Anal. Chem. 1892, 64, 1455-1460

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Collisional Activation with Random Noise in Ion Trap Mass Spectrometry Scott A. McLuckey,' Douglas E. Goeringer, and Gary L. Glish Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6365

Random noise appiled to the end caps of a quadrupole ion trap is shown to be an effective means for the collisionalactivation of trapped ions independent of masshharge ratio and number of ions. This technique Is compared and contrasted with conventionalsingle-frequency coiiislonaiactivatlonfor the molecular ion of N,Kdimethyianiiine, protonated cocaine, the molecular anion of 2,4,6-trinitrotoiuene, and doubly protonated neuromedln U-8. Collisional activation with noise tends to producemore extensivefragmentationthan the conventional approach due to the fact that product ions are also kinetically excited In the noise experlment. The efficiency of the noise experiment in producing detectable product ions relative to the conventional approach ranges from being equivalent to being a factor of 3 less efficient. Furthermore, discrimination against low masslcharge product ions is apparent in the data from muitlpiy charged blomolecuies. Nevertheless, coliislonai actlvation with random noise provldes a very slmpie means for overcoming problems associated with the dependence of single-frequency collisional activation on masshharge ratio and the number of ions In the ion trap.

INTRODUCTION The quadrupole ion trap is currently undergoing rapid development as an analytical mass ~pectrometer.l-~ Although the device was invented by Paul and colleagues over two decades ag0:,5 attention from the analytical mass spectrometry community was largely directed elsewhere until Stafford and co-workersintroduced the mass-selectiveinstability mode of operation and the use of helium bath gas to enhance mass resolution and sensitivitya6 Since then, a variety of developments enhancing the analytical utility of the ion trap have been made including, inter alia, the capability for mass spectrometry/mass spectrometry (MS/MS),7MS" where n > 2,899 mass/charge range extension into the many tens of thousands,lOJ1interfacing of external ion s0urces,~~-~5 and

high mass resolution via slow ~cans.~6-18 Some of the most significant analytical capabilities of the ion trap are based on use of a supplementary radiofrequency (rf) signal applied to the end-cap electrodes to excite kinetically ions at their mass-to-charge dependent frequencies.19 At sufficiently large amplitudes of the supplementary rf signal, ions can absorb enough power to be ejected from the ion trap. This is the basis for so-called "resonance ejection" which is used in extended mass range experiments,lOJl in high mass resolution and in isolating ions.20 At relatively low supplementary rf signals, ions may not absorb enough power to exit the ion trap due to power dissipation via collisions with the background helium, typically present at about 1 mTorr. Under these circumstances, ions can undergo many collisions with the bath gas at kinetic energies that range from near zero to tens of electronvolts. This forms the basis for collisional activation (CA) in the ion trap and is the most common means for inducing fragmentation in ion trap MS/MS and MS" experiment^.^,^^ However, ion acceleration in the ion trap is dependent upon both the parent ion massicharge and the number of ions in the ion trap. As discussed further below, the use of a supplementary rf signal of a single frequency to effect CA limits the utility of ion trap MS/MS and MSn in some analytical applications. Several approaches can be made to address some or all of the limitations. For example, Yates et aLZ2have described the use of rapid frequency prescans over a narrow mass range (1.7 amu) to determine empirically the parent ion resonant frequency in an automated fashion. These workers have also described the use of a supplementary signal with a range in frequency of 10 kHz, equivalent to a mass/charge range of 1.7 at a qz value of 0.3.22 Both approaches were found to deal effectively with the space charge problem. The former was more complex to implement, required more time, and depended upon good reproducibility in the number of ions formed in successive ionization events, whereas the latter placed higher constraints on ion isolation. Another approach, of course, would be to borrow the sophisticated technique of

~

* To whom correspondence should be addressed.

