the ion can be ejected if the increased amplitude of motion for the ion is too large, a balance exists between excitation and ejection.14 Resonance excitation collision-induced dissociation (CID) is the most common means for achieving ion dissociation in the QITMS,15-17 but it does have some disadvantages. For example, product ions less than about one-third the m/z of the parent ion are not observed (for reasons discussed below), and CID is difficult to pair with fluctuating sources, such as MALDI, as the resonant frequency changes with the number of ions trapped. Boundary activated dissociation (BAD), another method to increase ion kinetic energy, can circumvent the frequencymatching problems but still results in the loss of low-m/z product ions and a tradeoff between ejection and dissociation. Most of the drawbacks of CID arise from the competition between ejection and excitation. Because the inherent motions of the trapped ions are being increased in amplitude, the motions must be kept sufficiently small to remain in the ion trap. However, the motions must also be increased enough that the energy of the collisions is sufficient to induce dissociation. To better illustrate the competition, ions can be considered as being trapped in a pseudopotential well. The depth of the well, D, can be approximated for qz < 0.4 by the Dehmelt pseudopotential well model and is proportional to the square of the amplitude of rf trapping frequency and inversely proportional to the square of the m/z.

D ∝ q2 ∝ (rf amplitude)2/(m/z)2

(2)

If the amplitude of an ion’s motion exceeds the depth of the well, the ion will be ejected. However, a certain minimum amplitude is necessary for dissociation. To decrease the likelihood of ejection, the well should be deep. Raising the rf amplitude increases the well depth, but it also raises the low-m/z cutoff. Raising the cutoff means that any low-m/z product ions that are formed are not trapped. As a compromise, a well depth is generally chosen such (11) Williams, J. D.; Cooks, R. G.; Syka, J. E. P.; Hemberger, P. H.; Nogar, N. S. J. Am. Soc. Mass Spectrom. 1993, 4, 792-797. (12) 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. (13) McLuckey, S. A. J. Am. Soc. Mass Spectrom. 1992, 3, 599-614. (14) Charles, M. J.; McLuckey, S. A.; Glish, G. L. J. Am. Soc. Mass Spectrom. 1994, 5, 1031-1041. (15) Splendore, M.; Londry, F. A.; March, R. E.; Morrison, R. J. S.; Perrier, P.; Andre´, J. Int. J. Mass Spectrom. Ion Processes 1996, 156, 11-29. (16) Biemann, K.; Martin, S. A. Mass Spectrom. Rev. 1987, 6, 1-76. (17) Gronowska, J.; Paradisi, C.; Traldi, P.; Vettori, U. Rapid Commun. Mass Spectrom. 1990, 4, 307-314.

