Anal. Chem. 1999, 71, 2067-2070
Accelerated Articles
Infrared Multiphoton Dissociation in an External Ion Reservoir Steven A. Hofstadler,* Kristin A. Sannes-Lowery, and Richard H. Griffey
Ibis Therapeutics, A Division of Isis Pharmaceuticals, 2292 Faraday Avenue, Carlsbad, California 92008
In this work we present a novel scheme for performing infrared multiphoton dissociation (IRMPD) external to the mass analyzer in an external ion reservoir consisting of an rf-only multipole and a pair of electrostatic lens elements. Ions generated by electrospray ionization (ESI) are accumulated in an rf-only hexapole and dissociated by irradiation at 10.6 µm from a CW CO2 laser in the source region of the mass spectrometer. This scheme is unique from other IRMPD schemes as dissociation occurs in a spatially distinct region of the spectrometer and is independent of the mass spectrometry platform used to analyze the fragment ions. The effectiveness of the technique is demonstrated with ESI IRMPD FTICR mass spectrometry of a 20-mer phosphorothioate oligonucleotide. A comparison of the external IRMPD scheme with nozzle-skimmer dissociation and conventional in-cell IRMPD reveals a significant improvement in signal-tonoise ratio and fragment yield, particularly for larger, more highly charged fragment ions.
Infrared multiphoton dissociation (IRMPD) has previously been shown to be an effective means for dissociating macromolecular ions providing sequence and/or structure information in conjunction with mass spectrometric detection. Following the earlier work of Beauchamp and co-workers1 and Eyler and coworkers,2 McLafferty and co-workers demonstrated that IRMPD, in conjunction with FTICR detection, is an effective method by which macromolecular ions, including proteins and oligonucleotides, can be dissociated and further characterized.3 Similarly, following the earlier work of March and co-workers4 and Yost and (1) Bomse, D. S.; Berman, D. W.; Beauchamp, J. L. J. Am. Chem. Soc. 1981, 103, 3967-3971. (2) Baykut, G.; Watson, C. H.; Weller, R. R.; Eyler, J. R. J. Am. Chem. Soc.1985, 107, 8036-8039. (3) Little, D. P.; Speir, J. P.; Senko, M. W.; O’Connor, P. B.; McLafferty, F. W. Anal. Chem. 1994, 66, 2809-2815. 10.1021/ac990176n CCC: $18.00 Published on Web 05/04/1999
© 1999 American Chemical Society
co-workers,5 Brodbelt and co-workers employed IRMPD in conjunction with SWIFT on a quadrupole ion trap mass spectrometer to characterize dissociation pathways of a variety of species such as macrolide antibiotics and hydrogen-bonded complexes6,7 and demonstrated that the production of fragment ions via IRMPD is significantly influenced by the pressure of bath gas in the ion trap. With helium bath gas pressures approaching 1 × 10-4 Torr, those researchers demonstrated that collisional quenching competes with photoactivation of analyte ions. If the rate of collisional deactivation approaches the rate of photoactivation, the net rate of photofragmentation is substantially reduced. While a number of researchers have incorporated methods based on IRMPD to enable improved tandem MS analysis, the technique has not been widely employed. IRMPD is generally performed on mass spectrometers based on Penning or Paul traps by combining an external optical scheme and modifications to the vacuum chamber and/or trapping electrodes which allow the beam to traverse the ion trapping volume. In some Penning/Paul trap configurations, the optical path through the central axis of the trap is obstructed by an external ion source8 or other elements such as a cell-mounted filament assembly.9 To effect photodissociation with such hindered configurations, additional cell-mounted optical components must be employed, which adds considerable complexity to the experimental scheme.10 In this work, we demonstrate that infrared multiphoton dissociation can be very effectively performed in an external ion (4) Hughes, R. J.; March, R. E.; Young, A. B. Can. J. Chem. 1983, 61, 834845. (5) Stephenson, J. L., Jr.; Booth, M. M.; Shalosky, J. A.; Eyler, J. R.; Yost, R. A. J. Am. Soc. Mass Spectrom. 1994, 5, 886-893. (6) Colorado, A.; Shen, J. X.; Brodbelt, J. Anal. Chem. 1996, 68, 4033-4043. (7) Goolsby, B. J.; Brodbelt, J. S. J. Mass Spectrom. 1998, 33, 705-712. (8) Tutko, D. C.; Henry, K. D.; Winger, B. E.; Stout, H.; Hemling, M. Rapid Commun. Mass Spectrom. 1998, 12, 335-338. (9) Wu, Q. Y.; Anderson, G. A.; Udseth, H. R.; Sherman, M. G.; Vanorden, S.; Chen, R.; Hofstadler, S. A.; Gorshkov, M. V.; Mitchell, D. W.; Rockwood, A. L.; Smith, R. D. J. Am. Soc. Mass Spectrom. 1996, 7, 915-922. (10) Dunbar, R. C.; Weddle, G. H. J. Phys. Chem. 1988, 92, 5706.
