Anal. Chem. 2001, 73, 1270-1276
Tandem Infrared Multiphoton Dissociation and Collisionally Activated Dissociation Techniques in a Quadrupole Ion Trap Brian J. Goolsby† and Jennifer S. Brodbelt*,‡
Motorola SPS, Inc. Digital DNA Laboratories, 3501 Ed Bluestein Boulevard, Austin, Texas 7872, and Department of Chemistry and Biochemistry, The University of Texas at Austin, Austin, Texas 78712
Tandem infrared multiphoton dissociation and collisionally activated dissociation methods are implemented in a quadrupole ion trap mass spectrometer and used to characterize an array of antibiotic ions generated by electrospray ionization. The tandem methods prove useful for probing fragmentation genealogies, evaluating the structures of lower mass fragment ions produced from higher mass molecular ions, and differentiating isobaric ions. The infrared multiphoton dissociation method is more efficient for producing an array of fragment ions over a large mass range, whereas collisionally activated dissociation is preferable for the analysis of lower m/z ions. Photodissociation (PD), particularly with an infrared laser, has been shown to be a viable alternative to collisionally activated dissociation (CAD) for probing molecular structure,1-13 such as those of biological molecules. For example, infrared multiphoton dissociation (IRMPD) has been implemented in FTICR mass spectrometers to characterize the structures of proteins,7,8 DNA,6 saccharomicin antibiotics,6 and phosphorothioate oligonucleotides * Corresponding author: (phone) (512) 471-0028; (fax) (512) 471-8696; (e-mail)
[email protected]. † Motorola SPS, Inc. Digital DNA Laboratories. ‡ The University of Texas at Austin. (1) Vartanian, V. H.; Goolsby, B.; Brodbelt, J. S. J. Am. Soc. Mass Spectrom. 1998, 9. (2) Tonner, D. S.; McMahon, T. B. Anal. Chem. 1997, 69, 4735-4740. (3) Stephenson, J. L., Jr.; Booth, M. M.; Boue, S. M.; Eyler, J. R.; Yost, R. A. In Biochemical and biotechnological applications of electrospray ionization mass spectrometry; Snyder, A. P., Ed.; American Chemical Society: Washington, D.C, 1996; p 601. (4) Shi, S. D.-H.; Hendrickson, C. L.; Marshall, A. G.; Siegel, M. M.; Kong, F.; Carter, G. T. J. Am. Soc. Mass Spectrom. 1999, 10, 1285-1290. (5) Nuwaysir, L. M.; Castoro, J. A.; Wilkins, C. L. Org. Mass Spectrom. 1991, 26, 721-722. (6) Little, D. P.; Aaserud, D. J.; Valaskovic, G. A.; McLafferty, F. W. J. Am. Chem. Soc. 1996, 118, 9352-9359. (7) Little, D. P.; Speir, J. P.; Senko, M. W.; O’Connor, P. B.; McLafferty, F. W. Anal. Chem. 1994, 66, 2809-2815. (8) Li, W.; Hendrickson, C. L.; Emmett, M. R.; Marshall, A. G. Anal. Chem. 1999, 71, 4397-4402. (9) Hofstadler, S. A.; Sannes-Lowery, K. A.; Griffey, R. H. Anal. Chem. 1999, 71, 2067-2070. (10) Goolsby, B. J.; Brodbelt, J. S. J. Mass Spectrom. 2000, 35, 1011-1024. (11) Colorado, A.; Shen, J. X.; Vartanian, V. H.; Brodbelt, J. Anal. Chem. 1996, 68, 4033-4043. (12) Goolsby, B. J.; Brodbelt, J. S. J. Mass Spectrom. 1998, 33, 705-712. (13) Castoro, J. A.; Nuwaysir, L. M.; Ijames, C. F.; Wilkins, C. L. Anal. Chem. 1992, 64, 2238-2243.
