Application of Multishot Acquisition in Fourier Transform Mass

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Application of Multishot Acquisition in Fourier Transform Mass Spectrometry Peter B. O’Connor* and Catherine E. Costello

Mass Spectrometry Resource, Department of Biochemistry, Boston University School of Medicine, 715 Albany Street, R806, Boston, Massachusetts 02118

A new method of ion injection and trapping is discussed wherein ions are accumulated over several laser shots in the FT-ICR cell prior to detection. This allows accumulation of ion signal without accumulating noise so that the signal/noise ratio is much improved provided that the “space-charge” limit of the total number of ions in the cell is not exceeded. “In-cell” ion accumulation allows selected ion accumulation by simply sweeping unwanted ions out of the cell prior to subsequent ion trapping events and also allows shifted ion accumulations to correct for time-of-flight distortions in the ion abundance distributions. Fourier transform mass spectrometry (FTMS) provides unrivaled mass resolution, mass accuracy, and flexibility of experiments compared to all other types of mass spectrometers. With the ability to store ions in the cell and irradiate them with many types of EM fields, mass spectrometrists can fragment them to almost any degree desired,1-5 remeasure them in the cell for improved sensitivity,6,7 and reaxialize them to hold them in the cell indefinitely.8 The limiting factor to the complexity of the experiment is typically the flexibility of the data system to allow more involved experimental functionality. Matrix-assisted laser desorption/ionization (MALDI) was first reported in 1987 using time-of-flight (TOF) mass analyzers,9 and the MALDI-TOF combination is quickly becoming a routine tool for biochemical laboratories. MALDI has been utilized extensively * Corresponding author: (phone) (617) 638-6705; (fax) (617) 638-6761; (email) [email protected]. (1) 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. (2) Little, D. P.; Speir, J. P.; Senko, M. W.; O’Connor, P. B.; McLafferty, F. W. Anal. Chem. 1994, 66, 2809-2815. (3) Williams, E. R.; Henry, K. D.; McLafferty, F. W.; Shabanowitz, J.; Hunt, D. F. J. Am. Soc. Mass Spectrom. 1990, 1, 413-416. (4) Gauthier, J. W.; Trautman, T. R.; Jacobson, D. B. Anal. Chim. Acta 1991, 246, 211-225. (5) Senko, M. W.; Speir, J. P.; McLafferty, F. W. Anal. Chem. 1994, 66, 28012808. (6) Speir, J. P.; Gorman, G. S.; Pitsenberger, C. C.; Turner, C. A.; Wang, P. P.; Amster, I. J. Anal. Chem. 1993, 65, 1746-1752. (7) O’Connor, P. B.; Speir, J. P.; Wood, T. D.; Chorush, R. A.; Guan, Z.; McLafferty, F. W. J. Mass Spectrom. 1996, 31, 555-559. (8) Schweikhard, L.; Guan, S.; Marshall, A. G. Int. J. Mass Spectrom. Ion Processes 1992, 120, 71-83. (9) Karas, M. I.; Bachmann, D.; Bahr, U.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1987, 78, 53-68. 10.1021/ac0005565 CCC: $19.00 Published on Web 09/08/2000

