Anal. Chem. 1902, 64, 2879-2001
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Signal-to-Noise Enhancement of Neutral-Ion Correlation Measurements for Tandem Time-of-Flight Mass Spectrometry F. H. Strobel and D. H. Russell’ Department of Chemistry, Texas A&M University, College Station, Texas 77843
In this paper we report a method for improving the S/N ratio by reducing the background signal, e.g., reducing N. Tandem time-of-flight (TOF) mass spectrometry is a The method used to reduce N is taken from nuclear chemistry developing area with excellent potential for ultra-highand is generally referred to as “pile-up rejection.”* In the sensitivity analysis and structural characterization of complex neutral-ion correlation experiment, the occurrence of a biological molecules. Pulsed ionization methods such as dissociation reaction of the mass-selected ion is indicated by pulsed Cs+ liquid-SIMS, are not directly compatible with the neutral product striking a detector positioned behind the scanninginstruments, but such methods are compatible with reflectron,5?6and the ion is detected by a detector positioned tandem TOF. In addition, several laboratories have demto receive the reflected ions. Thus a dissociation reaction onstrated that pulsed ionization methods, specificallypulsed yields two products, e.g., an ion and a neutral, that must be liquid-SIMS,’ multiphoton ionization (MPI), and laser decorrelated. As with all event correlation experiments it is sorption ionization,especiallymatrix-assisted laser desorption important to keep the flux of the events low in order to ionization (MALDI),2are inherently more sensitive than minimize the number of “false” correlation events, e.g., continuous ionization methods, and recent work has dembackground signals from uncorrelated events. The devices onstrated that MALDI combined with tandem TOF mass used for this experiment and data obtained in this manner spectrometry is an ultra-high-sensitivity analysis m e t h ~ d . ~ are described in the following sections. The added sensitivity of pulsed ionization is particularly advantageous to tandem mass spectrometry and the develEXPERIMENTAL SECTION opment of ultra-high-sensitivity (10-18 to 10-15M) biological The apparatus used in this study has been described previmass spectrometry.3 On the other hand, instrumentation for 0us1y.~Briefly, the instrument consists of a Kratos MS-50highperforming tandem TOE’ mas8 spectrometry experiments is resolution mass spectrometer that has been modified by addition still early in the development stages: and optimum methods of a reflectron TOF that acts as MS-I1 for tandem mass for performing such experiments have not been established. spectrometry experiments. The Kratos MS-50 operates as supplied by the vendor,and the R-TOF is a single-stage reflectron In a recent paper we described a magnetic sectodreflectrondesigned specifically for tandem mass spectr~metry.~ The TOF TOF instrument under development in our laboratory5 and electronics are standard LeCroy TDC components that are reported data obtained using neutral-ion correlation meainterfaced to an IBM-PC compatible computer by using a surements.6 The neutral-ion correlation experiment permits National Instruments, Inc. GPIB card. The TOF spectra are a continuous ionization source to be used on the magnetic displayed using software compiled in our laboratory. sector/reflectron-TOFinstrument. Although we successfully The samples used in this study were obtained from Sigma Chemical Co. and were used without purification or preparation demonstrated the instrument concept, the signal-to-noise(S/ steps. The samples were ionized by continuous Cs+ ion liquid N) ratio of the collision-induced dissociation (CID).spectra SIMS,and all other aspects of the experiments were performed was disappointing. The major factor limiting the S/N ratio as described in our earlier report5 of the CID spectra is the background of neutrals formed by neutralization of the incident ions upon collision with the RESULTS AND DISCUSSION target gas. The neutrals are formed by either endothermic charge-transfer reactions between the incident ion and the To illustrate the neutral-ion correlation/pile-up rejection target gas7 or by CID to form product ions that are not sampled experiment, three spectra for Leu-enkephalin are contained by MS-11, e.g., low m/z fragment ions such as H+that are not in Figure 1. Figure 1A contains the metastable ion spectrum efficiently transmitted by MS-I1 or CID reactions involving obtained by using neutral-ion correlation, and Figure 1B large scattering angles and the fragment ions are scattered contains the metastable ion spectrum obtained by using outside the acceptance angle of the spectrometer. The latter neutral-ion correlation/pile-up rejection. The fragment ions process seems an unlikely explanation because both the ion are identified using the notation suggested by Roepstorff and and neutral formed by CID would be scattered, and the Fohlman.10 The spectrum contained in Figure 1A required efficiency for neutral collection should also be low, which it 60 OOO dissociation events to yield a S/N ratio of 201 (based is not. on signal amplitude and noise defined by the background fluctuations) for the most abundant fragment ion. The spectrum contained in Figure 1Brequired 10 OOO dissociation (1)Tecklenburg, R. E., Jr.; Castro, M. E.; Russell, D. H. Anal. Chem. 1989,61,153. (b) Olthoff, J. K.; Cotter, R. J. Nucl. Instrum. Methods events to yield a S/Nratio of >100:1for the most abundant Phys. Res., Sect. B 1987, B26, 566. fragment ion; however, the uncertainty of the peak height is (2)Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T. Anal. Chem. determined by W l 2 ,where N is the number of counts l991,63,1193A. (3)Strobel, F.H.; Solouki, T.; White, M. A.;Russell, D. H. J.Am.SOC. associated with the peak height. Thus neutral-ion correlation/ Mass Spectrom. 