Anal. Chem. 2004, 76, 1545-1549
Accelerated Articles
Ion “Threshing”: Collisionally Activated Dissociation in an External Octopole Ion Trap by Oscillation of an Axial Electric Potential Gradient Melinda A. McFarland,† Christopher L. Hendrickson,† and Alan G. Marshall*,†
Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Tallahassee, Florida 32310
We have implemented efficient and rapid collisionally activated dissociation (CAD) external to an ICR cell by use of a novel axial electric potential gradient mounted in an external ion accumulation octopole. The gradient is produced by eight tilted (in the axial direction) wires mounted between the rods of the octopole. Rapid switching of the wire potential between a positive and negative value drives the ion axial motion back and forth and, in the presence of nitrogen gas at suitable pressure, induces dissociation. A fragmentation period on the order of tens of milliseconds is typical. Precursor ion isolation is achieved in a quadrupole mass filter mounted between the electrospray source and the accumulation octopole. A scan rate of >1 Hz is possible with resolving power and mass accuracy equivalent to direct infusion experiments (for equivalent detection period for 1 scan). The method is thus sufficiently rapid for MS/MS with on-line LC sample introduction. Moreover, compared to CAD in the ICR cell, external CAD improves mass accuracy, producing thermal on-axis fragment ions for detection.
Electrospray ionization (ESI)1 Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry2-5 has become a powerful technique in tandem mass spectrometry because of its high mass accuracy and mass resolving power. The addition of external ion * To whom all correspondence should be addressed. E-mail: marshall@ magnet.fsu.edu. † Also members of the Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32310. (1) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F. Mass Spectrom. Rev. 1990, 9, 37-70. (2) Marshall, A. G.; Hendrickson, C. L.; Jackson, G. S. Mass Spectrom. Rev. 1998, 17, 1-35. (3) Winger, B. E.; Hofstadler, S. A.; Bruce, J. E.; Udseth, H. R.; Smith, R. D. J. Am. Soc. Mass Spectrom. 1993, 4, 566-577. 10.1021/ac035208s CCC: $27.50 Published on Web 02/19/2004
© 2004 American Chemical Society
accumulation6 to the FT-ICR instrument makes possible the mass selection and accumulation of ions of limited abundance prior to transfer to the ICR cell, thereby increasing the dynamic range.7,8 Accumulation of ions prior to the ICR cell also bridges the gap between the continuous electrospray ionization source and the necessarily pulsed detection of ICR by allowing subsequent ions to be stored while previous ions are being detected. The nearly 100% duty cycle and higher scan rate accomplished by the addition of external accumulation has furthered the utility of this technique by making it compatible with on-line techniques such as liquid chromatography.6,9 The need for a short experiment period (e.g., imposed by on-line chromatography experiments) has increased the need for rapid fragmentation techniques, such as infrared multiphoton dissociation (IRMPD)10-12 and electron capture dissociation.13,14 Traditional collisionally activated dissociation (CAD)15,16 (4) Beu, S. C.; Senko, M. W.; Quinn, J. P.; Wampler, F. M., III; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 1993, 4, 557-565. (5) Hendrickson, C. L.; Emmett, M. R. Annu. Rev. Phys. Chem. 1999, 50, 517536. (6) 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. (7) Hendrickson, C. L.; Quinn, J. P.; Emmett, M. R.; Marshall, A. G. Quadrupole Mass Filtered External Accumulation for Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. In 48th ASMS Conference on Mass Spectrometry and Allied Topics; Long Beach, CA, 2000; CD-ROM. (8) Belov, M. E.; Nikolaev, E. N.; Anderson, G. A.; Auberry, K. J.; Harkewicz, R.; Smith, R. D. J. Am. Soc. Mass Spectrom. 2001, 12, 38-48. (9) Palmblad, M.; Tsybin, Y. O.; Ramstrom, M.; Bergquist, J.; Hakansson, P. Rapid Commun. Mass Spectrom. 2002, 16, 988-992. (10) Woodlin, R. L.; Bomse, D. S.; Beauchamp, J. L. J. Am. Chem. Soc. 1978, 100, 3248-3250. (11) Little, D. P.; Speir, J. P.; Senko, M. W.; OConnor, P. B.; McLafferty, F. W. Anal. Chem. 1994, 66, 2809-2815. (12) Li, W.; Hendrickson, C. L.; Emmett, M. R.; Marshall, A. G. Anal. Chem. 1999, 71, 4397-4402. (13) Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. J. Am. Chem. Soc. 1998, 120, 3265-3266. (14) Zubarev, R. A.; Horn, D. M.; Fridriksson, E. K.; Kelleher, N. L.; Kruger, N. A.; Lewis, M. A.; Carpenter, B. K.; McLafferty, F. W. Anal. Chem. 2000, 72, 563-573.
