Anal. Chem. 1993, 65, 1043-1047
Tandem Time-of-Flight Mass Spectrometer Timothy J. Cornish and Robert J. Cotter' Department of Pharmacology and Molecular Sciences, Middle Atlantic Mass Spectrometry Laboratory, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
A compact, laser desorption tandem tlmeof-flight ma88 spectrometer b deocrlbed. The Instrument Incorporates two dualetago reflectron analyzers and a collirlon region for produclng product' Ions by collldon-Induced dissociation. Selection of Ions of a particular mass Is accomplished by deflection of Ions from stable trajectory angles entering the second reflectron, while the use of a pulsed valve for lntroductlon of the colHsion gas obvlatesthe needfor differential pumping of the collldon region. Inltlal results are presented, as well as a dlscuscllon for optlmlzlng the performance of tandem tlme-of-fllght instruments.
INTRODUCTION Tandem mass spectrometry (MS/MS) has, over the last 10 years, proven to be a most effective method for elucidating molecular structure, especially for the analysis of biological compounds. In most cases, structural information is provided by collision-induced dissociation (CID)in the region between the two mass analyzers. High-energy collisions are generally used in high-performance four-sector instruments (EBEB), while low-energycollisionsare common for triple-quadrupole (QqQ) and hybrid (EBqQ) tandem mass spectrometers. Instrumentation, particularly in the case of four-sector mass spectrometers, is extremely costly and prohibitively cumbersome for routine use as an analytical technique in all but a few biochemically-orientedresearch centers. For thisreason, there have been a number of attempts to introduce timeof-flight mass analyzers into either hybrid or stand-alone tandem configurations with the common goal of simplifying the complex instrumentation and reducing the size and expense of the MSIMS technique. One such example is the hybrid instrument under development by Russell and co-workers,lP2 in which a doublefocusing sector mass analyzer is used to mass select the ion of interest at high resolution, followed by collision-induced dissociation and analysis of the product ions in a reflectron3 time-of-flight mass analyzer. More commonly, MS/MS spectra have been obtained directly from reflectron timeof-flightinstruments. In the instrument described by Duncan et al.,4 electrically-gatedions were photodissociated by a laser focused at the turn-around region in the rear of a reflectron, producing product ion mass spectra of tin clusters with reasonable resolution. In another instrument developed by Schlag et al.,5 mass selected ions are tightly space-focused at a point in the first linear region of a reflectron mass spectrometer where they intersect the beam of a photodissociation laser. Product ion mass spectra of benzene and its (1) Strobel, F. H.; Solouki, T.;White, M. A.; Russell, D. H. J.Am. SOC. Mass Spectrom. 1990,2,91-94. (2) Strobel, F. H.; Preston, L. M.; Washburn, K. S.; Russell, D. H. Anal. Chem. 1992,64, 754-762. (3) Mamyrin,B. A.; Karatajev,V. J.; Shmikk, D. V.; Zagulin, V. A. Sou. P h p . JETP 1973, 37,45-48. (4) Cornett, D. S.;Peschke, M.; LaiHing, K.; Cheng, P. Y.; Willey, K. F.; Duncan, M. A. Reu. Sci. Instrum. 1992,63, 2177-2186. (5) Weinkauf, R.; Walter, K.; Weickhardt, C.; Boesl, U.; Schlag, E. W. 2. Naturforsch. 1989,44a, 1219-1225. 0003-2700/93/0365-1043$04.00/0
fragments were generated by scanning the reflectron voltages to maintain resolution through the mass range. The correlated reflex technique reported by LeBeyec and co-workers6 and later used by Standing et al.7 produces product ion mass spectra by recording reflected ions coincident with neutral species detected behind the reflectron. Ion gating is not necessary in this approach, which is restricted to ionization schemes in which a very few ions are produced on each timeof-flight cycle and can be recorded using time-to-digital conversion (TDC) techniques. In addtiion, the method has been used exclusively for obtaining spectra resulting from metastable (unimolecular) decomposition. Cooks et al.8 described a tandem instrument incorporating two linear time-of-flight mass analyzers, using surfaceinduced dissociation (SID)to produce the product ions. In a later version of their