Tandem Time-of-Flight Mass Spectrometry with a Curved Field

Feb 21, 2004 - to enable the recording of mass-selected product ions formed by ... ticular, the curved-field reflectron enables the use of the full 20...
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Anal. Chem. 2004, 76, 1976-1981

Tandem Time-of-Flight Mass Spectrometry with a Curved Field Reflectron Robert J. Cotter,* Ben D. Gardner, Sergei Iltchenko, and Robert D. English

Middle Atlantic Mass Spectrometry Laboratory, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205

The unique focusing properties of the curved-field reflectron provide a simple solution to the problem of compensating for the broad range of energies of product ions produced postsource in a time-of-flight mass spectrometer. This has been shown previously for the technique known as postsource decay, but in this report we demonstrate its use for tandem time-of-flight mass spectrometry using a high-performance MALDI time-of-flight instrument modified by the addition of a collision chamber to enable the recording of mass-selected product ions formed by collision-induced dissociation (CID). In particular, the curved-field reflectron enables the use of the full 20-keV kinetic energy provided by the ion source extraction voltage as the collision energy in the laboratory frame and obviates the need to reaccelerate the product ions, using a second “source” or “lift” cell. Results are presented for the collision-induced dissociation of fullerenes over a range of collision gas pressures and precursor ion attenuation. In addition, CID tandem mass spectra are obtained for several peptides. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is well established as the method of choice for the analysis of many biologically relevant high molecular weight compounds. The minimal fragmentation typically observed in the MALDI process greatly facilitates molecular weight determination. More recently, interest is gaining in the development of tandem instruments using MS/MS analyses to characterize the structures (or sequences) of these high molecular weight compounds. While a number of tandem and hybrid instruments (ion traps, quadrupole-TOFs, etc.) now routinely provide MS/MS analyses using relatively low energy collisions, there is a particular interest in the tandem time-of-flight (TOF/ TOF) configuration as a means for providing high-energy collisioninduced dissociation. High-energy collisions are particularly important for the analysis of very high mass, singly charged ions. The primary challenge in recording the mass spectra resulting from high-energy collisions in a TOF/TOF mass spectrometer is the requirement for broad energy range focusing of the product ions. While the energy distribution of ions formed in the initial * Corresponding author. E-mail: [email protected].

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MALDI process is relatively small in relation to the total energy, the range of energies (E2) of product ions formed in any postsource process is broad, spanning the range from a few hundred electronvolts up to the precursor ion energy (E1) and scaling with the product ion mass:

E2 ) (m2/m1)E1

where m1 and m2 are the precursor and product ion masses, respectively. When a fragment or product ion is formed postsource, its velocity and its flight time remain the same as that for its precursor unless disturbed by an additional accelerating or decelerating field. Thus, for example, postsource decay (PSD) mass spectra are commonly obtained on reflectron instruments but not from linear (nonreflecting) mass analyzers. The problem then is that the range of energies encompassed by either PSD or collision-induced dissociation (CID) product ions exceeds the energy-focusing bandwidths of the common single-stage or dualstage reflectrons (Figure 1). As described further below, the solutions to that problem have included scanning or stepping the reflectron voltage (Figure 1a), reaccelerating product ions (Figure 1b), or using nonlinear reflectrons with wider bandwidth (Figure 1c). This problem was encountered earlier during our own development of a dual-reflectron TOF/TOF mass spectrometer in 1993. The first instrument was composed of two dual-stage reflectron mass analyzers and a collision region.1,2 Protonated molecular ions, formed by MALDI were mass-selected at the focal point of the first reflectron and just before entering the collision region where they were dissociated by collisions with an inert gas introduced via a pulsed valve system. Pulsing the collision gas pressure enabled very high attenuations of the molecular ion beam, while maintaining high vacuum throughout the mass spectrometer without differential pumping. The resultant product ions were then mass analyzed by a second reflectron analyzer. Unfortunately, though providing higher order focusing than the single-stage reflectron, the dual-stage reflectron focuses only a very narrow range of energies. This means that the product ion mass range that could be focused in the tandem was also limited and in fact (1) Cotter, R. J.; Cornish, T. J. A Anal. Chem. 1993, 65, 1043-1047. (2) Cotter, R. J.; Cornish, T. J. Tandem Time-of-Flight Mass Spectrometer. U.S. Patent 5,202,263, April 13, 1993. 10.1021/ac0349431 CCC: $27.50