Analytical Chemistry, Vol. 76, No. 6, March 15, 2004 1545
in an FT-ICR instrument requires the use of a collision gas in the ICR cell and subsequent pumpdown delay such that the experimental event sequence is much longer than for other MS/MS techniques and is thus incompatible with on-line separations. Alternatively, the pumpdown time delay can be circumvented in hybrid instruments by fragmentation in the accumulation region external to the ICR cell. Multipole storage assisted dissociation (MSAD)17,18 and IRMPD have been demonstrated. However, MSAD is not widely used because it requires very high ion density (which cannot be routinely accumulated in an acceptable time frame). In fact, it is quite difficult to facilitate in our octopole traps, presumably due to the flatter pseudopotential well and larger inner diameter compared to the hexapole traps used by the discoverers. IRMPD in an external trap19,20 is similarly unpopular, because high pressure in the external trap increases the rate of internal energy dissipation and reduces dissociation efficiency (for example, no protein or peptide IRMPD has been shown in an external trap). More effectively, the two most common forms of CAD have been implemented in hybrid ICR instruments. The first form induces fragmentation through collisions between precursor ions and inert gases (such as nitrogen or argon) within a linear multipole. The collision energy is defined by the dc offset on the multipole rods, as in triple-quadrupole instruments.21 The second induces dissociation by resonant excitation (as in quadrupole ion traps), in which resonantly accelerated precursor ions undergo multiple collisions with small inert gas molecules (typically helium).22,23 In this experiment, product ions are not resonant and do not undergo subsequent fragmentation. Although external accumulation in an external rf linear multipole trap does provide rf focusing and thermalization by collisional cooling prior to transfer to the ICR cell, the essentially flat potential along the length of the multipole makes ion extraction too slow for efficient capture in the ICR cell. We have previously reported the modification of our external accumulation octopole by the addition of a novel axial electric potential gradient. The gradient is produced by eight tilted (in the axial direction) wires mounted between the rods of the octopole.24 During ion extraction from the modified rf linear octopole ion trap, the dc potential on (15) McLuckey, S. A. J. Am. Soc. Mass Spectrom. 1992, 3, 599-614. (16) Senko, M. W.; Speir, J. P.; McLafferty, F. W. Anal. Chem. 1994, 66, 28012808. (17) Sannes-Lowery, K. A.; Griffey, R. H.; Kruppa, G. H.; Speir, J. P.; Hofstadler, S. A. Rapid Commun. Mass Spectrom. 1998, 12, 1957-1961. (18) Håkansson, K.; Axelsson, J.; Palmblad, M.; Hakansson, P. J. Am. Soc. Mass Spectrom. 2000, 11, 210-217. (19) Hofstadler, S. A.; Sannes-Lowery, K. A.; Griffey, R. H. Anal. Chem. 1999, 71, 2067-2070. (20) Drader, J. J.; Hannis, J. C.; Hofstadler, S. A. Anal. Chem. 2003, 75, 36693674. (21) Danell, R. M.; Easterling, M.; Orden, S. V.; Berg, C. B.; Anderson, J.; Meier, J.; Speir, P., Automated Protein Identification Using Data-Dependent Q-FTMS. In 51st ASMS Conference on Mass Spectrometry and Allied Topics; Montreal, PQ, Canada, 2003. (22) Syka, J. E. P.; Bai, D. L.; Jr, G. C. S.; Horning, S.; Shabanowitz, J.; Hunt, D. F.; Marto, J. A. A Linear Quadrupole Ion Trap Fourier Transform Mass Spectrometer: A New Tool for Proteomics. In 49th ASMS Conference on Mass Spectrometry and Allied Topics; Chicago, IL, 2001. (23) Syka, J. E. P.; Bai, D. L.; Jr, G. C. S.; Horning, S.; Shabanowitz, J.; Hunt, D. F.; Marto, J. A. A Linear Quadrupole Ion Trap Fourier Transform Mass Spectrometer: Performance Characterization and Applications to Proteomics. In 50th ASMS Conference on Mass Spectrometry and Allied Topics; Orlando, FL, 2002. (24) Wilcox, B. E.; Hendrickson, C. L.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 2002, 13, 1304-1312.