1991,2,91. pile-up rejection improves the S/N by a factor of 5 and reduces (4)Stults,J. T.; Enke, C. G.;Holland, J. F. Anal. Chem. 1983,55,1323. the required number of events by a factor of 6. The collision(b) Stults, J. T.; Holland, J. F.; Watson, J. T.; Enke, C. G. Znt. J.Mass Spectrom. Ion Processes 1986,71,169.(c) Glish, G.L.; Goeringer, D. E. induced dissociation spectrum for the [M + HI+ion of LeuAnal. Chem. 1984,56,2291.(d) Glish, G.L.; McLuckey, S. A.; McKown, enkephalin is shown in Figure 1C. This spectrum was H. S. Anal. Instrum. 1987,16,191. (5)Strobel, F. H.;Preston, L. M.; Washburn, K. S.; Russell, D. H. (8) Williams, 0. M.; Sandle, W. J. J. Phys. E 1970,3,741. Anal. Chem. 1992,64,754. (9)Strobel, F. H.;Russell, D. H. Manuscript in preparation. (6)Della-Negra,S.;Le Beyec,Y. Anal. Chem. 1985,57,2035.(b) Tang,
INTRODUCTION
X.;Ens, W.;Standing, K. G.; Westmore, J.B. Anal. Chem. 1988,60,1791. (7) Bricker, D.L.; Russell, D. H. J. Am. Chem. SOC. 1986,108,6174. (b) Bricker, D. L.; Russell, D. H. Anal. Chim.Acta 1985,178,117.
(10)Roepstorff, P.; Fohlman, J. Biomed. Mass Spectrom. 1984,11, 601. (11)Holmes, J. L. Mass Spectrom. Reu. 1989,8, 513.
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greatly improved over that obtained without using pile-up rejection.5 To understand the advantages of pile-up rejection for neutral-ion correlation measurements, it is instructive to examine the factors that limit the S/N for Figure 1A. As the mass-selectedion (massselected by MS-I) traverses the fieldfree region between MS-I and MS-11, fast neutrals are produced by two processes, reactions 2 and 3.
u.
ml+
WZ
B
y3
u
w/2
M/Z
Flgure 1. Metastable ion (A and B) and collision-induced dlssociatlon (C) spectra for the [M + H]+ ion of Leu-enkephailn obtained by neutralion correlation (A) and neutral-ion correlation/pilauprejection (B and C).
NEUTRALS
I ONS
-
M
obtained by using neutral-ion correlation/pile-up rejection and an attenuation of the incident ion beam of 30%. Note that the CID spectrum has a higher noise level than the metastable ion spectrum. The noise level of the CID spectrum is greater because fewer dissociation events are averaged due to a high number of background events that contribute false correlations. Nonetheless, the S/N for the CID spectrum is
c
Fi’ + FY m10
+ mg+
(1)
(2)
(3)
Reaction 1 corresponds to collisional activation (CAI and subsequently leads to collision-induced dissociation (CID; reaction 2), thus producing a fragment ion (Fi+)and a fragment neutral (FjO) of the incident ion, denoted ml+. Reaction 3 produces an ion (the original target atom) and a neutral (mlO) but the ion has near-thermal kinetic energy and is not extracted from the collision cell; thus the only detectable product (detectable in the tandem mass spectrometry experiment) of reaction 3 is the fast-neutral. (m10 denotes a neutral species, and whether the neutral is an intact molecule or a fragment of the molecule is now known; however, results from neutralization-reionization experiments suggest that both intact molecules and fragments are formed.”) Reaction 2 yields a fast-ion and a fast-neutral, and both fragments retain the initial velocity of ml+. In the neutral-ion correlation experiment detection of FO j is the “start” signal for the timeof-flight clock for the ion detector. The arrival time of Fi+ at the ion detector is delayed in time relative to the neutral because the flight path of the ion is greater; thus the difference in arrival time of the neutral and ion can be used for TOF analysis of the ionic products of reaction 2. The occurrence of reaction 2 is confirmed by correlation of an ion with a neutral, whereas fast-neutrals formed by reaction 3 do not correlate with an ion. The primary source of background signal associated with coincidence measurements arises when the count rates for “true”coincidence signals are low and the count rate of “false” coincidence signals is high. For example, false coincidence gives rise to signal distortion if a neutral formed by reaction 3 strikes the detector (a start signal for the ion TOF clock) and within the ion acquisition time window a second neutral formed by reaction 2 strikes the detector, the TOF measurement for the ion is in error by At, the difference in arrival time between neutral 1and neutral 2 (Figure 2). Because At is random, the end result is noise in the ion TOF spectrum. In addition, the probability for background signal in a coincidence experiment is directly proportional to the time window allowed for detection of Fi+, which is determined by the flight time of the ion. Typically, time windows of 10-100 FS are required for acquisition of the full range MS-I1 mass spectrum for ion energies of 1-8 keV and m / z values of 1005000 for ml+. Such a large time window for the MS-I1 mass spectrum limits the maximum allowable ion current for ml+ to approximately 10 000-50 000neutrals/s. Data acquisition rates of 10-50 kHz exceeds the capabilities of the instrument hardware;thus it is necessary to acquire a statistical sampling of the actual number of neutrals formed. Because the number of products formed by reaction 3 far exceeds the number of products from reaction 2, signal-averagingis not an efficient means for improving the S/N ratio. Although the S/Nratio can be improved by summing together narrow segments of the TOF spectrum, e.g., sampling a narrow time window, this method increases the total data acquisition time for the experiment. In addition, this procedure is practical only if
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Flgure 2. Pulse sequence for neutrals and ions showing arrival of two neutrals (1 and 2) that ylelds false correlation signals.