instrument? a reflectron configuration was used as the second mass analyzer (MS2). More recently, Jardine et al.10 have described a tandem mass spectrometer using a floating collision cell and two linear time-of-flight mass analyzers for observingthe products of charge exchange reactions and collision-induced dissociation. In this paper, we report our fiit results in obtainingproduct ion mass spectra in a tandem time-of-flight(TOF/TOF) using high-energy collision-induceddissociationand two dual-stage reflectron analyzers. High-energy collision-induced dissociation has become a reliable and effective method for the structural (amino acid) analysis of peptides, while photodissociation and surface-induced dissociation have been utilized primarily for small organic compounds and metal clusters. At the same time, the task of introducing a highpressure region into an instrument whose ion transmission advantage is derived (in part) from the absence of slits or small orifices is not straightforward and is the major reason that other investigators have generally opted for laser beams or surfaces to induce fragmentation. However, our goal has been to develop a compact, inexpensive tandem instrument forthestructuralanalyaisofpeptideawithunitmassresolution up to 3000 Da. By using high-energy collisions, such an instrument would, in fact, provide amino acid sequencing capabilities similar to those that are now obtained on foursector tandem instruments. In a recent paper,ll we introduced this compact time-offlight mass spectrometer and demonstrated its operation in the single reflectron mode. We have since completed the installation of the second reflectronsector,the collision region, and the mass gating optics. Here we present our initial results using the instrument in the double reflectron mode (no collisions) and as a tandem instrument. (6) Della-Negra, S.; Le Beyec, Y. A d . Chem. 1985,57, 2036. (7) Standing, K. G.;Beavis, R.; Bollbach, G.; Ens, W.; Lafortune, F.; Main, D.; Schueler, B.; Tang, X.; Westmore, J. B. AM^. Instrum. 1987, 16, 173. (8)Schey, K. L.; Cooks, R. G.; Grix, R.; Wollnik, H. Int. J. Mass Spectrom. Zon Processes 1987, 77, 4 H 1 . (9) Schey, K. L.; Cooks,R. G.; Kraft, A,; Grix, R.; Wollnik, H. Int. J. Mass Spectrom. Zon Processes 1989,94,1-14. (10)Jardine, D. R.; Morgan, J.; Alderdice, D. S.; Derrick, P. J. Org. Mass Spectrom. 1992,27, 1077-1083. (11) Cornish, T.;Cotter, R. J. Rapid Commun. Mass Spectrom. 1992, 6, 242-248. 0 1993 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 85, NO. 8, APRIL 15, 1993
P T I P L 2500 N Ilrogen L o s e r
loour
I i
i
digital oscilloscopeand a Stanford Research Systems (Palo Alto, CA) Model DG 535 delay generator which serves as the timing device for the mass selection gate pulse generator. The ion source voltage was 4 kV while both flight tubes and the collision chamber were generally set at ground potential, providing collision energies of 4 keV. In the second reflectron, the grid terminating the first stage was 1600 V while the final grid was set at 4020 V to ensure that ions with energies up to 4 keV are reflected. The instrument shown in Figure 1is equipped with two grids placed after the collision chamber for postaccelerating the product ions. In one of the experiments described below, -500 V was applied to the second of these grids, to the second flighttube, and to the entrance grid of the secondreflectron to improve product ion extraction. Additionally, the final reflectron grid voltage was reduced to 3950 V to improve mass resolution for the product ions. The spectra shown represent between 20 to 30 single-shot spectra add/averaged by the digital oscilloscope prior to downloading to the host computer. a-Cyano-4-hydroxycinnamicacid, gramicidin S, and caffeic acid (used as the sample matrix) were obtained from Sigma (St. Louis, MO). Rhodamine 590 was obtained from Exciton (Dayton, OH). For comparison with the tandem time-of-flightinstrument, a collision-induceddissociation mass spectrum for rhodamine was also obtained on a Kratos (Manchester,U.K.)CONCEPT 1H double-focusing mass spectrometer using fast atom bombardment (FAB) ionization.
I'. W .,. ! ".; AVTECH AVR-GC
1 GATE
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Flgurr 1. Diagram of the tandem timeof-fllght mass spectrometer.