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Figure 1. Experimental configuration for performing IRMPD in an external ion reservoir. The unfocused beam of a 25-W CO2 laser is aligned to be coaxial with the central axis of the rf-only hexapole. Ions are accumulated and dissociated in the high pressure (5 × 10-6 mBarr) prior to being transferred to the FTICR trapped ion cell where they are mass analyzed.
reservoir by passing an unfocused laser beam through the ion volume during an external ion accumulation event. Marshall and co-workers previously demonstrated that, prior to mass analysis by FTICR, ions generated by electrospray can be accumulated in an external ion reservoir composed of an rf-only multipole bounded by electrostatic elements.11 This approach not only improves the ionization duty cycle to near unit efficiency but affords the opportunity to manipulate the ion ensemble prior to mass analysis. For example, we recently demonstrated that under the appropriate conditions electrospray-generated ions can be dissociated with high efficiency prior to mass analysis by employing extended accumulation intervals in the external ion reservoir.12 Similarly, Douglas and co-workers demonstrated that m/z-selective isolation and dissociation can be effected in a linear ion trap by superimposing a small dipolar excitation on the quadrupolar field of a linear rf ion trap13 and subsequently detected by TOF. In addition to providing a simple, platform-independent IRMPD scheme, this method generates an improvement in fragment ion abundance and sequence coverage compared to conventional “incell” IRMPD schemes.14 EXPERIMENTAL SECTION Instrumentation. All experiments were performed on a Bruker Daltonics (Billerica, MA) Apex 70e Fourier transform ion cyclotron resonance mass spectrometer. The spectrometer is equipped with an Analytica (Branford, CT) electrospray source utilizing a grounded ESI emitter, a countercurrent drying gas, a glass desolvation capillary, a single skimmer cone, and an rf-only hexapole ion guide. Ions are accumulated in the external ion reservoir for 500 ms and pulsed into the Infinity trapped ion cell where they are analyzed by FTICR. All aspects of the experiment including data acquisition, processing, and plotting were performed using Bruker XMASS version 4.0 software running on a Silicon Graphics R5000 workstation. A 17 µM solution of Isis 2302, a 20-mer phosphorothioate oligonucleotide (5′-GCC CAA GCT GGC ATC CGT CA-3′) was electrosprayed from a 50:50 2-propanol/water solution containing 0.1% tripropylamine through an off-axis electrospray probe at a flow rate of 1.5 µL/min. IRMPD. As shown in Figure 1, the external ion accumulation region is composed of a biased skimmer cone, an rf-only hexapole (11) Senko, M. W.; Hendrickson, C. L.; Emmett, M. R.; Shi, S. D. H.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1997, 8, 970-976. (12) Sannes-Lowery, K.; Griffey, R. H.; Kruppa, G. H.; Speir, J. P.; Hofstadler, S. A. Rapid Commun. Mass Spectrom. 1998, 12, 1957-1961. (13) Campbell, J. M.; Collings, B. A.; Douglas, D. J. Rapid Commun. Mass Spectrom. 1998, 12, 1463-1474. (14) Hofstadler, S. A.; Griffey, R. H.; Pasa-Tolic, L.; Smith, R. D. Rapid Commun. Mass Spectrom. 1998, 12, 1400-1404.
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operating at 5 MHz (500 Vpp), and an auxiliary “gate” electrode at the low-pressure end of the hexapole. In this work, the capillary exit voltage is maintained at -68 V to avoid fragmentation due to conventional nozzle-skimmer dissociation or -180 V to induce nozzle-skimmer dissociation. For operation in the negative ionization mode, the potential of the skimmer cone is typically held at -15 V while the gate electrode toggles between -15 V during accumulation and 0.2 V during injection; the polarity of these electrodes is reversed for operation in the positive ionization mode. A 25-W CW CO2 laser (Synrad Inc., Mukilteo, WA) operating at 10.6 µm was used for all IRMPD experiments. IRMPD of ions in the external ion reservoir was effected by irradiation during the 500-ms ion accumulation event. A laboratory-built aluminum optical bench was positioned approximately 1.5 m from the actively shielded superconducting magnet such that the laser beam was aligned with the central axis of the magnet. Using standard IR-compatible mirrors and kinematic mirror mounts, the unfocused 3-mm laser beam was aligned to traverse directly through the 3.5-mm holes in the trapping electrodes of the Infinity trapped ion cell and longitudinally traverse the hexapole region of the external ion guide finally impinging on the skimmer cone. Alignment was accomplished by a preliminary visual alignment with a visible diode laser such that light could be seen exiting the glass desolvation capillary at the source end of the spectrometer. Subsequent alignment was optimized by the mass spectral fragmentation response with the IR laser triggered during the ion accumulation interval. The laser was operated at an output of 28 W as measured at the entrance to the mass spectrometer; no attempt was made to measure the actual laser power in, or beyond, the hexapole. RESULTS AND DISCUSSION In the instrumental configuration shown in Figure 1, an unfocused laser beam traverses the central axis of both the trapped ion cell and the external ion reservoir. This configuration allows IRMPD to be performed either in the trapped ion cell or in the external ion reservoir (or both, for example, in a temporally separated MS3 experiment). As we demonstrate below, this external source IRMPD scheme has several attributes which may be exploited for determination of macromolecular sequence. Ion dissociation occurs in a spatially distinct region of the spectrometer and is decoupled from the method by which the fragment ions are mass analyzed. This scheme can be used with any MS platform that is directly compatible with a pulsed ionization source (including quadruple ion traps and time-of-flight detectors) and perhaps adapted to accommodate m/z scanning instruments as well.