that qz ) 0.2-0.3 and ions of less than about one-third the m/z of the parent ion are not trapped and, thus, not observed. Another potential difficulty with resonance excitation CID is in matching the secular frequency. The number of ions trapped affects the secular frequency. A high density of trapped ions effectively creates a dc potential in the QITMS. This potential changes the stability and the secular frequencies of the ions.18 If the ion population changes from scan to scan, the frequencies will also change. This is generally not a problem with a stable ion source. However, for ion sources with large variations in current, such as matrix-assisted laser desorption/ionization (MALDI), ion intensity can fluctuate widely from scan to scan, so the ion population and the resonant frequencies do not stay fixed. Infrared multiphoton photodissociation (IRMPD) can avoid all of these problems associated with CID. With IRMPD, the internal energy of an ion is increased by photon absorption rather than by energetic collisions induced by raising the kinetic energy of the ions. By absorbing multiple photons of the IR radiation, an ion gains enough internal energy to dissociate. Because the ion’s motion (its kinetic energy) is not being increased, the frequencies of motion and well depths are not relevant for IRMPD. The resonant frequency does not need to be determined, and the dissociation is not affected if the frequency fluctuates. The well depth does not affect the MS/MS efficiency, so the low-m/z cutoff need not be raised, and small-m/z product ions can be trapped and observed. While IRMPD has been used in ion cyclotron resonance (ICR) instruments with success,19-21 IRMPD in the QITMS does present some difficulties. ICR instruments are operated at an ultrahigh vacuum of less than 10-9 Torr, as the best resolution is achieved in an ICR at the lowest pressure. However, a helium bath gas is used in the QITMS at 10-3 Torr, which improves both resolution and sensitivity.22 In the QITMS, ions constantly undergo low-energy collisions with the helium bath gas. These collisions can serve to transfer internal or kinetic energy from the ion to the bath gas.23 This process is described as collisional cooling and helps to trap the ions and to confine the ions to the center of the trapping volume. However, as an ion is irradiated by the IR laser, if the rate at which the internal energy is removed via collisions exceeds the rate at which it is added via photons, the ion will not dissociate. IRMPD has been used successfully in the QITMS.24-27 However, it must be performed at lower pressures where the cooling effects are lessened. Unfortunately, the performance of the QITMS is significantly decreased at these lower pressures. (18) Vedel, F.; Andre´, J. Phys. Rev. A 1984, 29, 2098-2101. (19) Little, D. P.; Speir, J. P.; Senko, M. W.; O’Connor, P. B.; McLafferty, F. W. Anal. Chem. 1994, 66, 2809-2815. (20) Hofstadler, S. A.; Sannes-Lowery, K. A.; Griffey, R. H. Anal. Chem. 1999, 71, 2067-2070. (21) Peiris, D. M.; Yang, Y.; Ramanathan, R.; Williams, K. R.; Watson, C. H.; Eyler, J. R. Int. J. Mass Spectrom. Ion Processes 1996, 157/158, 365-378. (22) Stafford, G. C. J.; Kelley, P. E.; Syka, J. E. P.; Reynolds, W. E.; Todd, J. F. J. Int. J. Mass Spectrom. Ion Processes 1984, 60, 85-98. (23) Goeringer, D. E.; McLuckey, S. A. J. Chem. Phys. 1996, 104, 2214-2221. (24) Colorado, A.; Shen, J. X.; Vartanian, V. H.; Brodbelt, J. Anal. Chem. 1996, 68, 4033-4043. (25) Boue´, S. M.; Stephenson, J. L.; Yost, R. A. Rapid Commun. Mass Spectrom. 2000, 14, 1391-1397. (26) Goolsby, B. J.; Brodbelt, J. S. J. Mass Spectrom. 1998, 33, 705-712. (27) Stephenson, J. L.; Booth, M. M.; Shalosky, J. A.; Eyler, J. R.; Yost, R. A. J. Am. Soc. Mass Spectrom. 1994, 5, 886-893.

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Using a heated bath gas to “assist” the dissociation process can alleviate the collisional cooling problem. It has been suggested that the internal temperature of ions in the QITMS is the same as that of the bath gas.23 Heated bath gas (g180 °C) can assist dissociation using CID and can alone cause dissociation in some cases.28 Using a heated bath gas, the ions should not transfer as much internal energy to the bath gas after gaining energy from the IR irradiation as they would to cooler gas atoms. The combination of a heated bath gas with IRMPD is shown here to produce significant dissociation at higher pressure (1 × 10-3 Torr) than previously observed. A characteristic of IRMPD is that, unlike CID, it is not selective. Any ion in the path of the laser that absorbs the radiation may be dissociated. Not only are parent ions dissociated but the product ions will also continue to absorb photons and can dissociate further. While the product ion spectrum can be richer, genealogical information is lost. However, the work presented here shows that some control can be exercised over which ions are irradiated in the QITMS. The motions of selected ions can be increased via resonance. The effect is that these ions spend less time in the center of the trap and less time in the path of the laser. This technique will be referred to as axial expansion, as ion motion is increased in the axial direction. Results are presented to demonstrate the selectivity of this method. EXPERIMENTAL SECTION All experiments were performed on a modified Finnigan ITMS controlled with ICMS software.29 Peptide ions are generated via a custom-built nanoelectrospray source. Peptides were obtained from Sigma Chemical Co. and used without purification. Peptides are prepared as 10-100 µM solutions in 75:20:5 CH3OH/H2O/ CH3COOH. Base pressure in the QITMS is 2.5 × 10-5 Torr. Helium buffer gas of is added via a leak valve to give a constant pressure during the experiment. Pressures range from 2.5 × 10-5 to 1.75 × 10-3 Torr. CID is achieved using resonant excitation as described previously.30 A qz value of 0.2 is used for all CID experiments resulting in a m/z cutoff of 122 for [YGGFL + H]+ and 127 for [YGGFL + Na]+. A Synrad 50-W IR laser is used for IRMPD. A ZnSe window allows the laser beam to enter the vacuum housing. The ring electrode has been modified by drilling a 0.125-in. hole through the center, perpendicular to the axial direction. The laser beam is directed through this hole and passes through the center of the trapping volume in the radial direction. The laser is triggered by a TTL pulse from the ITMS electronics. Irradiation times vary from 30 to 700 ms, and the power ranges from 8 to 50 W. Unless otherwise indicated, the laser output is set to 100% (50 W), and the irradiation time is adjusted for optimal efficiency, as resonance excitation voltage would be for CID, typically 50-150 ms. A Lesker 1000-W stab-in bakeout heater inserted near the trapping electrodes is used to heat the bath gas and electrodes. The temperature is measured by a platinum resistance thermometer located ∼1 in. away from the trapping electrodes. The ambient temperature of 25-30 °C is raised to 160-170 °C for thermally assisted IRMPD. For the experiments described herein, the low-m/z cutoff (28) Asano, K. G.; Goeringer, D. E.; McLuckey, S. A. Int. J. Mass Spectrom. 1999, 185/186/187, 207-219. (29) Yates, N.; Yost, R. Unpublished, University of Florida, Gainesville, FL, 1992. (30) Asam, M. R.; Glish, G. L. J. Am. Soc. Mass Spectrom. 1997, 8, 987-995.

