Anal. Chem. 2006, 78, 4609-4614
Thermally Assisted Collision-Induced Dissociation in a Quadrupole Ion Trap Mass Spectrometer Alawee H. Racine, Anne H. Payne, Philip M. Remes, and Gary L. Glish*
Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599
Thermally assisted collision-induced dissociation (TACID) provides increased dissociation in comparison with CID performed at ambient temperature in a quadrupole ion trap mass spectrometer. Heating the bath/collision gas during CID increases the initial internal energy of the ions and reduces the collisional cooling rate. Thus, using the same CID parameters, the parent ion can be activated to higher levels of internal energy, increasing the efficiency of dissociation and the number of dissociation pathways. The increase in the number of dissociation pathways can provide additional structural information. A consequence of the increase in initial internal energy is the ability to use less power to effect collisional activation. This allows lower qz values to be used and, thus, a greater mass range of product ions to be observed. TA-CID alleviates the problems associated with traditional CID and results in more available information than traditional CID. Quadrupole ion trap mass spectrometers have become increasingly common among today’s mass spectrometers. Ion traps are tandem in-time instruments with many advantages, including the capability of multiple stages of MS (MSn) in a single analyzer, high sensitivity, and ruggedness. Although there are currently several ways to effect dissociation in an ion trap, traditionally, MSn has been achieved with collision-induced dissociation, (CID).1,2-4 Performing CID in a quadrupole ion trap mass spectrometer (QITMS) requires the ions to be trapped in the instrument before manipulation. The stability of an ion in the trapping volume is dependent on several variables, including the dimensions of the trap, the mass-to-charge ratio of the ion, and the ac and dc voltages applied to the electrodes. An ion’s motion can be described by a second-order differential equation. The solution to this equation can be used to determine the ac and dc voltages, resulting in a stable ion trajectory in a QITMS.5 Typically, the QITMS is * To whom correspondence should be addressed. Department of Chemistry, CB #3290 Venable Hall, University of North Carolina, Chapel Hill, NC 27514. E-mail:
[email protected]. (1) Cody, R. B.; Burnier, R. C.; Cassady, C. J.; Freiser, B. S. Anal. Chem. 1982, 54, 2225-2228. (2) Louris, J. N.; Brodbelt-Lustig, J. S.; Cooks, R. G.; Glish, G. L.; VanBerkel, G. J.; McLuckey, S. A. Int. J. Mass Spectrom. Ion Processes 1990, 96, 117137. (3) McLuckey, S. A.; Glish, G. L.; VanBerkel, G. J. Int. J. Mass Spectrom. Ion Processes 1991, 106, 213-235. (4) Glish, G. L. Analyst 1994, 119, 533-537. (5) 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, 1995; Vol. I, pp 25-48. 10.1021/ac060082v CCC: $33.50 Published on Web 05/16/2006
© 2006 American Chemical Society
operated with an ac voltage applied to the ring electrode to trap ions and no dc voltage. In this case, the parameter of interest is the qz parameter defined in eq 1.
qz ) -2qr )
8eV m(r0 + 2z02)Ω2 2
(1)
An ion’s qz value is an important parameter in describing the manipulation of an ion. In eq 1, V represents the amplitude of the ac voltage applied to the ring electrode and is the most common parameter used to manipulate ions in a quadrupole ion trap. This ac voltage is typically in the radio frequency range and, thus, is called the rf voltage. The mass of the ion is represented by m (kg), and the charge on the ion is represented by e. Ω represents the angular frequency of the rf voltage. Typically, the r0, z0, and Ω values are fixed, and the magnitude of the ac rf voltage is used to control the stability of an ion’s trajectory. For any combination of these values that result in a qz < 0.908, an ion has a stable trajectory and is trapped in the ion trap. Because the qz value is inversely proportional to mass-to-charge ratio, all ions above the mass-to-charge ratio at qz ) 0.908 are trapped. This lowest massto-charge ratio trapped is commonly referred to as the low mass cutoff (LMCO). The low mass cutoff can be changed by changing the amplitude of the rf voltage. Changing the rf voltage changes the LMCO and the qz values of all the ions. Manipulation of ions, including the trapping of ions, and the general performance of an ion trap mass spectrometer are improved with the addition of bath gas to the trapping volume. As ions experience low-energy collisions with the bath gas, some of the ions’ kinetic energy is transferred to internal energy, and some is transferred to the neutral bath gas. Collisional damping or cooling of the ion’s kinetic energy causes a smaller trajectory for the ion and results in damping of the ion’s motion to the center of the trapping volume. Ion trap pressures of ∼1 mTorr are commonly used to improve resolution, sensitivity, detection limits, and ion trapping efficiency.5-8 Trapped ions have a stable periodic motion termed the secular frequency. An ion’s kinetic energy in the z dimension of the ion trap can be increased by applying a supplementary ac voltage to the endcap electrodes with the same frequency as the ion’s secular (6) March, R. E.; Todd, J. F. J. Practical Aspects of Ion Trap Mass Spectrometry; CRC Press: New York, 1997. (7) 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. (8) Goeringer, D. E.; McLuckey, S. A. J. Chem. Phys. 1996, 104, 2214-2221.