EXPERIMENTAL SECTION The compact time-of-flightmass spectrometer shown in Figure 1has been described in detail previous1y.l' The vacuum housing
is a 32 in. (length) X 8.4 in. (width) X 6 in. (depth) welded aluminum coffin chamber, pumped by a Balzers (Hudson, NH) Model TPH270, 270 L/s turbomolecular pump. The mass spectrometer includes two dual-stage reflectron analyzers connected to one another through a collision region, consisting of several deceleratingand acceleratinglenses. The ionization region utilizes single-stageextraction of ions in a uniform high-voltage field defined by a backing plate and extraction grid, followed by a three-element Einsel lens system for focusing and a steering lens. The stainless steel sample probe surface is flush with the backing plate, and the laser beam is incident on this surface at a shallow angle of 6O. The detector is a matched pair of Galileo (Sturbridge, MA) 1-in. channelplates, fitted with a shielded, conical anode to improve detector rise time and reduce ringing. Ions are formed using a PTI (PhotonTechnologyInternational, Ontario, Canada) Model PL2300 1.2 mJ, 600 pS pulsed nitrogen laser. The laser beam is attenuated using a neutral density optical wedge and focused with a 2.5-cm quartz lens. The analog signal is captured and digitized by a Tektronix (Beaverton, OR) Model TDS-5401G sample/s transient recorder/digitaloscilloscope with a bandwidth of 500 MHz. The transient recorder is triggered by the output from an Electro-Optics Technology (Freemont, CA) Model ET2010 fast photodiode placed in the unfocused beam of the nitrogen laser. Spectra were downloaded to a 486/33 MHz PC and summedand plotted using TOFWARE,a data acquisition system for time-of-flightmass spectrometers availablefrom ILYS Software (Pittsburgh, PA). Selection of ions of a given mass is provided by a 200-V deflection pulse, obtained from an AVTEC (Ogdensburg, NY) Model AVR-G-CVpulse generator and applied to one of the four steering elements adjacent to the collision cell. While the amplitude of this pulse is not sufficient to exclude high-energy ions from the collision chamber and the second mass analyzer, the resultant deflection angle gives rise to unstable trajectories for these ions in the reflectron, so that they are not recorded by the detector. Thus, this approach obviates the need for narrow slits or orifices to provide mass selection. The collision gas is introduced by an NRC (Fountain Valley, CA) Model BV 100pulsedvalve. The nozzle orifice was modified by attaching a needle extension to delivera high density of neutral argon directly at the collision site. The pulsed jet has a duration (FWHM) of approximately 200 ps and is sufficiently short to eliminate the need for differential pumping to prevent significant increases in analyzer pressure, even at 100% attenuation of the ion beam. The timing sequence begins with the firing of the pulsed valve. A BNC (Berkeley, CA) Model 810 pulse generator then provides a delay of approximately 100 ps before firing the nitrogen laser. The laser pulse is detected by the photodiode which triggers the
THEORY OF OPERATION The time-of-flight ( t )of an ion is generally approximated by the time that it spends in a field-free drift region:
t = [m/2eV]'/2 L = km1I2
(1) where m is the mass of the ion, e is the charge on an electron, V is the accelerating voltage, and L is the length of the drift tube. When the time-of-flight mass spectrometer includes a reflectron, the ions decelerate and re-accelerate, each with times corresponding to t = [2m/eV]'/2 d = kJrn'l2
(2) where d is the depth of the reflectron. In effect, ions travel through two drift regions (LIand Lz,where L = L1 + L2) and twice through the reflectron distance (d), so that their total flight times
+
t = [m/2eV]'/2 (L, L, + 4d) = k"m1/2
(3) still follow a square root dependence upon mass. Thus, empirical equations for calibrating the mass scale:
+
t = am'I2 b (4) where the constants a and b are determined from the flight times of known masses are generally valid for reflectron as well as linear instruments. In the tandem time-of-flight mass spectrometer described here, the ions arrive at the collision chamber at times that are predicted by eq 3, which can be used to determine the time for application of the ion gate pulse. As they leave the collision chamber, both precursor and product ions will continue to have the same velocities in the drift region, but will have different kinetic energies: KE, = [mdm,]eV (5) where ma is the mass of the precursor ion, and mb is the mass of the product ion. Because they enter the reflectron with different energies, they will penetrate the retarding field at different depths before turning around d = m,V/m,E (6) where E is the field strength (V/d) in the reflectron. Thus,