© 2000 American Chemical Society

on FTMS instruments.10-25 MALDI-FTMS has had success primarily because one is able to both achieve high mass accuracy and carry out tandem mass spectrometry experiments with the same instrument. It has the advantage of being a pulsed ion source operating with an instrument that is inherently a pulsed mass detector so that, at least theoretically, 100% of the ions created can be trapped and detectedsin contrast to coupling a TOF or FT mass analyzer to a continuous ion beam whereupon the beam must be sampled and the instrument is duty cycle limited. The sensitivity of the FTMS is similar to that of TOF mass spectrometers26,27 in that both can detect ions in the attomole ranges (the FTMS from the Fellgett advantage, the TOF from the high sensitivity of electron multipliers). Ion accumulation on FTMS instruments has become increasingly interesting of late with the external accumulation schemes recently developed.24,28-32 Ion accumulation in an external ion trap (10) Speir, J. P.; Gorman, G. S.; Amster, I. J. In Laser Desorption, Chemical Ionization, and Laser Desorption/Chemical Ionization Applications with FTMS; Gross, M. L., Ed.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1992; pp 199-212. (11) Speir, J. P.; Amster, I. J. Anal. Chem. 1992, 64, 1041-1045. (12) Castoro, J. A.; Wilkins, C. L. Anal. Chem. 1993, 65, 2621-2627. (13) McIver, R. T., Jr.; Li, Y.; Hunter, R. L. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4801. (14) Stemmler, E. A.; Buchanan, M. V.; Hurst, G. B.; Hettich, R. L. Anal. Chem. 1995, 67, 2924-2930. (15) Yao, J.; Dey, M.; Pastor, S. J.; Wilkins, C. L. Anal. Chem. 1995, 67, 36383642. (16) Dey, M.; Castoro, J. A.; Wilkins, C. L. Anal. Chem. 1995, 67, 1575-1579. (17) Martinovic, S.; Kezele, N.; Shevchenko, S. M.; Klasinc, L. IMSC Bodapest 1994. (18) O’Connor, P. B.; Duursma, M. C.; Rooij, G. J. v.; Heeren, R. M. A.; Boon, J. J. Anal. Chem. 1997, 69, 2751-2755. (19) Wood, T. D.; Schweikhard, L.; Marshall, A. G. Anal. Chem. 1992, 64, 14611469. (20) Solouki, T.; Russell, D. H. Appl. Spectrosc. 1993, 47, 211-217. (21) Hettich, R. L.; Buchanan, M. V. Int. J. Mass Spectrom. Ion Processes 1991, 111, 365-380. (22) Castoro, J. A.; Ko ¨ster, C.; Wilkins, C. Rapid Commun. Mass Spectrom. 1992, 6, 239-241. (23) Pasa-Tolic, L.; Huang, Y.; Guan, S.; Kim, H. S.; Marshall, A. G. J. Mass Spectrom. 1995, 30, 825-833. (24) Baykut, G.; Jertz, R.; Witt, M. Rapid Commun. Mass Spectrom. 2000, 10, 1238-1247. (25) Muddiman, D. C.; Bakhtiar, R.; Hofstadler, S. A.; Smith, R. D. J. Chem. Educ. 1997, 74, 1288-1292. (26) Li, Y. Z.; McIver, R. T. Rapid Commun. Mass Spectrom. 1994, 8, 743-749. (27) Green, M. K.; Medforth, C. J.; Muzzi, C. M.; Nurco, D. J.; Shea, K. M.; Smith, K. M.; Lebrilla, C. B.; Shelnutt, J. A. Eur. Mass Spectrom. 1997, 3, 439-451. (28) Pope, R. M.; Shen, N. Z.; Nicoll, J.; Tarnawiecki, B.; Dejsupa, C.; Dearden, D. V. Int. J. Mass Spectrom. Ion Processes 1997, 162, 107-119. (29) 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.