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Figure 1. TA-IRMPD, irradiated 100 ms at qz ) 0.065 (a) compared with CID at qz ) 0.2 (b) of [YGGFL + H]+.

of 40 used during ion accumulation was also used during IRMPD, and qz values fell at 0.065 for [YGGFL + H]+ and 0.062 for [YGGFL + Na]+. These qz values are relevant only insofar as the low-mass cutoff they imply for observation of the product ions and do not affect the efficiency of the IRMPD. Stored waveform inverse Fourier transform (SWIFT) is implemented using LabVIEW (Version 4.0.1, National Instruments, Austin, TX) to synthesize the time-domain waveform, and an arbitrary waveform generator (Sony/Tektronix, Beaverton, OR, model AWG2020, partially upgraded to AWG2021 functionality) is used to apply the waveform to the end-cap electrodes, as described previously.31 RESULTS AND DISCUSSION IRMPD versus CID. As was observed previously, IRMPD product ion spectra resemble those resulting from CID, although the former often have more extensive dissociation (Figure 1). CID of [YGGFL + H]+ has been shown to follow a complex dissociation pathway involving a cyclic rearrangement.32 The C-terminal residue from [an - NH3]+ ion is transferred to the N-terminus in this rearrangement. These ions are denoted here with the oneletter abbreviations of the residues making up the product ion. The new linkage from the transferred residue to the N-terminus is marked with an asterisk (*).33 All other ions are labeled according to conventional nomenclature.34 The same product ions, (31) Asam, M. R.; Ray, K. L.; Glish, G. L. Anal. Chem. 1998, 70, 1831-1837. (32) Vachet, R. W.; Bishop, B. M.; Erickson, B. W.; Glish, G. L. J. Am. Chem. Soc. 1997, 119, 5481-5488. (33) Asam, M. R.; Glish, G. L. J. Am. Soc. Mass Spectrom. 1999, 10, 119-125. (34) Roepstorff, P. Biomed. Mass Spectrom. 1984, 11, 601.