Analytical Chemistry, Vol. 78, No. 13, July 1, 2006 4609
frequency.9 The gain in kinetic energy results in an increase in the ion’s motion in the axial direction. If the ion gains enough kinetic energy, its motion will exceed the bounds of the trap, and it will be ejected. Ejection of an ion by application of a supplementary resonance voltage is called resonance ejection. If the ion gains less kinetic energy than is required for ejection, the ion’s motion will increase in the axial direction but the ion will remain trapped. An ion that has gained kinetic energy from the supplementary voltage but remains trapped is said to undergo resonance excitation. Because the stability of the trajectory of the ion, that is, whether it is ejected or just excited, is dependent on the amount of kinetic energy the ion acquires, there is a competition between resonant ejection and resonant excitation. Both processes are useful in experiments with a QITMS. Resonance excitation is commonly used to effect CID in the QITMS.9-12 CID is accomplished by colliding ions with the collision gas, usually the bath gas, to convert the kinetic energy of the ion to internal energy. The process of dissociating ions with CID in a QITMS involves several steps. Typically, the ion is isolated by ejecting all other ions from the trapping volume. The ion of interest, the parent ion, is resonantly excited to increase its kinetic energy. The parent ion collides with the bath gas, converting some of the ion’s kinetic energy into internal energy.9,13 Multiple collisions occur between the ion and bath gas, each of which can result in an increase in the ion’s internal energy. Ion dissociation results in various bonds being broken, each of which requires a certain amount of energy to break. When an ion’s internal energy exceeds the critical energy of dissociation for one of the possible dissociation pathways, the ion dissociates into product ions. The efficiency of CID is a common way to describe the degree of dissociation. CID efficiency is defined as the sum of the product ion intensities divided by the total initial parent ion intensity. CID efficiency is also the product of collection efficiency and fragmentation efficiency. Collection efficiency is defined as the sum of the parent ion and product ion intensities after dissociation divided by the total initial parent ion intensity. Collection efficiency should decrease at higher qz values because of ion loss caused by the low mass cutoff. Fragmentation efficiency is the sum of the product ions divided by the sum of the product ions and the parent ion in the CID spectrum. Fragmentation efficiency should decrease at lower qz values because less activation occurs. Therefore, at lower qz values, CID efficiency suffers because of the low fragmentation efficiency; however, at higher qz values, CID efficiency suffers because of the poor collection efficiency. Hence, a compromise is made, and CID is typically performed at a qz of 0.25. At a qz value of 0.25, the low mass cutoff results in product ions with a mass-to-charge ratio 0.15. For WHWLQL at a qz value of 0.25, the CID efficiency at ambient temperature was 55.4%; however, CID efficiency increased to 74.7 and 93.7% at 100 and 160 °C, respectively. Overall, CID efficiency was greater at 100 and 160 °C than at ambient temperature for the peptides studied at all three qz values investigated. Additional Trends Observed. The formation of internal product ions is enhanced at elevated temperature. In many, but not all, cases, more internal product ions were observed at elevated temperature than at ambient temperature. WHWLQL illustrated a pattern similar to the ALILTLVS qz 0.15 data discussed earlier. At qz ) 0.35, one internal fragment was identified at ambient temperature for WHWLQL, but two and three internal fragments were observed at 100 and 160 °C, respectively. The increase in internal product ions indicates an increase in consecutive dissociation. The increase in internal energy of the parent ion at elevated temperature increases the probability that the parent ion will have enough internal energy to form a product ion that will dissociate further. Because TA-CID increases the internal energy of an ion, the predominate dissociation pathway observed at elevated temperature may differ from that observed at ambient temperature. Experiments are currently being conducted to determine if the predominant dissociation pathway observed differs at elevated temperature, as compared to ambient temperature.29 Internal Energy Calculations. The increase in internal temperature, ∆E, resulting from raising the bath gas temperature is found by simply taking the difference in thermal energies at elevated and ambient temperatures. These energies were calculated using eqs 3 and 4 as described above and are shown in Table 3. The values in Table 3 can be viewed as the amount of internal
(29) Jue, A.; Racine, A. H.; Glish, G. L. Nashville, TN, May 2004.