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in an external ion reservoir coupled to an FTICR mass spectrometer.9 IRMPD has also been a versatile alternative to CAD in quadrupole ion trap applications, such as for the characterization of oligonucleotides,3 penicillin and cephalosporin antibiotics,12 tetracycline antibiotics,1 erythomycin,11 and aminoglycoside antibiotics.10 In most of the cases mentioned above, IRMPD proves to be equally or more efficient relative to CAD. This advantage results from the fact that IRMPD does not appreciably alter the kinetic energies or stable orbits of the trapped ions. Collisional techniques, however, often lead to ion scattering during energetic collisions intended to excite them internally. Furthermore, IRMPD is effective regardless of trapping potentials, such as the rf voltage applied to the ring electrode of a quadrupole ion trap. The rf voltage influences the energetics of activation and defines the range of mass-to-charge ratios of ions that can be trapped. Thus, for IRMPD, the low-mass cutoff can be maintained sufficiently low that fragments over a wide mass range can be stored and detected. Because infrared photoabsorption is universal if an appropriate wavelength is selected, it may be applied to any trapped ion without requiring resonant tuning. The possibility of performing MSn by extending the irradiation period has been shown to increase the degree of fragmentation and thus can be used to map genealogies of dissociation, albeit without great specificity. However, the universal absorption feature of IR radiation also means that some of the fragment ions may not be readily observable because of ongoing IR absorption and subsequent dissociation prior to detection. The selectivity of fragmentation can be enhanced by implementation of tandem methods, including IRMPD/IRMPD, IRMPD/CAD, and CAD/IRMPD. Each of these tandem combinations offers advantages for certain applications, especially in comparison to traditional CAD/CAD methods, as illustrated in this report. IRMPD/IRMPD methods have been reported by two groups using FTICR mass spectrometry. Wilkins et al. used an excimer laser for UV/visible PD/PD experiments of porphyrins.5,13 Very high sequential dissociation efficiencies were noted when the analyte possessed a suitable chromophore. IRMPD/IRMPD was reported by Tonner and McMahon2 for the proton-bound dimer of diethyl ether, and again the high efficiency of IRMPD was noted as an advantage when mapping fragmentation genealogies in which losses of ions at each stage of activation could be minimized relative to the losses incurred upon collisional activation. 10.1021/ac001161o CCC: $20.00
© 2001 American Chemical Society Published on Web 02/14/2001
In the present study, tandem IRMPD/CAD methods are implemented in a quadrupole ion trap mass spectrometer and used to characterize an array of antibiotics generated by electrospray ionization. The structures of lower mass primary fragments produced from larger mass ions are probed, fragmentation genealogies are evaluated, and the identities of primary fragment ions possessing the same mass-to-charge ratios from different precursors are investigated with the aim of providing class-specific dissociation information. EXPERIMENTAL SECTION An in-house-built quadrupole ion trap system that utilized modified Finnigan ITD electronics and software with an ESI source and interface modeled after the Oak Ridge National Laboratory design was used for all experiments.14 A SWIFT system controlled by TTL triggers in the scan function was used for resonant ejection and excitation (CAD) purposes and was described in detail previously.11 Photodissociation experiments were performed with a model 575 Apollo CW CO2 laser used in conjunction with a Uniblitz shutter. The center of the trap was irradiated through a ZnSe window in