Figure 2. Representative ESI-FTICR of a 20-mer phosphorothioate oligonucleotide in which (a) ions were accumulated for 500 ms in the external ion reservoir and transferred to the trapped ion cell where they were mass analyzed, (b) IRMPD was performed in the FTICR trapped ion cell following a 500-ms ion accumulation event in the external ion reservoir, and (c) IRMPD was performed in the external ion reservoir concurrent with a 500-ms ion accumulation event with subsequent ion transfer and detection.
The effectiveness of the method is illustrated in Figure 2. Figure 2a contains a typical ESI-FTICR spectrum of a 20-mer phosphorothioate oligonucleotide obtained by externally accumulating ions for 500 ms prior to injection and detection in the FTICR cell. Figure 2b contains an ESI-FTICR spectrum obtained from in-cell IRMPD effected by externally accumulating ions for 500 ms in the external ion reservoir, transferring them to the trapped ion cell, and then irradiating them for 100 ms. Figure 2c contains an IRMPD ESI-FTICR spectrum obtained from externally accumulating ions for 500 ms concurrent with IR irradiation in the external reservoir. Following the accumulation/irradiation interval, the ions are transferred to the trapped ion cell where they are mass analyzed. While a significant degree of fragmentation is observed from the in-cell IRMPD as shown in Figure 2b, the majority of the assignable fragment ions correspond to relatively low molecular weight singly charged fragments consistent with w- and a-base ions15 and their respective decomposition products including internal neutral base loss and dehydration. As previously described,14 a subpopulation of the ion ensemble does not undergo dissociation even when extended irradiation intervals are employed due to a nonzero magnetron radius which results in the ion ensemble precessing around the impinging laser beam. This phenomenon is particularly important when “sidekick” trapping16 is employed as the trapping event can result in an ion population with a substantial magnetron radius. As will be described in detail elsewhere, this phenomenon can be exploited to effect m/z-selective IRMPD in a Penning trap. From the collection of fragment ions detected, only six bases from the 5′ terminus and six bases from the 3′ terminus of the analyte can be assigned unambiguously. Very few fragment ions are present at multiple charge states and there are insufficient fragment ions (15) McLuckey, S. A.; Habibi-Goudarzi, S. J. Am. Chem. Soc. 1993, 115, 1208512095. (16) Caravatti, P.; Allemann, M. Org. Mass Spectrom. 1991, 26, 514-518.
Figure 3. Expanded view of the 625-660 m/z range from the data shown in Figure 2 in which (a) IRMPD was effected in the trapped ion cell and (b) IRMPD was effected in the external ion reservoir prior to ion transfer. The external IRMPD configuration provides improved fragment ion abundance, sequence coverage, and signal-to-noise ratio compared to the in-cell scheme. Table 1. Fragment Ions Observed for a 20-Mer Phosphorothioate Oligonucleotide Using Three Dissociation Techniques: Nozzle-Skimmer (∆NS), IRMPD in the Trapped Ion Cell of a FTICR Mass Spectrometer (In-Cell IRMPD), and IRMPD Effected in the External Ion Reservoir with Subsequent Detection by FTICR (Hexapole IRMPD)a IRMPD
IRMPD
fragment ∆NS in-cell hexapole fragment ∆NS in-cell hexapole a2-base a3-base a4-base a5-base a6-base a7-base a8-base a9-base a10-base a11-base a12-base a13-base
1 1 1 1 1 1
1 1 1, 2 1, 2 2 2
1 1 1, 2 1, 2 2, 3 2, 3 2, 3 2, 3 3, 4 4 3, 4
w2 w3 w4 w5 w6 w7 w8 w9 w10 w11 w12 w13
1 1 1 2 2 2
1 1, 2 2 2 2, 3
1 1, 2 1, 2 2, 3 3 2, 3, 4 2, 3, 4 2, 3, 4 3, 4 3, 4
a The numbers in the columns refer to the (negatively charged) ions observed for each species. Note that the external IRMPD scheme provides improved coverage of the oligonucleotide sequence and provides multiple charge states for most fragments.