© 2004 American Chemical Society Published on Web 02/21/2004

Figure 1. Approaches used to match the broad kinetic energy range of product ions to the bandwidth of the reflectron mass analyzer. (a) In PSD instruments, the dissociation of 20-keV molecular ions produces product ions with energies ranging from a few to 20 keV (shown as a series of parallel horizontal lines). Because this range of energies is broader than the reflectron energy bandwidth (shown in gray), the energy acceptance window is scanned or stepped by changing the voltage at the back of the reflectron analyzer. (b) In TOF/TOF mass spectrometers it is more common to address this problem by carrying out collisions at lower (1-2 keV) energy, either by using a lower acceleration voltage or by slowing the ions down prior to collision. The resultant lower product ion bandwidth can then be focused by additionally accelerating the ions before entering the reflectron. (c) Nonlinear reflectrons, such as the curved field reflectron, can provide higher bandwidth, thus eliminating the need for scanning the acceptance window or compressing the product ion energy range by deceleration and reacceleration of the ions.

included only those ions within the top 10% of the product ion mass range, that is: those that penetrated the second reflectron stage. A later version of this instrument3 used two single-stage reflectrons and had the advantage that product ions could be recorded over a broader mass range (and on a linear mass scale), though again, product ions with masses close to the precursor mass were better focused than lower mass ions. We note that, although pulsed extraction was not used in this instrument, mass resolutions as high as 1 part in 11 000 (gramicidin, m/z 1142) were realized for molecular ions focused through both reflectrons. In 1993, Enke and co-workers4,5 had also designed a tworeflectron tandem time-of-flight mass spectrometer but used photodissociation to form the product ions. They addressed the product ion focusing problem by first decelerating the primary ions prior to dissociation in order to reduce the range of product ion energies (Figure 1b) and then reaccelerating the product ions. In this way, the final kinetic energies could be accommodated by the bandwidth of the reflectron. This approach in some form is used in current commercial TOF/TOF instruments; however, it was not advantageous in our instrument in which it was intended to use the full, initial kinetic energy (in this case 4 keV) for effective collision-induced dissociation. Thus, in 1993 we developed (3) Cornish, T. J.; Cotter, R. J. Org. Mass Spectrom. 1993, 28, 1129-1134. (4) Seterlin, M. A.; Vlasak, P. R.; Beussman, D. J.; McLane, R. D.; Enke, C. G. J. Am. Chem. Soc. 1993, 4, 751-754. (5) Beussman, D. J.; Vlasak, P. R.; McLane, R. D.; Seeterlin, M. A.; Enke, C. G. Anal. Chem. 1995, 67, 3952-3957.