the vacuum chamber aligned with a 6-mm hole drilled radially in the ring electrode.11 The unfocused laser beam creates a 6-mm-diameter beam path through the ion trap, meaning that the ions probably remain nearly continuously in the path of the laser beam. Solutions (5 × 10-4 M) of each compound were made in methanol. Electrospray of the solutions was typically undertaken at a needle voltage of 4.5 kV and a flow rate of 3 µL/min. The chamber pressure was typically 0.1 mTorr (uncorrected), and addition of a bath gas to aid collision activation for dissociation was unnecessary. CAD and IRMPD were achieved under similar experimental conditions, with the precursor ion isolated before activation. SWIFT CAD waveforms were applied to the end-cap electrodes and ranged from 1.5 to 5 Vp-p. The IRMPD laser irradiation period was varied (10-200 ms) as needed to achieve MS/MS fragmentation at a flux of 30 W/cm2 unless otherwise noted. RESULTS AND DISCUSSION Several variations of the tandem IRMPD/CAD experiments were implemented, including IRMPD/IRMPD, IRMPD/CAD, and CAD/IRMPD, and in some cases compared to CAD/CAD results. Compounds of interest were selected from classes of antibiotics: aminoglycosides, tetracyclines, cephalosporins, and erythromycins (Figure 1). These classes have an array of complex structures, and thus, the versatility of the tandem IRMPD/CAD modes for solving specific structural problems is highlighted in the following sections. Photodissociation Complemented by Collisional Activation. Photodissociation as the first step of a tandem experiment offers a highly efficient way to produce a broad array of fragment ions. CAD as the second step allows m/z-specific activation, a feature that is not always possible with the nonspecific photoactivation process. For example, infrared multiphoton dissociation of protonated penicillin has been reported previously as a viable alternative to CAD.12 In Figure 2, the two techniques are combined (14) Van Berkel, G. J.; Glish, G. L.; McLuckey, S. A. Anal. Chem. 1990, 62, 1284-1295.
Scheme 1. Proposed Fragmentation of Protonated Penicillin
Scheme 2. Proposed Breakdown of the Cephalosporin Core
to elucidate the genealogies of two primary fragments. The initial IRMPD step produces a fragment at 160+ (Figure 2A), which subsequent CAD converts to m/z 114 (Figure 2B). IRMPD of (penicillin G + H)+ also produces the fragment ion at 114+, which upon isolation and CAD predominantly dissociates by loss of 27 u, likely as HCN. This sequence of fragmentation is proposed in Scheme 1, as supported by the spectra in Figure 2. Tandem IRMPD/CAD is also useful for comparing the fragmentation genealogies of isobaric fragment ions formed by a class of compounds. For example, cefadroxil, cephalexin, and cephradine are three cephalosporins that have an identical β-lactam core (see Figure 1). The formation of a fragment ion at m/z 158 is observed upon IRMPD of each of these protonated cephalosporins (Figure 3A), and this is the only fragment that is common to all three. Thus, the fragment at m/z 158 is a potential diagnostic ion in a selected reaction monitoring strategy aimed at identifying cephalosporins. The CAD spectra of the m/z 158 ions are shown in Figure 3B, and the three fragmentation patterns are nearly identical, thus confirming the core β-lactam structure of the cephalosporins. Scheme 2 illustrates the proposed assignments for the secondary fragments at m/z 67 and 140. The tandem IRMPD technique is especially useful for probing the structures of low-mass fragment ions, ones that are not efficiently formed by CAD due to the trapping conditions imposed by using rf levels that are low enough to store ions of low m/z Analytical Chemistry, Vol. 73, No. 6, March 15, 2001
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Figure 1. Compounds studied by tandem dissociation methods.