(30) Turecek, F. Org. Mass Spectrom. 1991, 26, 1074-1081.
Figure 2. CID spectra of ALILTLVS at a qz ) 0.15 and various temperatures. The spectra show that an increase in temperature results in an increase in dissociation: (A) ambient temperature (B) 100 °C, and (C) 160 °C.
Analytical Chemistry, Vol. 78, No. 13, July 1, 2006
4613
Table 2. Ratio of the Number of Residues from Which Product Ions Were Observed to Total the Total Number of Residues qz ) 0.15
qz ) 0.25
qz ) 0.35
peptide
30 °C
100 °C
160 °C
30 °C
100 °C
160 °C
30 °C
100 °C
160 °C
ALILTLVS FLLVPLG WHWLQL LLFGYPVYV YGGFLR YGGFMK
2/8 4/7 4/6 5/9 2/6 2/6
5/8 7/7 6/6 9/9 6/6 3/6
8/8 5/7 6/6 9/9 6/6 6/6
3/8 5/7 4/6 6/9 3/6 3/6
2/8 7/7 6/6 4/9 6/6 6/6
8/8 7/7 6/6 9/9 6/6 4/6
2/8 4/7 6/6 3/9 4/6 2/6
6/8 7/7 6/6 7/9 4/6 3/6
8/8 4/7 6/6 2/9 6/6 3/6
Table 3. Increase in Parent Ion Internal Energy versus Ambient Temperature Operation
ature, which means more parent ions are activated and dissociated before they can be ejected.15
∆E (eV) peptide
100 °C
160 °C
ALILTLVS FLLVPLG WHWLQL LLFGYPVYV YGGFLR YGGFMK
1.08 1.16 1.03 1.46 0.87 0.83
1.93 2.01 1.86 2.52 1.56 1.49
energy that does not need to be added by collisional activation at elevated temperatures versus ambient temperature. Although dissociation energetics are not known for the peptides used here, BIRD and thermal dissociation experiments measuring Arrhenius activation parameters for leucine enkephalin and bradykinin determine Ea values in the 1.0-1.3 eV range.16,31-33 Previous results from our lab indicate that the critical energy for dissociation does not change for a given product ion type from peptide to peptide, but that the total internal energy increases with peptide size (degrees of freedom), as would be expected.34 So although the added energy from the heated bath gases is sufficient to cause dissociation if one waits long enough, more internal energy needs to be added to the parent ion to cause it to dissociate in the 25ms time window of typical quadrupole ion trap experiments. At elevated temperatures, the amount of internal energy needed from collisions to induce dissociation is less than at ambient temper(31) Asano, K. G.; Goeringer, D. E.; McLuckey, S. A. Int. J. Mass Spectrom. 1999, 185/186/187, 207-219. (32) Schnier, P. D.; Price, W. D.; Jockusch, R. A.; Williams, E. R. J. Am. Chem. Soc. 1996, 118, 7178-7189. (33) Schnier, P. D.; Price, W. D.; Strittmatter, E. F.; Williams, E. R. J. Am. Soc. Mass Spectrom. 1997, 8, 771-780. (34) Vachet, R. W.; Winders, A. D.; Glish, G. L. Anal. Chem. 1996, 68, 522526.
4614 Analytical Chemistry, Vol. 78, No. 13, July 1, 2006
CONCLUSIONS The effectiveness of collision-induced dissociation in a quadrupole ion trap has traditionally been limited by the low mass cutoff and the competition between ion ejection and ion activation. However, the implementation of thermally assisted collisioninduced dissociation can effectively reduce the traditional limitations associated with CID. By heating the bath gas and electrodes and, thus, increasing the internal energy of the ions, it is possible to access dissociation pathways that are inaccessible at ambient temperature. TA-CID allows CID to be implemented effectively at qz values lower than 0.25, which alleviates problems related to the low mass cutoff. TA-CID increases the internal energy of the parent ion prior to the excitation and dissociation event, which in turn results in more product ions being formed at lower qz values. An ion with greater initial internal energy does not need as much collisional activation to dissociate. Therefore, the ions are dissociated before their motion becomes unstable and they are ejected from the quadrupole ion trap. More information is obtained at elevated temperature, as compared with ambient temperature, because of the increase in the dissociation efficiency and retention of lowmass ions at the low qz. TA-CID results in an increase in the number of product ions formed from protonated peptides. More backbone bond cleavages are seen at elevated temperatures than at ambient temperature. An increase in structurally relevant dissociation products results in an increase in information.
Received for review January 12, 2006. Accepted March 31, 2006. AC060082V