1546
Analytical Chemistry, Vol. 76, No. 6, March 15, 2004
the wires is pulsed positive (for cations). The imposed dc potential in the octopole results in much more rapid ejection of the ions from the octopole, for increased sensitivity, signal-to-noise ratio, and scan rate. In this paper, we report the implementation of CAD in an accumulation octopole external to the ICR cell, by periodic reversal of the axial potential gradient to induce energetic ionneutral collisions (ion “threshing”). This technique is a further advantage of instruments equipped with an ion accumulator modified with an axial field, which has already been shown to facilitate at least a 10-fold increase in sensitivity relative to unmodified accumulation multipoles. A fragmentation period on the order of 10 ms makes ion threshing amenable to experiments requiring rapid scan rate, such as LC-MS/MS. The extent of ion activation and dissociation is efficient and tunable by adjustment of the frequency, amplitude, and, primarily, the number of field reversals of the applied dc potential, all parameters that are well suited to automated control. The ability to control the degree of fragmentation, as well as induce secondary fragmentation, can be used to elucidate a greater amount of structural information. METHODS Sample Preparation. Bovine ubiquitin, melittin, and LHRH were purchased from Sigma (St. Louis, MO) and used without further purification. For electrospray, stock solutions were diluted to a concentration of 1 µM for ubiquitin and 2 µM for melittin and LHRH in 1:1 methanol (Baker, Philipsburg, NJ)/water with 2.5% acetic acid. Melittin and LHRH were electrosprayed from a mixture containing 2 µM each of a total of four standard peptides and small proteins. Accumulation times ranged from 2 to 5 s. The Erythrina corallodendron lectin (Sigma) was tryptic digested by addition of 100 pmol of modified trypsin (Promega, Madison, WI) at 38 °C for 4 h. Peptides were concentrated and purified by use of a C18 Zip Tip (Millipore, Bedford, MA) and diluted to a final concentration of ∼5 µM in methanol/water with 2.5% acetic acid. Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. All experiments were performed with a 9.4-T passively shielded ESI-Q-FT-ICR mass spectrometer equipped with a microelectrospray ionization source.25 Samples were infused at a flow rate of 0.3 µL/min through a 50-µm-i.d. fused-silica capillary with a tip mechanically ground to a uniformly thin wall.26 For external accumulation (Figure 1)7,27 ions are held in a focusing octopole (370 Vp-p, 1.6 MHz, dc offset -9 V, CL1 9 V), then passed through a quadrupole mass filter (front octopole dc offset +4 V, CL1 0 V) for mass selection, and subsequently deposited in an accumulation octopole (CL2 2 V, CL3 +9 V, accumulation octopole dc offset -9 V), both containing nitrogen at ∼1 × 10-3 Torr for collisional cooling of the ions. The nitrogen pressure is constant for the duration of the experiment. The accumulation octopole (rod length 14 cm, i.d. 0.48 cm, 370 Vp-p, 1.6 MHz) has been (25) Senko, M. W.; Hendrickson, C. L.; PasaTolic, L.; Marto, J. A.; White, F. M.; Guan, S. H.; Marshall, A. G. Rapid Commun. Mass Spectrom. 1996, 10, 1824-1828. (26) Quinn, J. P.; Emmett, M. R.; Marshall, A. G. A Device for Fabrication of Emitters for Low-flow Electrospray Ionization. In 46th ASMS Conference on Mass Spectrometry and Allied Topics; Orlando, FL, 1998; pp 1388-1388. (27) Hendrickson, C. L.; Quinn, J. P.; Emmett, M. R.; Marshall, A. G. MassSelective External Ion Accumulation for Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. In 49th ASMS Conference on Mass Spectrometry and Allied Topics; Chicago, IL, 2001; CD-ROM.