while performing collisional activation of much higher mass precursors (a significant problem only in quadrupole ion traps, not FTICR traps). An excellent example is provided by the fragmentation genealogy of protonated members of the tetracycline class (including tetracycline and its analogues doxycycline, minocycline, chlortetracycline, demeclocycline, methacycline, oxytetracycline, and anhydrotetracycline). These compounds uniformly produce a key structure-specific fragment ion at m/z 98 upon IRMPD that is not apparent by CAD at optimal storage potentials (i.e., q ) 0.3), below which storage becomes inefficient during the CAD experiment.1 Elucidation of the m/z 98 fragment could provide important evidence to identify members of this class of compounds. Two possible structures for this fragment have been proposed,1 as shown in Scheme 3. CAD of this fragment ion at m/z 98, after isolation from the IRMPD spectrum of protonated tetracycline shown in Figure 4A, results in the spectrum shown in Figure 4C. The losses of water, 28 u, and 30 u are consistent with the proposed structure 1 in Scheme 3, and the specificity of this IRMPD/CAD fragmentation pattern would be a suitable way for identifying the class of tetracyclines. For comparison to the IRMPD/CAD method, the same experiment was carried out using two discrete CAD steps. Figure 5 shows that CAD/CAD produces a similar dissociation spectrum but suffers from a poor signal-to-noise ratio due to the 1272
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Scheme 3. Possible Fragments Formed by Tetracycline Dissociation
lower intensity of 98+ produced or trapped when collisional activation is used to dissociate protonated tetracycline. Another example illustrating the utility of the IRMPD/CAD method involves the characterization of macrolide antibiotics such as erythromycin. Protonated erythromycin dissociates predominantly to form an ion at m/z 158 (Figure 6A), a fragment that is characteristic of the macrolide class and is also observed upon dissociation of protonated oleandomycin and related analogues.15 (15) Goolsby, B. J.; Brodbelt, J. S., manuscript in preparation.
Figure 2. (A) IRMPD of (penicillin G + H)+ followed by CAD of fragment ions: (B) m/z 160 and (C) m/z 114.
This key fragment ion is not observed upon CAD of protonated erythromycin due to the inefficient CAD caused by trapping a mass range wide enough to observe both the m/z 734 precursor ion and the diagnostic fragment at m/z 158. Thus, CAD/CAD experiments are not viable for probing the fragmentation pattern of m/z 158. However, collisional activation is successfully used to dissociate the ion at m/z 158 after its formation by IRMPD and subsequent isolation (Figure 6B and C). The fragmentation pattern obtained for m/z 158 is characteristic of the desosamine sugar, thus providing an effective way to decipher this fragment ion and identify the type of macrolide. The tandem IRMPD/CAD method is also a convenient way to compare the fragmentation patterns of primary fragment ions with identical m/z values that are produced from singly versus doubly charged precursor ions. A relevant example emerged in a recent study of the IRMPD of protonated aminoglycosides.10 Many of the protonated and doubly protonated aminoglycosides produced fragment ions with the same m/z values, suggesting that the fragment ions could have similar structures and indicating that the doubly protonated precursors underwent a charge loss process during dissociation. Examples of the IRMPD spectra of protonated and doubly protonated tobramycin are shown in Figure 7A and C, respectively, with each having a dominant fragment ion at m/z 324. This fragment ion was proposed to result from the loss of one of the terminal sugars (designated the C sugar in Figure 1) from protonated tobramycin or the same terminal (protonated C) sugar from the doubly protonated precursor. The tandem IRMPD/CAD experiment allows interrogation of the ion at 324, as shown in Figure 7B and D. The resulting fragmentation patterns are closely matched, giving ions at m/z 163 and 205, assigned as the B-ring and the B-ring plus the sCHdCHOH portion of the A-ring, respectively. This specific dissociation
Figure 3. (A) IRMPD of protonated cefadroxil, cephalexin, and cephradine illustrating the common formation of m/z 158. (B) CAD of the fragment at m/z 158 formed by the IRMPD of protonated cefadroxil, cephalexin, and cephradine.
sequence indicates that the singly and doubly protonated molecules produce identical fragment ions. Furthermore, it is significant to note that the tandem dissociation promotes cross-ring cleavage, which is more desirable than simple breakage of the glycosidic linkages for compound identification. Collisional Activation Followed by Photodissociation. Reversing the order of the dissociation steps described above to accomplish CAD/IRMPD provides another means of probing ion structure. CAD can be tuned to a specific m/z to produce a fragmentation spectrum, and IRMPD can be applied to a particular fragment to produce secondary fragments, which can be selectively formed by varying the irradiation time. Figure 8 illustrates Analytical Chemistry, Vol. 73, No. 6, March 15, 2001
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Figure 4. (A) IRMPD of protonated tetracycline to produce a fragment at m/z 98, followed by (B) isolation of m/z 98, and (C) CAD of m/z 98.