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of some type has many advantages including higher duty cycle for chromatographic experiments, external fragmentation, and higher trapping efficiency by use of gated ion trapping. In addition, selected ion accumulation in the FTMS ion cyclotron resonance (ICR) cell33 is a method wherein ions of a particular m/z are accumulated while other ions are rejected. In this paper, we will discuss “in-cell” accumulation of MALDI ions in a Fourier transform mass spectrometer using comparatively simple pulse sequences. Significant improvements in signal/ noise ratio, selected ion accumulation, and m/z bandwidth are possible with this method. EXPERIMENTAL SECTION These experiments were performed using an IonSpec Ultima Fourier transform mass spectrometer with a 7-T active-shielded superconducting electromagnet. This instrument has two separate vacuum systems, each with three turbomolecular pumps (Pfeiffer TMU 260), analog electronics (24 DAC voltages, 16 TTL triggers, rf quadrupole circuit, high-power excitation amplifier, mixer/ preamplifier for detection), dual pulsed valve inlet system, two ionization guages (Granville Phillips model 274 Bayard-Alpert type), quadrupole ion guide, and capacitively coupled closed cylindrical cell (similar to the design by Beu and Laude,34 but with additional external trapping plates). One vacuum system has an Analytica of Branford electrospray ion source, the other has an IonSpec MALDI ion source. The two systems share the magnet (Cryomagnetics 7-T active-shielded superconducting magnet with a 5-in. warm bore; 5-G line is ∼10 cm from the face of the magnet) and the data system [with a 40-MHz frequency synthesizer, an Arbitrary waveform generator (40 MHz, 1 megapoint), voltage sequence programmer (for controlling DACs as a function of time through the pulse sequence), pulse sequence generator (1 µs timing resolution), and transient digitizer (2 megapoints in acquisition and accumulation buffers)] and computer (pentium III 600 computer with 128 MB RAM, 8 GB hard drive). The MALDI mass spectrometer uses an IonSpec 10 faceted sample probe, a 377-nm N2 laser (Laser Science, Inc., Franklin, MA) with a motor-driven beam steering mirror (Newport Optics, Irvine, CA), a motor-driven focusing lens (Newport Optics, Irvine, CA), an iris, and a manually adjusted gradient filter for adjusting the laser spot power. The spot is imaged with a CCD camera (Panasonic model GP-KR222), and the signal is displayed on a color monitor (Sony model PVM-14N2M). The sample spots are deposited on the facets of the probe by depositing a 1-µL drop of saturated 2,5-dihydroxybenzoic acid (DHB) or 6-azo-2-thiothymine (ATT) matrix in methanol and allowing it to dry before adding a 1-µL spot of analyte typically dissolved at 10 µM in water with 0.1% trifluoroacetic acid. The plume generated by the laser desorption emerges normal to the target surface and at 45° to the entrance orifice to the mass spectrometer. The ions are then (30) Hofstadler, S. A.; Sannes-Lowery, K. A.; Griffey, R. H. Rapid Commun. Mass Spectrom. 1999, 13, 1971-1979. (31) Hofstadler, S. A.; Wu, Q. Y.; Bruce, J. E.; Chen, R. D.; Smith, R. D. Int. J. Mass Spectrom. Ion Processes 1995, 142, 143-150. (32) Maziarz, E. P.; Baker, G. A.; Lorenz, S. A.; Wood, T. D. J. Am. Soc. Mass Spectrom. 1999, 10, 1298-1304. (33) Bruce, J. E.; Anderson, G. A.; Hofstadler, S. A.; Vanorden, S. L.; Sherman, M. S.; Rockwood, A. L.; Smith, R. D. Rapid Commun. Mass Spectrom. 1993, 7, 914-919. (34) Beu, S. C.; Laude, D. A., Jr. Anal. Chem. 1992, 64, 177-180.

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extracted through the orifice (the sample surface is physically grounded, the “right” and “left” electrodes are typically biased at +50 Vdc for positive ions, and the “extraction” electrode is biased at -10 Vdc), into the rf-only quadrupole ion guide35-37 (biased at -40 Vdc, 100-300 Vbp, ∼1 MHz), where they traverse the magnetic field gradient and enter the high magnetic field region of the mass spectrometer. The ions are trapped using gated trapping with the outer trapping plates of a capacitively coupled closed cylindrical cell. For gated trapping, the trap plates typically are pulsed from ground potential to the trapping voltage of 20 V after a delay of approximately 50-100 µs from the laser pulse, varied according to the mass range expected, while the inner trapping rings are held constant at 0.5 Vdc. A gas pulse is then injected into the mass spectrometer through a pulse valve system (pressure in the analyzer stage of the vacuum system rises to ∼1 × 10-6 Torr for 2 s) for cooling the ions to the center of the cell. The ions are then held in the cell for 5-10 s while the pressure pumps back down to a base pressure of ∼1 × 10-10 Torr prior to excitation (frequency sweep from m/z 200 to 2500 in 4 ms at 60 V amplitude) and detection (acquisition rate of 1 MHz into 512k 12-bit data points). The signal is then apodized (quarter-sine), zero-filled, and magnitude mode fast-Fourier transformed. DISCUSSION Multishot Acquisition. For the multishot MALDI experiments (Figure 1B), the laser is pulsed at ∼10 Hz with the gated trapping event occurring ∼500-1500 µs after each pulse, depending on the required mass range. Instead of the front trapping plate being dropped to ground potential as is typically done, it is only dropped from the gated trapping potential of 20 to 1 Vdc so that ions trapped in previous events are not allowed to escape. With the pressure kept at ∼1 × 10-6 Torr during this time, the ions cool to the cell center prior to the next gating event. This method therefore accumulates ions in the cell over a series of MALDI events, effectively signal summing in the cell without adding additional noise. Growth in signal with the number of shots varied with the number of ions in the cell and will be discussed below. Stitched Pulse Sequences. As the commercial software we use to control the instrument does not allow more than 10 events for any TTL or DAC, we are limited to 9 shots plus the initialization event for each DAC in each pulse sequence. However, as the data system holds the state of any DAC or TTL at its final state at the end of a sequence, a series of pulse sequences can be devised to cover any conceivable application. In the simplest case, we used three pulse sequences (Figure 1). First, a “quench” sequence (Figure 1A) was used to dump any ions remaining in the cell and initialize the state of all TTLs and DACs; second was a “cellloading” pulse sequence (Figure 1B), which performed the multipulse MALDI ionization event and trapping as described above; and, finally, there was an “excitation/detection” pulse sequence (Figure 1C), which simply ramped the trapping voltages to 0.5 V, excited the ions into coherent cyclotron motion, and detected them. Figure 1C, however, also has several ion isolation (35) McIver, R. T., Jr. Int. J. Mass Spectrom. Ion Processes 1990, 98, 35-50. (36) McIver, R. T., Jr.; Hunter, R. L.; Bowers, W. D. Int. J. Mass Spectrom. Ion Processes 1985, 64, 67-77. (37) Huang, Y. L.; Guan, S. H.; Kim, H. S.; Marshall, A. G. Int. J. Mass Spectrom. Ion Processes 1996, 152, 121-133.