Figure 1. Schematic diagram (not to scale) of the NHMFL 9.4-T ESI-Q-FT-ICR instrument. The electrospray ion source, the ion optics, and the ICR cell are shown.
modified for improved ion extraction to include tilted wires that produce an axial potential gradient.24 Fragmentation experiments take advantage of that adjustable (approximately exponential) potential to manipulate ions for collisional activation while ions are trapped in the accumulation octopole (see below). Following CAD, ions are transferred through an octopole ion guide to an open-ended cylindrical Penning cell28 for frequency-sweep excitation and broadband detection (512 Kword data points). The digitized time-domain transient signal is Hanning apodized and Fourier transformed to yield a magnitude-mode frequency spectrum that is converted to a mass-to-charge ratio spectrum by the usual quadrupolar approximation.29,30 The experimental event sequence is controlled by a modular ICR data acquisition and analysis system.31 CAD PROCEDURE AND RESULTS In an unmodified octopole ion trap, the electric potential acting on the ions varies steeply at either end but is essentially flat in the center, making manipulation of trapped ions difficult. The addition of tilted wires lessens this problem by adding an axial potential gradient.24 Under conditions for which fragmentation is not desired, the dc potentials on the angled wires and octopole rods are pulsed positive relative to conductance limit 3 (CL3; see Figure 1) as the trapped ions are released, for more efficient transfer of ions to the ICR cell for detection. For external collisionally activated dissociation, the ions are first accumulated and trapped in the accumulation octopole. Prior to ejection of ions to the ICR cell, the conductance limits remain positive, here +10 V, while the tilted wires are pulsed to a positive dc potential followed by a negative dc potential (typically (30 V; see Figure 2), each pulse lasting hundreds of microseconds. The periodic reversal of the polarity of the field is repeated some tens of times, depending on the desired degree of parent ion fragmentation. The alternating, approximately exponential axial potential drives the ions from one end of the octopole to the other (ion (28) Beu, S. C.; Laude, D. A., Jr. Int. J. Mass Spectrom. Ion Processes 1992, 112, 215-230. (29) Ledford, E. B., Jr.; Rempel, D. L.; Gross, M. L. Anal. Chem. 1984, 56, 2744-2748. (30) Shi, S. D.-H.; Drader, J. J.; Freitas, M. A.; Hendrickson, C. L.; Marshall, A. G. Int. J. Mass Spectrom. 2000, 195/196, 591-598. (31) Senko, M. W.; Canterbury, J. D.; Guan, S.; Marshall, A. G. Rapid Commun. Mass Spectrom. 1996, 10, 1839-1844.
Figure 2. Schematic representation of the motions of ions trapped in the accumulation octopole relative to the axial electric potential of the wires. Polarity reversal of the inset wires reverses direction of the ion, thereby increasing the number and energy of collisions with the buffer gas. Without the gradient wires, the potential in the middle of the octopole is flat and the ions cannot be efficiently manipulated.