Figure 6. (A) IRMPD of protonated erythromycin A, followed by (B) isolation of m/z 158 and (C) CAD of m/z 158.
Figure 5. CAD/CAD of protonated tetracycline: (A) isolation of m/z 98 formed by CAD of protonated tetracycline and (B) CAD of m/z 98.
an example of the CAD/IRMPD technique for protonated erythromycin A. The protonated precursor is dissociated collisionally and a primary fragment, m/z 576, is isolated. The fragment is then irradiated for 5 and 30 ms in Figure 8C and D, respectively. In this manner, the ratio of secondary fragments at m/z 158 and 113 can be controlled, i.e., a type of energy-resolved experiment. Tandem IRMPD for Elucidating Fragment Genealogies. It has been noted in reports of IRMPD that the potential for inadvertent MSn at extended irradiation times, while sometimes beneficial, can lead to fragments of unknown origin.12 For example, a low-mass fragment from a large molecular ion might have originated from the molecular ion or from a primary fragment undergoing further dissociation due to the efficient absorption of IR photons by most trapped ions. Due to the importance placed on the ability to track fragmentation pathways, techniques have developed to confirm the origins of the fragment ions. Double resonance is sometimes used to remove a primary fragment 1274 Analytical Chemistry, Vol. 73, No. 6, March 15, 2001
Figure 7. (A) IRMPD of protonated tobramycin, followed by (B) isolation of m/z 324 and CAD of m/z 324, and (C) IRMPD of doubly protonated tobramycin, followed by (D) isolation and CAD of m/z 324.
during IRMPD. If other fragments disappear as a result, it is likely that they originated from the primary fragment. Time-resolved IRMPD has also been used to determine genealogies. By plotting the intensities of spectral elements as a function of irradiation time, genealogical relationships can be deduced. IRMPD/IRMPD can be used in a manner that is similar to the double-resonance technique. Instead of ejecting a fragment during photodissociation as would be the case in a double-
Figure 8. (A) Isolation of protonated erythromycin A, (B) CAD of protonated erythromycin A, and isolation and IRMPD of fragment 576+ for (C) 5 and (D) 30 ms.
resonance experiment, a primary IRMPD fragment is isolated for further analysis. A subsequent IRMPD event reveals the fragments that are direct descendents of the primary fragment. An example of this is shown in Figure 9, in which a minor primary fragment, 616+, from protonated midecamycin is isolated and photodissociated. From spectrum D, it is clear that the 616+ fragment represents a pathway for the formation of the secondary fragment at 174+. Infrared multiphoton dissociation is not always the preferred activation method relative to collisional activation in tandem experiments. For the structural characterization of protonated cephradine, collisional activation efficiently dissociates the fragment at m/z 158 (Figure 3B) whereas IRMPD was ineffective at dissociating this ion. In fact, no fragments could be produced upon IRMPD of the ion at m/z 158. Likewise, an IRMPD/IRMPD experiment was attempted for the m/z 154 fragment ion derived from protonated tetracycline (Figure 10). Photodissociation of m/z 154 was largely ineffective and produced only a slight amount of secondary fragmentation at m/z 98 (Figure 10A) despite the large abundance of this ion formed upon IRMPD of protonated tetracycline, whereas the analogous IRMPD/CAD sequence was highly efficient (Figure 10B). The poor efficiency seen in these two IRMPD/IRMPD examples, both involving activation of relatively low-mass fragment ions, may be due in part to the larger oscillations made by low-mass ions in the ion trap, which can carry the ions outside the path of the IR laser, or due to more effective radiative cooling of the ions or lack of efficient IR absorption modes. CAD proved to be much more efficient at producing secondary fragmentation, demonstrating that CAD may be the better choice for activation of low-m/z ions.
Figure 9. IRMPD/IRMPD of midecamycin, showing (A) IRMPD of protonated midecamycin, (B) blowup of low-intensity fragments, (C) isolation of fragment 616+, and (D) IRMPD of 616+.