Figure 1. Pulse sequences: (A) a quench, (B) the typical multishot sequence used in this experiment, and (C) the excite/detect.

events and a sustained off-resonance irradiation (SORI)38 event defined, but with amplitude of 0 Vbp so that if one wishes to do MS/MS with the accumulated ions, one simply has to adjust the excitation voltage for the isolation and SORI pulses. Note that the first two sequences each contain a dummy excitation and detection pulses wherein the amplitude of the excite is zero and a minimal number of noise data points is acquired since the data system requires that a detection event exists in every pulse sequence. It is essential that the state of all DAC voltages and TTL triggers in the system not change between pulse sequences. In a typical experiment, the operator would load the quench sequence and run it one time. Then the operator would load the cell-loading sequence and run that a variable number of times (the maximum in the experiments reported here was a 900-shot loads9 shots/ sequence run 100 times), and finally the operator would load the excitation/detection pulse sequence to acquire the data. Signal averaging is typically done by averaging scans in the computer after acquisition. Thus, for n scans, one gets an increase of n in the signal with a concomitant n1/2 increase in noise from uncorrelated noise sources (i.e., white noise) for a total signal/ noise gain of n1/2. Since the FTMS stores ions in the trapped ion cell prior to excitation/detection, one can accumulate ions in the cell over time to improve the signal/noise ratio. The multishot experiment described above allows accumulation of ions in the cell prior to excitation for detection. This will, in theory, allow linear increase in the S/N ratio with number of laser shots, as signal is growing linearly, but the noise is constant (since it is (38) Gauthier, J. W.; Trautman, T. R.; Jacobson, D. B. Anal. Chim. Acta 1991, 246, 211-225.

only one “scan”). Figure 2 shows this effect with the peptide melittin (sequence: GIGAVLKVLTTGLPALISWIKRKRQQ-NH2). The single-shot experiment (Figure 2a) was done with the laser attenuated to approximately half its normal power to limit the number of ions per shot, while the 9-shot experiment used the exact same laser attenuation and setup, but simply fired the laser 9 times with 9 gated trapping events. In this case, the S/N ratio improved 20-fold for the 9 shots (much higher than the expected linear improvement in signal/noise ratio, as will be discussed below). Interestingly, the three peaks around m/z 3500 (marked with an asterisk), which are radio frequency interference signals from the turbo pumps, are remarkably constant in amplitude over time and hence provide a useful internal intensity calibrant. In Figure 3, the same multishot comparative experiment is done with the tryptic digest of a mutant form of the serum protein transthyretin,39-42 and the improvement is 2-fold for the same 9 shots, but in a condition where the single-shot spectrum already had almost saturated the cell. These data then suggest that multishot “in cell” signal averaging is very helpful for experiments where the initial ion number is very low and will improve signal very rapidly to the “space-charge” limit of the cell, but no farther. In Figure 2, it is also noted that the signal/noise ratio increased much more than the linear growth predicted with a 2.2n improvement. If consecutive pulse events were completely uncoupled, one would predict a linear improvement in signal, so this much higher improvement in signal/noise ratio simply means that having trapped ions in the cell prior to ion injection greatly increases trapping efficiency. Also, note that the signal growth was exactly the same on four replicate experiments that duplicated these results, indicating that shot-to-shot variability in MALDI is negligible, as expected from experience with this kind of sample at low signal level. This nonlinear improvement in trapping efficiency is very likely due to a “Coulombic collision” between the ions trapped in the cell and the newly arriving ions, but the actual mechanism of increase in trapping efficiency has two probable interpretations. One interpretation is that this increased efficiency is due to a mechanism similar to that used in the Bruker “Infinity” cell’s sidekick experiment43,44 in which the path length of the ions in the cell is lengthened and the chance of a collision with background gas is increased by addition of a magnetron moment to the ion motionsdue to a “glancing blow” collision that displaces the ions radially. Alternatively, the Coulombic collision may simply transfer a fraction of the momentum from the newly arrived ions to the trapped ions, which, if the trapping potentials are sufficiently high, will trap them both. Most likely, the true mechanism is a combination of the two. Shifted Multishot Experiments. A MALDI-FTMS is limited in m/z window by the time-of-flight effect.18 As the ions are created (39) Theberge, R.; Connors, L.; Skinner, M.; Skare, J.; Costello, C. E. Anal. Chem. 1999, 71, 452-459. (40) Theberge, R.; Connors, L.; Skare, J.; Skinner, M.; Falk, R. H.; Costello, C. E. Amyloid 1999, 6, 54-58. (41) Connors, L. H.; Theberge, R.; Skare, J.; Costello, C. E.; Falk, R. H.; Skinner, M. Amyloid 1999, 6, 114-118. (42) Theberge, R.; Connors, L. H.; Skinner, M.; Costello, C. E. J. Am. Soc. Mass Spectrom. 2000, 11, 172-175. (43) Caravatti, P.; Allemann, M. Org. Mass Spectrom. 1991, 26, 514-518. (44) Caravatti, P., U.S. Patent 4,924,089, issued 8 May, 1990.