threshing), so that the ions undergo rapid suprathermal collisions with the nitrogen buffer gas and dissociate. Following fragmentation, the tilted wires are pulsed to +30 V (for positive ions), the octopole rods are pulsed from -9 (for ion confinement) to ∼1 V, conductance limit 3 is pulsed to a negative potential, and the ions are thereby transferred to the ICR cell for detection. The effect of ion population, as well as collision gas, has been examined (data not shown). We do not observe a strong correlation between ion density and dissociation efficiency. Comparison of the effects of different collision gases led to the choice of nitrogen, because helium did not cause sufficient energy transfer to induce fragmentation, and collisions with argon were too energetic, yielding excessive secondary fragmentation. Increasing the pressure (above optimal) tended to stabilize the ions before they could be collisionally activated. Figure 3 shows a single-scan mass spectrum of the doubly protonated charge state of the peptide luteinizing hormone releasing hormone (LHRH) after 8 ms of axial electric potential controlled collisionally activated dissociation (ion threshing). Energetic collisions with nitrogen are induced by pulsing the dc potential on the tilted wires of the accumulation octopole to +30 V for 200 µs, then -30 V for 200 µs, and repeating the oscillation 20 times for a total CAD duration of 8 ms. An rms mass accuracy of 0.3 ppm was achieved, based on calibration from the parent and four fragment peaks, for a single scan. An important advantage of external ion CAD is that fragment ions are subjected to rf focusing prior to arrival at the ICR cell so that the resultant mass accuracy is comparable to that for direct MS mode. In contrast, fragmentation in the ICR cell can increase ion cyclotron and magnetron radii prior to excitation/detection, thereby degrading mass accuracy. Fragmentation of a larger peptide is shown in Figure 4 (single scan). Threshing of the 4+ charge state of melittin yielded 64% sequence coverage and an rms mass accuracy of 0.25 ppm, based on calibration from the parent and four fragment peaks, for a single scan. The dc voltage on the wires was pulsed to +30 V for 200 µs and then -30 V for 200 µs. The cycle was repeated 10 times for Analytical Chemistry, Vol. 76, No. 6, March 15, 2004
1547
Figure 3. Single-scan fragment ion mass spectrum of doubly protonated LHRH following 8 ms of axial electric potential controlled collisionally activated dissociation (ion threshing). The dc voltage on the wires was pulsed to +30 V for 200 µs and then -30 V for 200 µs. The cycle was repeated 20 times. The rms mass accuracy is 0.8 ppm. Notation for a, b, and y fragment ions is standard.33
Figure 5. Fragment ion mass spectrum of the 9+ charge state of bovine ubiquitin following 10 ms of axial electric potential controlled collisionally activated dissociation. The dc voltage on the wires was pulsed to +30 V for 200 µs and then -30 V for 200 µs, with the cycle repeated 25 times. The spectrum is a sum of 30 scans.
Figure 4. Single-scan fragment ion mass spectrum of the 4+ charge state of melittin following 4 ms of threshing. Sixty-four percent sequence coverage and an rms mass accuracy of 0.253 ppm are achieved. The dc voltage on the wires was pulsed to +30 V for 200 µs and then -30 V for 200 µs. The cycle was repeated 10 times.
Figure 6. Fragment ion mass spectrum of an N-glycosylated peptide that was a minor component of a tryptic digest of a lectin. The isolated parent was accumulated in the octopole to create a sufficient parent population prior to 45 ms of oscillation of the exponential potential for fragmentation. The dc voltage on the wires was pulsed +30 V for 300 µs and then -30 V for 300 µs, with the cycle repeated 75 times. The spectrum is a sum of 50 scans.