+ mg
[ml+l + m g
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ION DETECTOR
I m B I T CLUB
I
1 CLUBTDC
Ftgurr 8. Schematic drawing for the clrcuitry to eilmlnate false correlated slgnals and Improve the slgnal-to-noise ratio for the metastable ion and collision-Induced dissociation spectra.
the signal for ml+ is constant in time; e.g., often times the liquid-SIMS spectrum changes (in terms of analyte versus matrix background signal)12as the sample is depleted, and thus the time-averaged spectra are composite MS-I1 spectra for analyte and matrix background ions. The pile-up rejection S/N enhancement can be accomplished in two ways: (i) acquire and store all the neutral-ion arrival events and process the data off-line,'s or (ii) develop a hardwired system for performing the experiment on-line. We chose to use an on-line system because this permits evaluation of the tandem mass spectrometry data as the data are being acquired. On-line evaluation of the data is important because some samples yield acceptable S/N ratios with minimum acquisition times, either because the dissociation efficiencyis high or a large amount of sample is available,but other samples require much longer acquisition times to achieve acceptable S/N levels. The on-line experiment is performed by the circuitry shown in Figure 3. This circuitry signals the computer that valid data have been obtained. Valid data are defined by (i) a signal for ion arrival within the ion flight time window and (ii) a second neutral not detected within the ion flight time. The signals from: the neutral and ion detectors are used as inputs to two constant-fraction discriminators (CFD) (Canberra 2126 and 2128). Each CFD has four outputs (two NIM and two TTL). One output on the neutral CFD is used to start the TDC (LeCroy 4208), and one output of the ion detector is used as the TDC stop. The TDC then sends a signal through its "busy" output which starts a gate generator (LeCroy 2323A). The signal from the two CFD's are "ANDed" with the gate generator output. At the end of the gate a signal would normally clear the TDC; however, if a signal is obtained by the ion detector during the gate period, (12)Caldwell, K.A.; Gross, M. L. Proceedings of the 39th American SocietyforMosa Spectrometry Annual ConferenceonMass Spectrometry and Allied Topics,Nashville, TN, May 19-24,1991;ASMS: East Lansing, MI, 1991;p 122-123. (13)Huang, L. Q.;Conzemius, R. J.; Junk, G. A.; Houk, R. S. Znt. J. Moss Spectrom. Ion Processes 1989,90,85.(b) Parks,M. A.; Gibson, K. A,:Quinones. L.: Schweikert. E. A. Science 1990,248,988.(c) Conzemius, R.'J:; Svec, H. J. Znt. J. Mass Spectrom. Ion.Processes 1990,103, 57.
two events occur. The first process sends a signal to the TDC, which indicates valid data have been received. The second process inhibits the signal which clears the TDC. If a second neutral is received, the gate is stopped so that no signals from the ion detector are processed as valid data.
CONCLUSIONS The studies to date clearly illustrate the potential for tandem TOF mass spectrometry for ultra-high-sensitivity analysis3 and structural characterization of biological molecules.5 Although neutral-ion correlation methods can be used for acquisition of metastable ion and collision-induced dissociation (CID) spectra, the background signal from neutralization of the incident ion by collisions with residual gases or the CID target gas reduces the S/N ratio for the product ions. Consequently, neutral-ion correlation/pile-up rejection, a method for rejection of spectral data from events where false coincidence signals limit the S/N ratio, dramatically improves the data and reduces the time required for data acquisition. For example, neutral-ion correlation/pileup rejection improved the S/N ratio for the metastable ion spectrum of the [M + HI+ion of Leu-enkephalin by a factor of 5. In addition, because the acquisition rates are limited by the computer (50 Hz)and not the rate of dissociation events (1000-50 OOO Hz),the acquisition time is reduced by a factor of 6. A comparable improvement is also obtained for the CID spectrum of Leu-enkephalin [M + HI+ions.
ACKNOWLEDGMENT This work is sponsored by the U.S. Department of Energy, Division of Chemical Sciences,Office of Basic Energy Sciences (Grant No. DE-FG05-85ER-13434),and the US. Department of Energy, University Research Instrumentation Program (Grant No. DE-FG05-89ER75502). RECEIVED for review May 1, 1992. Accepted August 20, 1992. Registry No. Leu-enkephalin, 58822-25-6.