to determine the entire sequence of the oligonucleotide. As evidenced by the expanded view of the spectra in Figure 3, the spectrum acquired with the external IRMPD scheme is rich in fragment ions corresponding to a wide range of charge states (1to 5-) and molecular weights. Full coverage of the oligonucleotide sequence is observed with most w- and a-base ions observed at multiple charge states (see Table 1). In addition to an improvement in fragment ion abundance and sequence coverage, the spectrum acquired with the external IRMPD scheme exhibits improved resolving power and signal-to-noise ratio relative to the spectrum acquired by utilizing in-cell IRMPD. Additional classes of fragment ions are observed, particularly c-H+ ions resulting from cleavage of an axial P-O bond. Analytical Chemistry, Vol. 71, No. 11, June 1, 1999
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We postulate that the expanded range of fragment ions observed is the result of at least two contributing factors. First, performing IRMPD during the external ion accumulation event means that ions accumulated in the hexapole reservoir can experience a range of irradiation intervals if the laser is activated concurrently with ion accumulation in the reservoir. For example, if we consider a 500-ms accumulation/dissociation interval, an ion trapped within the first 10 ms of the event will have the opportunity to be irradiated for nearly 500 ms while an ion trapped near the end of the event may be exposed to the laser beam for only a few milliseconds. Additionally, performing the dissociation in the highpressure region of the hexapole allows collisional focusing concurrent with collisional stabilization of energetically metastable fragments and mitigates the potential for a spatially defocused ion cloud in the trapped ion cell. Employing the standard McLuckey nomenclature for oligonucleotide fragmentation,15 Table 1 compares the fragment ions observed for the 20-mer phosphorothioate oligonucleotide employing three different dissociation techniques, nozzle-skimmer dissociation, in-cell IRMPD, and external IRMPD, all acquired under otherwise similar conditions. Note that while both nozzleskimmer dissociation (spectrum not shown) and in-cell IRMPD (Figure 2b) result in 5′ fragments extending only to the a7-base ion, the external IRMPD scheme provides multiple charge states of fragment ions extending to the a13-base ion. Similarly, from the 3′ end of the molecule, nozzle-skimmer and in-cell IRMPD provide fragment ions out to the w9- and w7-species, respectively, while the external IRMPD scheme provides w-ions as large as the w13-ion. It is also worth noting that in general the external IRMPD scheme provides more charge states of each fragment than the other methods. For example, while nozzle-skimmer dissociation produces the 2- charge state for the w7-, w8-, and w9-species, the external IRMPD scheme produces the 2-, 3-, and 4- charge state for each of these species, respectively, providing further confirmation of these sequence-specific ions. Additionally, we have observed cn-H+ ions with external IRMPD (Figure 3b) that result from initial cleavage of the axial 5′-PO bond with concomitant or subsequent abstraction of a proton by the
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departing oxygen anion. Under higher energy dissociation conditions, the resulting 3′-metaphosphate species would lose HPO3 leading to an a-ion. However, we detect several c-H+ ions, suggesting a collisionally deactivated product ion. None of the three dissociation methods employed yielded the a9-base or w11ions that would correspond to cleavage at the 5′-TG-3′ moiety. We have observed the lack of fragmentation at this TG site on a number of oligonucleotides with various backbone modifications and will describe the phenomenon in detail in a future publication. CONCLUSIONS In this work, we demonstrated a novel scheme for performing IRMPD in which dissociation is effected in an external ion reservoir. During ion accumulation in the external ion reservoir and prior to injection and mass analysis, an unfocused laser beam is passed through the ion reservoir causing dissociation of molecular ions. The fragment ion yield and sequence coverage is significantly improved with this scheme as ions experience a range of irradiation times depending on when they first entered the accumulation region. Additionally, it is likely that the large number of low-energy collisions that occur in the high-pressure external ion reservoir stabilize metastable fragment ions that would otherwise undergo further dissociation and go undetected. In this scheme, the dissociation occurs in a spatially distinct region of the spectrometer and is decoupled from the method by which the ions are analyzed. Thus, this scheme should be directly applicable to other MS platforms that are directly compatible with a pulsed ionization source (including quadruple ion traps and timeof-flight detectors). ACKNOWLEDGMENT This work was supported in part by a NIST Advanced Technology Program grant (97-01-0135) awarded to the Ibis Therapeutics Division of Isis Pharmaceuticals. Received for review February 11, 1999. Accepted April 9, 1999. AC990176N