ANALYTICAL CHEMISTRY, VOL. 85, NO. 8, APRIL 15, 1993
their flight times (t’) in the reflectron are given by
I
1045
I
= 2[2rn,d/e~]’/~= 2 [ 2 m ~ ~ / e ~ ~ m=, km, ] ’ / ~ (7) As they exit the reflectron, both precursor and product ions will continue to have their same, original velocities although they will be separated in time and space. Thus, both precursor and product ion spend the same time (t”) in the two drift regions. A mass scale can then be established between the time of the ion gate pulse and the appearance of the precursor ion: t’b
tb
= [mdm,lt’,
+ t”,
m
lbo
120
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(8)
which shows a linear dependence on mass. In the tandem time-of-flight instrument reported here, the two reflectrons are used differently. The first reflectron provides energy focusing, while the second provides both energy focusing and time dispersion of ions having different masses. In effect, the second reflectron behaves identically to that in a single reflectron instrument in which ions undergo metastable fragmentation in the first drift region.1° In the case where L = 4d, then t’ = t”, and eq 8 becomes
I
lb
lb
260
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1 7 * 1
(9) (where t = t’ + t”) as described by Standing et al.10 Equations 6-8 are derived for single-stage reflectrons in which ions are decelerated linearly. In our instrument, we have utilized dual-stage reflectrons which provide high mass resolution in a smaller sized reflectron. In the dual-stage reflectron ions are rapidly decelerated within the first 10% of the reflectron. Precursor ions all penetrate the second stage. However, product ions enter the reflectron with reduced energies (as described by eq 51, so that only those product ions with energies sufficient to penetrate the second stage are refocused at the detector. For those product ions which penetrate the second stage well beyond the second grid (approximatelythe top one-third to one-half of the mass range), the mass scale will be linear according to eq 8.
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Flgm2. (a)Matrixassisted laser dewrptlon/hizatbn mass spectrum of a-cyano-Chydroxycinnamic acid; (b) massspectrumafter selection of the fragment ion at mlz 172.1: (c) product ion mas8 spectrum of the precursor ion at m/z 172.1.
RESULTS AND DISCUSSION The mass spectrum of a-cyano-4-hydroxycinnamicacid
(MW 189.1) is shown in Figure 2a. The spectrumwas recorded using the detector at the end of the second mass analyzer, so that the ions have been transmitted through both dual-stage reflectrons. The peak at rnlz 190.05 corresponds to the protonated molecular ion, while peaks at 172.1and 146.1 may correspond to successive losses of H20 and CN, respectively. In Figure 2b, the pulsed ion gate has been activated for selection of the fragment ion at rnlz 172.1. While the ion gate transmits the entire isotopic cluster from rnlz 172 to 174, it is interesting to note that the mass resolution is not degraded by application of the ion gate pulse. The small peak appearing at approximately 20 ps does not correspond to ions transmitted by the ion gate but rather to metastable formation of product ions in the collision region andlor the drift regions between the two reflectrons. In Figure 2c, the pulsed valve has been activated, producing product ions at rnlz 146.1, 122.2, and 100.3. The unresolved signal appearing below rnlz 100.3 correspondsto low mass ions that have been reflected within the fiist stage of the dual-stage reflectron. The dual-stage reflectron can be operated as a single-stage reflectron by connecting the second grid to the first grid. In Figure 3, we compare the product ion mass spectra of the fragment ion at rnlz 172.1 obtained by single-stage reflection. While short single-stage reflection produces a spectrum that is less resolved than that obtained by dual-stage reflection, this spectrum was obtained to verify the mass assignments according to eq 8.
Slk
321s
W S
YYS
351s
AS
Figure 5. Product ion mass spectrum of a fragment ton from a-cyanoMydroxyclnnamic acid obtained using a single-stage reflectron.