the curved field reflectron as the second mass analyzer in the tandem time-of-flight instrument6,7 to enable focusing throughout the entire product ion mass range without the need for reacceleration of the ions (Figure 1c). (A similar instrument using a quadratic reflectron as the second mass analyzer has more recently been reported by Derrick et al.8) Following the introduction of the PSD technique by Spengler et al.,9 this high-bandwidth, nonlinear field reflectron was then used in single-reflectron mass spectrometers10 to enable focusing of PSD ions without scanning or stepping the reflectron voltage. Thus, the curved field reflectron addressed the focusing problem for both tandem CID and PSD. In a tandem mass spectrometer, it is not necessary that both mass analyzers be reflectrons. In fact, there is a long history of tandem TOF mass spectrometers that utilize linear mass analyzers. In 1992 Jardine et al.11 used two linear mass analyzers separated by a collision cell, where the product ions were reaccelerated to disperse their velocities and their flight times. An instrument reported earlier in 1989 by Cooks et al.12 used a linear TOF as the first mass analyzer, a surface for inducing collisions, and a second reflectron mass analyzer that was orthogonal to the initial flight direction. In 1992, Schlag et al.13 described and patented14 a tandem configuration in which the first mass analyzer was the drift region between the source exit and the second-order space focus plane, which then served as the focal point for a second (dual-stage reflectron) mass analyzer. Current commercial tandem time-of-flight instruments have substituted simple space focusing into a nonreflecting first mass analyzer with some form of pulsed extraction focusing and combined this with acceleration of the product ions into a reflecting mass analyzer as shown in Figure 1b. In the tandem instrument described by Vestal and co-workers15,16 and commercialized by Applied BioSystems (Framingham, MA), ions are formed by MALDI, focused by pulsed (or delayed) extraction, mass-selected by a timed ion gate, and then decelerated by a retarding lens to energies of 1-2 keV before entering a collision cell where they are dissociated. Product ions formed in the collision cell continue to have the same velocities as their massselected precursors, so that they all enter a second “source” at the same time, where they are accelerated into the reflectron mass (6) Cornish, T. J.; Cotter, R. J. Rapid Commun. Mass Spectrom. 1993, 7, 10371040. (7) Cornish, T. J.; Cotter, R. J. Non-Linear Field Reflectron. U.S. Patent 5,464,985, November 7, 1995. (8) Giannakopulos, A. E.; Thomas, B.; Colburn, A. W.; Reynolds, D. J.; Raptakis, E. N.; Makarov, A. A.; Derrick, P. J. Rev. Sci. Instrumen. 2002, 73, 21152123. (9) Kaufmann, R.; Kirsch, D.; Spengler, B. Int. J. Mass Spectrom. Ion Processes 1994, 131, 355-385. (10) Cornish, T. J.; Cotter, R. J. Rapid Commun. Mass Spectrom. 1994, 8, 781785. (11) Jardine, D. R.; Morgan, J.; Alderdice, D. S.; Derrick, P. J. Org. Mass Spectrom. 1992, 27, 1077-1083. (12) Schey, K. L.; Cooks, R. G.; Kraft, A.; Grix, R.; Wollick, H. Int. J. Mass Spectrom. Ion Processes 1989, 94, 11-14. (13) Boesl, U.; Weinkauf, R.; Schlag, E. W. Int. J. Mass Spectrom. Ion Processes 1992, 112, 121-166. (14) Boesl, U.; Schlag, E. W.; Walter, K., Weinkauf, R, MS-MS Time-of-Flight Mass Spectrometer. U.S. Patent 5,032,722, July 16, 1991. (15) Medzihradszky, K. F.; Campbell, J. M.; Baldwin, M. A.; Falik, A. M.; Juhasz, P.; Vestal, M. L.; Burlingame, A. L. Anal. Chem. 2000, 72, 552-558. (16) Yergey, A. L.; Coorssen, J. R.; Backlund, P. S. Jr.; Blank, P. S.; Humphrey, G. A.; Zimmerberg, J.; Campbell, J. M., Vestal, M. L. J. Am. Soc. Mass Spectrom. 2002, 13, 784-791.

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Figure 2. Diagram of the TOF/TOF mass spectrometer showing the location of the collision chamber, the mass selection ion gate at the focal point for the first mass analyzer, and the second mass analyzer with a curved field reflectron.

analyzer by pulsed extraction. Note that in order to accommodate the limited bandwidth of the reflectron, the kinetic energy of the precursor ions (and hence the collision energy in the laboratory frame) is kept at 1-2 keV, with the second “source” providing an additional 18 keV. The resultant range of kinetic energies from 18 to 20 keV can be accommodated by the reflectron. It should also be noted that fragment ions formed by PSD or other metastable processes prior to the retarding lens will not be passed by the gate (since their velocities are different), while those formed after the second source will have different flight times from their isomass counterparts formed in the collision chamber. These late-forming fragment ions interfere with the product ion mass spectrum and so are removed by a “metastable suppressor” or deflection system. In the instrument designed at Bruker Daltonics (Billerica, MA), initial kinetic energies (and laboratory collision energies) are in the range of 8 keV, with the additional acceleration of the product ions provided by raising the potential energy of the product ions as they pass through a “lift cell”.17 Because no retarding system is used in the precursor beam, ions formed by PSD in that region will have the same flight times as those formed by CID and so will contribute constructively to the product ion mass spectrum. Ions formed by metastable processes after leaving the lift cell must again be removed electrostatically. In this work, we have modified a commercial MALDI time-offight mass spectrometer equipped with a curved field reflectron in order to demonstrate that tandem mass analysis can be carried out without reaccelerating the product ions and that CID can be carried out at the full 20-keV collision energy (laboratory frame) provided by the source voltage. Because the curved field reflectron addresses both PSD and CID processes in the same way, there is no operational difference between PSD and tandem experiments with no collision gas. In this paper, we describe the design and basic features of the tandem instrument and report results on the collision-induced dissociation mass spectra of fullerenes and peptides. The fullerenes were studied in some detail in our 1993 instrument18 and have provided the opportunity to describe the (17) Schnaible, V.; Wefing, S.; Resemann, A.; Suckau, D.; Bu ¨ cker, A.; WolfKu ¨ mmeth, S.; Hoffman, D. Anal. Chem. 2002, 74, 4980-4988. (18) Cordero, M. M.; Cornish, T. J.; Cotter, R. J. J. Am. Soc. Mass Spectrom. 1996, 7, 590-597.