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Figure 2. Sensitivity improvement below the space-charge limit: (A) single-shot experiment; (B) 9-shot experiment for ion accumulation in the cell.

Figure 3. Sensitivity improvement near the space-charge limit: (A) single-shot experiment; (B) 9-shot experiment for ion accumulation in the cell.

effectively instantaneously within the plume and are accelerated to the ICR cell, ions of different masses travel with different velocities and hence arrive at the cell at different times. Since the geometry and acceleration potentials are generally constant, the width of this time-of-flight m/z window varies with experimental conditions but is primarily a function of the velocity distribution of the ions and the length of the trap. The two current methods to work around this problem are in-cell MALDI10-12,16,45-51 and shifted accumulations.16,18,52 In the latter method, the TOF window is shifted in time over several scans and the signals are combined 5128

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afterward. Using in-cell ion accumulation, one can accumulate a series of laser shots in which each TOF window is shifted slightly in time, allowing sequential trapping of different regions of the mass spectrum. This procedure is demonstrated in Figure 4 with a tryptic digest mixture of peptides from transthyretin. Figure 4A (45) Easterling, M. L.; Pitsenberger, C. C.; Kulkarni, S. S.; Taylor, P. K.; Amster, I. J. Int. J. Mass Spectrom. Ion Processes 1996, 158, 97-113. (46) Wilkins, C. L.; Weil, D. A.; Yang, C. L. C.; Ijames, C. F. Anal. Chem. 1985, 57, 520-524. (47) Brown, R. S.; Wilkins, C. L. Anal. Chem. 1986, 58, 3196-3199.

Figure 4. Shifted multishot advantage: (A) typical time-of-flight trapping window optimized at 1300 Da; (B) shifted multishot experiment through the mass range.