The time-of-flight mass spectrum of rhodamine dye is shown in Figure 4a. In this case, all ions were postaccelerated to an additional 500 eV of energy by placing the second grid after the collision chamber, the second flight tube, and the second reflectron entrance grid at -500 V to improve ion extraction from the collision region. (This technique must be used with caution, however, since it will result in considerable peak broadening in cases where dissociation reactions occur over a longer time frame than the collision time. For rhodamine, however, additional broadening waa
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 8, APRIL 15, 1993 45-
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Flgure 5. Mass spectrum of the molecular ion region gramicidin S.
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Flgufe4. (a)Timeof-flight mass spectrum of modaminedye;(b)tandem (CID) timeof-flight mass spectrum of rhodamine after selectionof the molecular ion at mlr 429.2; (c) product ion mass spectrum of mlz 429.2 obtained by linked (BlE) scan on a double-focusing mass spectrometer.
not observedwhen compared with spectra that did not involve postacceleration of the ions.) The molecular ion peak at mlz 429.2 was then mass selected by the deflection gate, and its collision-induced dissociation mass spectrum was obtained as shown in Figure 4b. In addition to postaccelerating the ions, the final reflectron grid voltage was reduced to 3950 V. This shifts the entire mass spectrum to longer times, since ions must penetrate the reflectron more deeply. It also reduces the precursor ion signal,since most ofthese ions have sufficient energy to pass through the final grid of the reflectron. More importantly, this places the optimal focus within the product ion mass region rather than a t the precursor ion mass and provides better overall mass resolution across the spectrum. Figure 4c shows the collision-induced dissociation mass spectrum of the molecular ion of rhodamine dye obtained on a Kratos CONCEPT 1H double-focusing mass spectrometer that was used to calibrate the tandem time-of-flight mass scale and to verify ita linearity. The mass spectrum in Figure 4c was obtained in the linked (B/E) mode which is linear in mass with respect to both the electric (E) and magnetic (B) sector fields. Using the peaks at m/z 341.1 and 429.2 as known masses, we assigned the masses of all other peaks in the spectrum in Figure 4b using a linear calibration carried out by the TOFWARE data system. In general, the masses obtained from the CID spectra produced by the tandem timeof-flight and the double-focusing instrument are in good
agreement and verify that the tandem time-of-flight mass spectrum shows good linearity within this mass range. Differences in the relative intensity distributions for the product ions obtained by both methods may reflect the fact that collisions with argon in the double-focusing instrument were carried out a t energies of 8 keV. It is interesting to note that the linearity in the mass scale in Figure 4b is maintained after postacceleration. Because the grid separating the two reflectron stages is maintained a t 1600 V, postaccelerated ions are decelerated to the same energy upon entering the second, longer stage as ions of the same mass that are not postaccelerated. Thus, ions of agiven mass spend the same amount of time in the second stage, where most of the mass dispersion takes place. The additional energy provided by postacceleration of the product ions will result in some mass dispersion within the linear regions of the second mass analyzer that follows a square root law. However, the effects upon the linearity of the mass scale in Figure 4b are minimal for low postacceleration voltages and over the top one-third of the mass range. Linearity is also maintained when the reflectron voltages are lowered. This is consistent with the suggestion by a number of investig a t o r ~ ' ~ -that ' ~ metastable ion spectra can be be obtained with good resolution throughout the mass range by changing the reflectron voltages to permit ions in several mass regions to enter the second stage. Such approaches could be utilized as well to focus product ions obtained by collisionalactivation throughout the entire mass range. The dual-stage reflectron was chosen for this instrument because it is possible to achieve high mass resolution over a compact distance. The partial mass spectrum of gramicidin S shown in Figure 5 where the MNa+ molecular ion cluster is resolved (FWHM) to better than one part in 3000 in the double reflectron mode.
CONCLUSIONS One of the major advantages of the time-of-flight mass analyzer is the ability to record ions of all masses from each ionization event, the so-called Felgett advantage. Thus, it is primarily compatible with pulsed techniques such as laser desorption but has been used as well with continuous (12)Tang,X.;Beavis,R.;Ens,W.;LaFortune,F.;Schueler,B.;Standing,
K.G. Znt. J. Mass Spectrom. Ion Processes 1988,85, 43-67. (13)Brunelle, A.; Della-Negra, S.; Depauw, J.; Joret, H.; Le Beyec, Y.