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effects of collision gas pressure on precursor ion attenuation and fragmentation. EXPERIMENTAL SECTION The TOF/TOF mass spectrometer was developed by modifying a Kratos Analytical (Manchester, England) AXIMA-CFR highperformance MALDI time-of-flight mass spectrometer equipped with a curved field reflectron. To cause the least disruption to the existing instrument, a collision chamber was mounted at the top of the ion source and ion focusing optics in the region ahead of a Bradbury-Nielsen19 mass selection gate (Figure 2). Because product ion velocities are not altered appreciably by collisions nor as the result of metastable decay, a precursor and its associated product ions will pass through the mass selection gate at the same time whether the collision chamber is located before or after the mass selection gate. This implies that the collision chamber can be placed in any location between the ion source exit and the entrance to the reflectron, but this location was chosen to enable easy insertion of a collision chamber mounted on a common and easily removable source flange. Because no precursor ion deceleration or product ion acceleration is utilized, the collision energy (in the laboratory frame) is derived from the full accelerating voltage (20 kV). In addition, PSD products produced in any location between the source and reflectron will have the same flight time as CID products and will contribute to the product ion spectrum. The collision chamber was constructed from a stainless steel cylinder (1.13 in. long, 0.2-in. i.d.) through which the ions pass. The collision gas is injected into the chamber through a long (2 m) 0.070-mm-i.d. glass capillary tube at a flow rate of 0-1 mL/ min. Because of the low conductance of the collision chamber, the collision gas density within the cylindrical chamber is ∼2 orders of magnitude higher than in the source region and sufficient to provide efficient activation, though the presence of the gas does raise the ambient pressure in both the source and analyzer regions. This does result in some loss of mass resolution but does provide the opportunity to demonstrate focusing of product ions across the full range of product ion energies (i.e., 1-20 keV). Because the current configuration did not include the capability for directly monitoring the pressure in the collision (19) Bradbury, N. E.; Nielsen, R. A. Phys. Rev. 1936, 49, 388-393.

chamber, both the collision gas back pressure (measured using a 0-80 psi regulator) and the source pressure (in Torr) were monitored. Buckminsterfullerene (C60) and R-cyano-4-hydroxycinnamic acid (ACHA) were obtained from Aldrich (Milwaukee, WI) and used without further purification. C60 samples were prepared as saturated solutions in benzene (Baker Laboratories, Phillipsburg, NJ), transferred directly to the sample plate and allowed to dry. The peptides bradykinin fragment 2-9 (MW 904.0) and substance P were obtained from Sigma Chemical (St. Louis, MO) and used without further purification. The 0.5-µL samples of peptide solutions in water were deposited on the sample plate and mixed with 0.5µL of a saturated solution of ACHA in a 1:1 mixture of acetonitrile and 0.1% TFA in water. High-purity helium (99.999%) was obtained from Connecticut Gas, Inc. (Stratford, CT). Mass spectra shown were obtained using 50 laser shots. RESULTS AND DISCUSSION In the instrument developed in 1993,1 the accelerating voltage was 4 kV, so that it was necessary to utilize the maximum available projectile ion energy as collision energy. This was possible using the curved field reflectron, which now provides an opportunity for utilizing up to 20-keV projectile energies. It should be noted that this defines the collision energy in the laboratory frame Elab. In the center of mass frame, the collision energy Erel depends on the masses of the projectile ion and the collision gas and is given by

Erel )