a total of 4 ms for the fragmentation event. The mass spectra shown in Figures 3 and 4 are similar in appearance to sustained off-resonance irradiation-CAD32 mass spectra of the same compounds (data not shown). Parent ion to fragment ion conversion is highly efficient and can be adjusted to eliminate parent ions completely by increasing the number of cycles of the fragmentation event. For example, the efficiency of conversion of melittin [M + 4H]4+ precursor ions to product ions was ∼42% for 10 ms and ∼55% for 20 ms threshing based on a S/N threshold of >3:1. These efficiencies reflect only the ion population that is transferred and trapped in the ICR cell during the transfer time of this experiment, which was chosen for maximum precursor ion signal at the expense of fragment ion populations on the edges of or outside that range. One difficulty with external fragmentation on
an ICR instrument is a diminished mass range due to a time-offlight effect in transit from the accumulation region to capture in the ICR cell. Relative magnitudes of peaks can be skewed based on choice of transfer time. Figure 5 shows a 30-scan CAD spectrum of the 9+ charge state of the protein, bovine ubiquitin, after 10 ms of fragmentation. Extensive b and y fragments33 are present, as well as some internal (secondary) fragmentation. Secondary fragmentation increases with longer fragmentation period and higher charge state and can be minimized by lowering the number of cycles. As for the smaller LHRH and melittin peptides, the dc voltage on the angled wires alternated between +30 and -30 V, with the same period of polarity reversal (200 µs). As for any low-energy ion “heating” experiment,34 the degree of ion dissociation depends on the rate and duration of excitation
(32) Gauthier, J. W.; Trautman, T. R.; Jacobson, D. B. Anal. Chim. Acta 1991, 246, 211-225.
1548 Analytical Chemistry, Vol. 76, No. 6, March 15, 2004
(33) Roepstorff, P.; Fohlman, J. Biomed. Mass Spectrom. 1984, 11, 601-601.
energy. Improper adjustment of the period or number of cycles of threshing yields little or no fragmentation. Preliminary analysis of the compounds presented in this paper as well as several other standards of various masses suggests that automation of threshing for ions of a given m/z range may be feasible. A 5-kDa N-glycosylated glycopeptide constituting a minor component of a tryptic digest of a lectin was also threshed (Figure 6), yielding results similar to IRMPD with the same instrument, including complete loss of the glycan as well as fragmentation at each of the glycosidic bondssinformation sufficient to map the branching pattern of the glycan “tree” (except for stereochemistry).35 As a minor component in a digest, with no prior chromatographic separation, this species required a long external accumulation period to create a sufficient parent ion population for MS/MS. The dc voltage on the wires was pulsed to +30 V for 300 µs and then -30 V for 300 µs, and the cycle was repeated 75 times. The spectrum is a sum of 50 scans. CONCLUSION We have achieved rapid CAD of electrosprayed ions as a fragmentation technique compatible with on-line LC ESI FT-ICR (34) McLuckey, S. A.; Goeringer, D. E. J. Mass Spectrom. 1997, 35, 461-474. (35) Håkansson, K.; Chalmers, M. J.; Quinn, J. P.; McFarland, M. A.; Hendrickson, C. L.; Marshall, A. G. Anal. Chem. 2003, 75, 3256-3262. (36) Blakney, G. T.; Chalmers, M. J.; Lam, T. T.; McFarland, M. A.; Emmett, M. R.; Hendrickson, C. L.; Marshall, A. G. High-Speed Data-Dependent HPLC FT-ICR MS/MS. In 51th ASMS Conference on Mass Spectrometry and Allied Topics; Montreal, PQ, Canada, 2003.
mass analysis.36 Fragmentation of each of a variety of standards indicates that automation of threshing for data-dependent massselected ions of a given m/z range is feasible.36 The degree of fragmentation is tunable for varying degrees of parent to fragment ion conversion. Further work will be needed to map out the energies associated with this technique as well as to establish the degree of charge-state dependence. Although other external CAD techniques are available, the presented threshing technique, made possible by the high ion ejection efficiency of our modified accumulation octopole, is particularly efficient and rapid, and thus well suited for on-line LC-MS/MS. ACKNOWLEDGMENT This work was supported by the NSF National High Field FTICR Facility (CHE-99-09502), Florida State University, and the National High Magnetic Field Laboratory in Tallahassee, FL. The authors also thank M. J. Chalmers, B. E. Wilcox, J. P. Quinn, and K. Håkansson for instrumentation development and helpful discussions.
Received for review October 13, 2003. Accepted January 21, 2004. AC035208S
Analytical Chemistry, Vol. 76, No. 6, March 15, 2004
1549