Rapid Commun. Mass Spectrom. 1991,5, 40-43. (14) Boesl, U.; Weinkauf, R.; Schlag, E. W. Int. J . Mass Spectrom. Ion Processes 1992, 112, 121-166.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 8, APRIL 15, 1993
ionization methods such as liquid SIMS,’5 electron impact,16J7 and electrospray ionization18using pulsed ion extraction and/ or ion storage techniques. In all cases, the duty cycle is considerably improved relative to scanning instruments, so that the possibilities exist for higher sensitivity and lower detection limits. Low mass resolution has often been cited as a particular disadvantageof time-of-flight mass spectrometers. However, of equal concern has been the inability to configure such instruments in tandem to enable the selection of specific componentsin a mixture and to provide structural information through collision-induced dissociation. In our view, reliance upon metastablefragmentationor the use of photodissociation or surface-induced dissociation will not provide the dissociation efficiency for biological molecules that has been achieved repeatedly on scanning instruments using collisioninduced diseociation. The hybrid instrument described by Russell and co-workers’ confirms the advantages of using collision-induced dissociation with time-of-flight mass spectrometry, but incorporates an expensive double-focusing instrument as the first mass analyzer. Double-focusing instruments maintain a distinct advantage in mass resolution (as much as one part in 100 OOO); however, such resolutions are rarely used for the structural analysis (e.g., amino acid sequencing) of biological molecules. Interestingly, in the tandem time-of-flight mass spectrometer, the Felgett advantage occurs only in the second mass analyzer, which records simultaneouslyall product ions from a given precursor. In the first mass analyzer, selection of an ion by electronic gating is functionally similarto mass selection (15) Emary, W. B.; Lys, I.; Cotter, R. J.; Simpson, R.; Hoffman, A. Anal. Chem. 1990,62, 1319-1324. (16) Wiley, W. C.; McLaren, I. H. Rev. Sci. Instrum. 1955,26, 1150. (17) Grix, R.; Kutacher, R.; Li, G.; Gruner, U.; Wollnik, H. Rapid Commun. Mass Spectrom. 1988,2, 83. (18) Dodenov, A. F.; Chemushevich, I. V.; Laiko, V. V. Presented at the 12th International Mass Spectrometry Conference, Amsterdam, Aug 26-30,1991. (19) Orlando, R.;Fenselau, C.;Cotter, R. J. J.Am. Soc. Mass Spectrom. 1991,2, 189-197.
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by a magnetic field. Thus, the tandem time-of-flight mass spectrometer is functionally equivalent to the hybrid instrument described by Russell,l except that scanning is not required (in the case of unknowns) to determine the masses of precursor ions. It is also functionally equivalent to foursector instrumentsequippedwith spatial array detectors. Such instruments are, however, both complex and expensive and motivated our efforts to develop a compact tandem timeof-flight mass spectrometer using high-energy collisioninduced dissociation. We have noted that the reflectron focuses only a portion of the mass range and that changes in the reflectron voltages can be used to bring different mass ranges into focus. This results from the fact that product ions carry different portions of the initial kinetic energy following high-energy collisions. Alternatively, selected precursor ions might be decelerated prior to entering the collision chamber, where they would undergo low-energy collision-induced dissociation. When reaccelerated, all ions would have approximately the same energy, KEb = eVz, and optimal focusing by the second reflectron would be achieved over the whole mass range. This approach would provide an opportunity to carry out reactive (endothermic) collisions at very low kinetic energy.19
ACKNOWLEDGMENT This work was supported by grants from the National Institutes of Health (GM 33967) and from the National Science Foundation (DIR 90-16567). Experiments were carried out at the Middle Atlantic Mass Spectrometry Laboratory, an NSF Shared Instrumentation Facility. We also acknowledge the help of Rob Streeper in obtaining CID mass spectra on the double-focusing mass spectrometer.
RECEIVED for review October 7, 1992. Accepted January 15, 1993.