1 mionmgas v 2 2 mion + mgas rel

where vrel is the relative velocity. Relative to the high kinetic energies of the ions, the thermal velocities of the inert gas molecules are negligible, so that the relative energy becomes

Erel )

1 mionmgas 2 v 2 mion + mgas

where v is the velocity of the projectile ion. The relative energy is then related to the laboratory energy by

Erel )

mgas E mion + mgas lab

The relative energy is small for a large molecule colliding with helium. In our previous 4-keV instrument, the collision energy for C60 with helium was only 11 eV. In the current instrument, the 20-kV accelerating potential produces a collision energy (in the center-of-mass frame) of 55.4 eV for the collision of fullerene with helium. Fullerenes. Figure 3 shows the helium-induced dissociation mass spectra obtained for C60. Figure 3a is the mass spectrum of the fullerene with no gas, and Figure 3b-f are spectra with

Figure 3. Tandem CID analysis of buckminsterfullerene C60: (a) spectrum of C60 following application of the ion gate to select m/z 720 with no helium gas; (b-f) increasing pressure of helium gas in the collision chamber, monitored by measurements of the backing pressure to the capillary inlet (in psi) and the ion source vacuum gauge (in Torr).

increasing amounts of helium added to the collision cell. The initial fragments that first appear in Figure 3b are the C2n+ series of Analytical Chemistry, Vol. 76, No. 7, April 1, 2004

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ions, with C44+ and C50+ being the dominant clusters.18,20-25 It has been shown by reionization of the neutral products that this series results from losses of large Cn neutrals rather than stepwise losses of C2.25 In that study, their observation of C28 as the largest neutralis consistent with our current observation that the smallest C2n+ cluster ion is the C32+ ion. Beginning with Figure 3d, a distribution of lower mass clusters is observed in the CID spectra, and these clusters differ by one carbon. From ion intensity measurements, we estimate that this spectrum corresponds to an attenuation of the molecular ion beam of ∼80%. These lower mass clusters increase in intensity in the mass spectra of Figure 3e and f, for which the estimated attenuations are 95 and 98%, respectively. The appearance of the low-mass Cn+ series at high attenuation suggests that these ions result from multiple or “catastrophic” collisions20 that result in a mass spectrum with predominant peaks at C11+, C15+, C19+, and C23+ that is similar to that observed in the laser ablation of graphite.26-29 In our previous work, we noted that multiple collisions were also consistent with the low-mass resolution we observed for the Cn+ series compared with the well-resolved C2n+ series.19 Ion kinetic energies were 4 keV in that instrument. In this case, the 20-keV ion kinetic energies are not appreciably altered by collisions with helium, so that resolution is maintained for the Cn+ series. An important feature of this and other MS/MS spectra obtained from this instrument is that the entire product ion mass range is focused by the curved field reflectron, though ion energies range from only a few hundreds of electronvolts to 20 keV. Peptides. Tandem CID mass spectra were also obtained for two peptides. Figure 4a shows the gated mass spectrum of substance P, that is: the protonated molecular ion has been massselected, there is no collision gas, and the laser power is sufficiently low that fragmentation from postsource decay is also low. Panels b-d of Figure 4 show the effects from increasing the amount of helium added to the collision chamber under the same laser power. The a and a-17 series dominate the CID mass spectrum, with the lower mass sequence ions increasing with increasing collision gas pressure. It is important to emphasize that some fragment ions (a4 for example) are observed in the CID spectrum only. Mass spectra for the peptide bradykinin fragment 2-9 are shown in Figure 5. The spectrum in Figure 5a is a normal ungated mass spectrum, where the lower mass ions are primarily fragments formed in the ion source. Figure 5b shows the massselected molecular ion spectrum, while panels c and d of Figure (20) Young, A. B.; Cousins, L. M.; Harrison, A. G. Rapid Commun. Mass Spectrom. 1991, 5, 226-229. (21) Doyle, R. J., Jr.; Ross, M. M. J. Phys. Chem. 1991, 95, 4954-4956. (22) Ross, M. M.; Callahan, J. H. J. Phys. Chem. 1991, 95, 5270-5273. (23) Weiske, T.; Bo¨hme, D. K.; Hrusˇa´k, J.; Kra¨tschmer, W.; Schwarz, H. Angew. Chem., Int. Ed. Engl. 1991, 30, 884-886. (24) Caldwell, K. A.; Giblin, D.; Gross, M. L. J. Am. Chem. Soc. 1992, 114, 37433756. (25) McHale, K. J.; Polce, M. J.; Wesdemiotes, C. J. Mass Spectrom. 1995, 30, 33-38. (26) Bloomfield, L. A.; Geusic, M. E.; Freeman, R. R.; Brown, W. L. Chem. Phys. Lett. 1985, 121, 33-37. (27) Heath, J. R.; O’Brien, S. C.; Zhang, Q.; Liu, Y.; Curl, R. F.; Kroto, H. W.; Tittel, F. K.; Smalley, R. E. J. Am. Chem. Soc. 1985, 107, 7779-7780. (28) O’Keefe, A.; Ross, M. M.; Baronavski, P. Chem. Phys. Lett. 1986, 130, 1719. (29) McElvany, S. W.; Creasy, W. R.; O’Keefe, A. J. Chem. Phys. 1986, 85, 632633.