Figure 10. (A) IRMPD and (B) CAD of isolated m/z 154 formed by IRMPD of protonated tetracycline.
Comparison of Tandem Dissociation Techniques. Comparison of the various tandem combinations (IRMPD/IRMPD, IRMPD/CAD, CAD/IRMPD, CAD/CAD) shows that all four methods are feasible in many situations and should be considered as alternatives depending on the specific goals of the analysis. Figure 11 shows side-by-side comparisons of the four tandem methods applied to doubly protonated tobramycin. The precursor ion (4692+) was dissociated using either IRMPD or CAD, and the primary fragment 163+, corresponding to the protonated C ring, was then selectively dissociated using either a second stage of IRMPD or CAD. CAD of the molecular ion was slightly less efficient at producing 163+; thus, the subsequent IRMPD or CAD dissociation steps provided lower signal overall. CAD/CAD gave the lowest signal intensity of the four tandem alternatives, whereas Analytical Chemistry, Vol. 73, No. 6, March 15, 2001
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Figure 11. Tandem dissociation of doubly protonated tobramycin (4692+) (all intensity scales the same). (A) IRMPD of (M + 2H)2+ followed by IRMPD of primary fragment 163+. (B) IRMPD of (M + 2H)2+ followed by CAD of primary fragment 163+. (C) CAD of (M + 2H)2+ followed by IRMPD of primary fragment 163+. (D) CAD of (M + 2H)2+ followed by CAD of primary fragment 163+.
IRMPD/CAD produced the most consistent fragment abundance over the mass range and the best S/N. Results from a similar set of experiments are shown in Figure 12, in which protonated midecamycin (m/z 814) is subjected to CAD or IRMPD followed by a second activation step for the dominant primary fragment, 174+, the central desosamine sugar species. Again, IRMPD is much more efficient at forming the primary fragment, 174+, which translates into greater overall signal in the two subsequent tandem options shown in Figure 12A and B. CAD more effectively dissociates the 174+, thus making IRMPD/CAD more efficient than IRMPD/IRMPD. Neither of the two tandem methods in which CAD was the first step was particularly effective, as illustrated by the low signal intensities in Figure 12C and D. For this example, IRMPD/CAD gave the best combination of efficiency, S/N, and signal intensity. CONCLUSIONS The utility of multiple, discrete dissociation steps is well established, but the application is usually limited to only one method of activation, especially in the case of collisional techniques. IRMPD and CAD can be combined in several versatile ways in the quadrupole ion trap. Common fragments formed by IRMPD of multiple charge states of a given molecule can be investigated by CAD without changing the dissociation parameters from sample to sample, similar to selected reaction monitoring in a multiquadrupole analyzer. The same can be said for investigating fragments common to multiple compounds from the same class. The tandem techniques can also be combined to overcome trapping limitations in the ion trap due to low-mass cutoff constraints as shown for larger molecules such as erythromycin
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Figure 12. Tandem dissociation of protonated midecamycin (m/z 814). (A) IRMPD of (M + H)+ followed by IRMPD of primary fragment 174+. (B) IRMPD of (M + H)+ followed by CAD of primary fragment 174+. (C) CAD of (M + H)+ followed by CAD of primary fragment 174+. (D) CAD of (M + H)+ followed by IRMPD of primary fragment 174+.
A or midecamycin. These tandem techniques enhance the versatility of IRMPD as an alternative dissociation method and increase the amount of diagnostic information attainable from IRMPD experiments. In general, IRMPD is more effective at generating fragment ions from relatively higher mass molecular ions, especially when key lower mass fragment ions are formed, and CAD is more effective for subsequent analysis of the lower mass fragment ions. ACKNOWLEDGMENT The authors gratefully acknowledge financial support from the Robert A. Welch Foundation (F1155). Received for review September 28, 2000. Accepted January 11, 2001. AC001161O