including the rearrangement ions, are seen in both the CID and IRMPD spectra. This implies that regardless of whether internal energy is added through collisions or by photons, it is quickly redistributed through the ion. However, there are some important differences. First, very low m/z product ions are seen in the IRMPD spectrum. These product ions are absent in the CID spectrum. This is in part due to raising of the low-m/z cutoff used during CID to put the ion in a sufficiently deep pseudopotential well. While the low-m/z cutoff was set to 122 for CID (qz ) 0.2), the cutoff was left at 40 (qz ) 0.065) for IRMPD. Because a lower m/z cutoff may be used with IRMPD, the low-m/z products are seen in the IRMPD spectrum. Additionally, many of these lowmass ions probably arise from consecutive dissociation pathways that are accessed via excitation of the first- or second-generation product ions. Such excitation does not occur in CID, as discussed below. Another difference in these two spectra is the relative intensities among the product ions. The lower m/z product ions are more abundant in the IRMPD spectrum than in the CID spectrum. This is due to the continued excitation of first-generation product ions by the IR irradiation. With CID, a voltage at a frequency matching the ion motions is used to resonantly excite and dissociate the parent ion. When a product ion is formed, it has an m/z different from that of the parent and a different frequency of motion. Therefore, the product ions are no longer excited by the resonant frequency, which is tuned to the parent ion. IRMPD is not a selective method of dissociation. Any ion within the path of the laser beam may absorb photons. A product ion will be further dissociated if the irradiation is continued. At short irradiation times, the product ion intensity is mostly limited to higher m/z, first-generation product ions. As the irradiation time is increased, these ions are further dissociated, and the intensity shifts to the lower m/z product ions. The effect of this nonselective excitation is evident in Figure 2, where ion intensity as a function of excitation time is shown for both CID and IRMPD. Sodium-cationized YGGFL is used to illustrate this due to its simple, consecutive dissociation pattern.35 The first-generation products upon MS/MS are dominated by the [b4 + Na + OH]+ ion. The main product upon MS3 of the [b4 + Na + OH]+ ion is the [b3 + Na + OH]+ ion. As CID times are increased (Figure 2a), the parent ion intensity decreases while the products increase. The products are dominated by the [b4 + Na + OH]+ ion, and subsequent dissociation is low. The product ion intensities level off when the parent ion population is completely dissociated. With IRMPD (Figure 2b), the parent ion intensity decreases as in CID. However, the product ion intensities also begin to decrease with increasing irradiation time as the firstgeneration products dissociate further. The [b4 + Na + OH]+ ion dominates the IRMPD spectrum at shorter irradiation times (25 ms) but has a decreased intensity as it is dissociated to the next generation product ion, the [b3 + Na + OH]+ ion, at longer irradiations (125 ms). IRMPD can be advantageous in that more product ions can be formed, which provides more structural information. However, these product ion dissociations can also complicate interpretation in that genealogical information is lost. (35) Lin, T.; Payne, A. H.; Glish, G. L. J. Am. Soc. Mass Spectrom. 2001, 12, 497-504.

Figure 2. Products of CID (a) and TA-IRMPD (b) of [YGGFL+Na]+ with varying excitation times. (a) CID product ion intensities do not decrease with time as only the parent ion is subject to excitation. (b) IRMPD product ions increase and then decrease with time as further dissociation occurs and smaller product ions appear. The close circle (b) represents [M + Na]+, the closed diamond ([) the [b4 + Na + OH]+ ion, the open square (0) the [a4 + Na - H]+ ion, and the open circle (O) the [b3 + Na + OH]+ ion.

To maintain this information, selective dissociation can be achieved using axial expansion, which will be discussed later. Effect of Pressure on IRMPD Efficiencies. The primary obstacle for using IRMPD in the quadrupole ion trap is the lack of efficient dissociation at normal operating pressures. At room temperature, the MS/MS efficiency (∑[product ion intensity]/ ∑[initial parent ion intensity]) drops off rapidly as the pressure is increased (Figure 3). Between 2 × 10-4 and 4 × 10-4 Torr, the efficiency decreases precipitously, and at 1 × 10-3 Torr, the normal operating pressure, the efficiency is almost zero. Decreasing IRMPD efficiency with increasing pressure has been noted by others.24,25 This is due to the increased number of collisions an ion will undergo at increased pressures during the time between IR photon absorptions. At ambient temperatures, the helium atoms of the bath gas are low energy relative to an IR-excited ion. As the IR-excited (“warm”) ion collides with the “cool” helium atoms, it is thermodynamically favorable for energy to be transferred away from the ion. The more collisions the ion undergoes, the more energy is lost. If an ion loses the internal energy gained from the IR photon before it absorbs another photon, the ion will not gain enough energy to dissociate. This occurs as the normal operating pressure is approached. Analytical Chemistry, Vol. 73, No. 15, August 1, 2001

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Figure 3. IRMPD efficiency of [YGGFL + H]+. Efficiency drops sharply with increasing pressure at ambient temperature (open circle, O) and is near zero at 1 × 10-3 Torr (standard operating pressure marked with the vertical dashed line). TA-IRMPD efficiency at 160 °C (closed circle, b) decreases much less rapidly and is still high (80%) at 1 × 10-3 Torr.

However, by heating the bath gas, the collisional energy loss can be mitigated. Two effects may contribute to this. First, as the trapping environment is heated, the ions gain thermal energy. It has been shown that thermal excitation alone can result in dissociation in some cases.28 The temperatures and trapping times used here (160 °C and