shows a typical single-shot mass spectrum with the TOF trapping range adjusted to center on the m/z value of the base peak in the spectrum. Figure 4B shows the same sample with 9 laser shots accumulated under conditions where the TOF trapping range is shifted through the m/z window. In this case, this means that the delay time between laser shot and raising the front trapping plate from 1 to 20 V is varied from 400 to 1200 µs in 100-µs increments, corresponding to the windows of m/z ∼500 to ∼3000. The m/z window has widened in this case from m/z 700-1700 to 450-2700 with a noted 2-fold drop in the signal/noise ratio of the base peakspresumably due to ion loss while the ions undergo Coulombic collisions with newly incoming ions. Although this experiment has not yet been done, it is likely that reversing the trap timing direction so that higher m/z ions are trapped first will decrease these losses as the newly incoming ions will have less momentum and the already trapped ions will have more inertia. Selected Ion Accumulation. In the past, selected ion accumulation in the FTMS has been done primarily by using quadrupolar excitation under high-pressure conditions to keep selected ions axialized while the rest of the ions drift out of the cell over time.33,53-55 This works well, but the multishot acquisitions shown above (Figure 2) have demonstrated the ability to (48) Brown, R. S.; Weil, D. A.; Wilkins, C. L. Macromolecules 1986, 19, 12551260. (49) Coates, M. L.; Wilkins, C. L. Anal. Chem. 1987, 59, 197-200. (50) Castoro, J. A.; Wilkins, C. L. Anal. Chem. 1993, 65, 2621-2627. (51) Sheng, L.-S.; Covey, J. E.; Shew, S. L.; Winger, B. E.; Campana, J. E. Rapid Commun. Mass Spectrom. 1994, 8, 498-500. (52) Sze, T. P. E.; Chan, T. W. D. Rapid Commun. Mass Spectrom. 1999, 13, 398-406. (53) Bruce, J. E.; Vanorden, S. L.; Anderson, G. A.; Hofstadler, S. A.; Sherman, M. G.; Rockwood, A. L.; Smith, R. D. J. Mass Spectrom. 1995, 30, 124133. (54) Huang, Y. L.; Jackson, G.; Kim, H. S.; Guan, S. H.; Marshall, A. G. Phys. Scr. 1995, T59, 387-391. (55) Li, G. Z.; Vining, V. A.; Guan, S. H.; Marshall, A. G. Rapid Commun. Mass Spectrom. 1996, 10, 1850-1854.

accumulate ions at reasonably high pressures without needing quadrupolar excitation. Thus, the stitched sequence script (Figure 1) can be trivially modified to yield a much simpler version of selected ion accumulation from MALDI (Figure 5) using a shaped frequency-domain power spectrum such as that provided by SWIFT.56-59 The correlated harmonic method60 or frequency sweeps should work as well. For MALDI, one applies a SWIFT pulse to the “cell-loading” pulse sequence so that, after every few shots, all the ions that are not of interest are ejected from the cell. With this straightforward method, selected ions are accumulated very rapidly to the space-charge limit of the ICR cell. The benefits of this for tandem mass spectrometry experiments are significant, as high ion abundance in the first stage is vital for increasing the practical number of stages of MSn and performing MSn on ions that dissociate into many different fragments. CONCLUSIONS FTMS has the ability to trap ions in the cell and to keep a population of ions in the cell as another population of ions is introduced. This allows accumulation of ions through several MALDI ionization events. Provided that the initial ion abundance is low (i.e., for samples requiring high sensitivity) In-cell ion accumulation can dramatically improve signal/noise for the ions trapped. In-cell ion accumulation demonstrates that having ions (56) Marshall, A. G.; Wang, T.-C. L.; Ricca, T. L. J. Am. Chem. Soc. 1985, 107, 7893-7897. (57) Marshall, A. G.; Wang, T.-C. L.; Chen, L.; Ricca, T. L. In New Excitation/ Detection Techniques in Fourier Transform Ion Cyclotron Resonance Mass Spectrometry; Buchanan, M. V., Ed.; ACS Symposium Series 359; American Chemical Society: Washington, DC, 1987; pp 21-33. (58) Chen, L.; Marshall, A. G. Int. J. Mass Spectrom. Ion Processes 1987, 79, 115-125. (59) Chen, L.; Marshall, A. G. Rapid Commun. Mass Spectrom. 1987, 1, 3942. (60) Dekoning, L. J.; Nibbering, N. M. M.; Vanorden, S. L.; Laukien, F. H. Int. J. Mass Spectrom. Ion Processes 1997, 165, 209-219.

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Figure 5. Selected ion accumulation: (A) broad-band distribution of tryptic fragments; (B) selected ion accumulation of the m/z 1302 peak.

in the cell prior to trapping a subsequent packet of ions greatly improves trapping efficiency. The ability to accumulate ions in the cell also allows one to shift the trapping window across the spectrum for a more complete distribution of ions. Thus, one can generate a broad-band distribution of ions from a technique that is limited by time-of-flight shifted ion distributions. This method also allows selected ion accumulation when combined with an ion isolation technique such as SWIFT.

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ACKNOWLEDGMENT This research was supported by NIH Grant P41 RR10888 for the Boston University Mass Spectrometry Resource. The authors express thanks to E. Mirgorodskaya for assistance in sample preparation techniques. Received for review May 15, 2000. Accepted July 30, 2000. AC0005565