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Figure 4. Tandem CID analysis of the peptide substance P: (a) spectrum of substance P following application of the ion gate to select m/z 1349 with no helium gas; (b-d) increasing pressure of helium gas in the collision chamber, monitored by measurements of the backing pressure to the capillary inlet (in psi).

5 show CID mass spectra upon increasing helium gas pressure. In Figure 5d, the molecular ion is considerably more attenuated relative to the product ions. CONCLUSIONS In the tandem instrument, which we reported in 1993,1 a pulsed valve was used to provide high collision gas density without the

exist for a continuous gas flow. With the high laser repetition rate (4-10 Hz) used in this instrument, a pulsed system such as this would be impractical. The simple cylindrical collision chamber used in this instrument provides high collision gas density under continuous gas flow, while maintaining good ion transmission and a minimal load on the vacuum system. At the highest collision gas pressure shown in Figure 3f, the attenuation of the molecular ion is more than 90%, while the vacuum is raised to 3.9 × 10-5 Torr. While this simple modification to the instrument provides an excellent demonstration of the ability of the curved field reflectron to focus the kinetic energies over a broad product ion mass range, it nonetheless suggests some specific improvements to mass resolution and ion transmission that might be realized on a well-engineered and optimized design. Specifically, the location of the collision chamber in the forward region of the first flight tube means that low-angle scattering resulting from collisions in the collision chamber will more adversely affect entry of the product ions into the reflectron than will a collision chamber located just before, or just after, the mass selection gate. In addition, the current design of the collision chamber itself relies on balancing the conductances without the benefit of differential pumping to achieve high attenuation in the chamber and high vacuum in the analyzer regions. Both of these aspects of the current design limit the product ion mass resolution, and in future work, we intend to address these problems through more aggressive modification to the instrument. In this effort we have used as the laboratory collision energy the maximum precursor ion kinetic energy available from acceleration from a 20-kV ion source. This is, of course, possible because there is no need to reaccelerate the product ions to meet the energy bandwidth requirements of the reflectron. It also provides the highest relative collision energy (in the center-ofmass frame) for larger ions. At the same time, we anticipate the use of lower collision energies, achieved by using lower accelerating voltages and again without the need for reacceleration. Additionally, it is planned to investigate the effects of collisions with heavier gases, such as argon and xenon. This instrument in fact provides an excellent opportunity to investigate high-energy collisions for biological molecules over a wider range than has been hitherto possible on a TOF/TOF mass spectrometer.

Figure 5. Tandem CID analysis of the peptide bradykinin fragment 2-9: (a) ungated reflectron mass spectrum, (b) mass spectrum of gated molecular ion, (c) CID mass spectrum using 10 psi helium, and (d) CID mass spectrum using 40 psi helium.

need for differential pumping to maintain high vacuum in the mass analyzer regions. The gas pulse width in that instrument was estimated to be ∼100 ms, and with a laser pulse repetition rate of 1 Hz, this reduced the gas load to ∼10% of the load that would

ACKNOWLEDGMENT This project has been funded in whole or in part with Federal funds from the National Heart, Lung and Blood Institute, National Institutes of Health, under Contract HV-28180 and by a grant R01GM/RR64402 to R.J.C. from the National Institutes of Heath.

Received for review August 12, 2003. Accepted December 12, 2003. AC0349431

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