(1)Todd, J. F.J. Mass Spectrom. Rev. 1991, 10, 3. (2) March, R. E. Org. Mass Spectrom. 1991, 26, 627. (3) Cooks,R. G.; Glish, G. L.; McLuckey, S. A.;Kaiser, R. E., Jr. Chem. Eng. News 1991,69, 26. (4) Paul, W.; Steinwedel, H. U.S.Patent 2,939,952, 1960. (5) Paul, W. Angew. Chem., Int. Ed. Engl. 1990,29, 739. (6) Stafford, G. C., Jr.; Kelley, P. E.; Syka, J. E. P.; Reynolds, W. E.; Todd, J. F. J. Int. J . Mass Spectrom. Ion Proc. 1984, 60,85. (7) Louris, J. N.; Cooks, R. G.; Syka, J. E. P.; Kelley, P. E.; Stafford, G. C., Jr.; Todd, J. F. J. Anal. Chem. 1987,59, 1677. (8)Louris, J. N.; Brodbelt-Lustig, J. S.; Cooks, R. G.; Glish, G. L.; Berkel, G. J.; McLuckey, S. A. Int. J . Mass Spectrom. Ion Proc. 1990, 96. .. 117. (9) McLuckey, S.A.; Glish, G. L.; Van Berkel, G. J. Int. J . Mass Spectrom. Ion Proc. 1991, 106, 213. (10) Kaiser, R. E.,Jr.; Cooks, R. G.; Moss, J.;Hemberger, P. H. Rapid Commun. Mass Spectrom. 1989, 3, 50. (11)Kaiser, R. E.,Jr.; Louris, J. N.; Amy, J. W.; Cooks, R. G. Rapid Commun. Mass Spectrom. 1990, 3, 225. (12) Louris, J. N.;Amy, J. W.; Ridley, T. Y.; Cooks, R. G. Int. J.Mass Spectrom. Ion Proc. 1989, 88, 97. I

(13) McLuckey, S. A.; Glish, G. L.; Asano, K. G. Anal. Chim. Acta 1989, 225, 25. (14) Pedder, R. E.;Yost, R. A.; Weber-Grabau, M. Proceedings of the 37th Conference on Mass Spectrometry and Allied Topics, Miami Beach, FL, May 1989, p 468. (15)VanBerkel, G. J.; Glish,G. L.; McLuckey, S. A. Anal. Chem. 1990, 62, 1284. (16) Schwartz, J. C.; Syka, J. E. P.; Jardine, I. J. Am. SOC.Mass Spectrom. 1991, 3, 198. (17) Williams,J.D.;Cox,K.A.;Cooks,R.G.;Kaiser,R.A.,Jr.;Schwartz, J. C.Rapid. Commun. Mass Spectrom. 1991, 5, 327. (18)Goeringer, D. E.;Whitten, W. B.; Ramsey, J. M.; McLuckey, S. A.; Glish, G. L. Anal. Chem., submitted for publication. (19) Fulford, J. E.;Hoa, D.-N.; Hughes, R. J.; March, E. E.; Bonner, R. F.; Wong, G. J. J. Vac. Sci. Technol. 1980, 17, 829. (20) McLuckey, S.A.; Goeringer, D. E.; Glish, G. L. J. Am. SOC.Mass Spectrom. 1991, 2, 11. (21) Johnson, J. V.;Yost, R. A.; Kelley, P. E.; Bradford, D. C. Anal. Chem. 1990,62, 2162. (22) Yates,N. A.;Yost,R.A.;Bradshaw,S.C.;Tucker,D. B.Proceedings of the 39th Conference on Mass Spectrometry and Allied Topics, Nashville, TN, May 1991, p 132.

0003-2700/92/0364-1455$03.00/0 0 1992 American Chemical Society

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stored waveform inverse Fourier transform (SWIFT)23used with Fourier transform ion cyclotron resonance instruments. This technique permits an almost unlimited combination of supplementary signals to be applied to the end caps during the CA period. Such an approach would represent the extreme in flexibility as well as cost and complexity. We report here random noise applied to the end caps as a means of “universal” collisional activation in an ion trap. There is precedence for the use of noise in mass spectrometry. Ijames and W i l k i n ~and ~ ~Marshall et alez5have evaluated noise as a means to excite ions as part of the detection step in Fourier transform mass spectrometry. The rationale behind our work was to find a simple and inexpensive approach to the limitations of single-frequency CA in the ion trap due to space charge effects, the need for frequency tuning, limited fragmentation, and the inability to collisionally activate parent ions over a range of madcharge values. This report compares and contrasts random noise CA with conventional single-frequency CA in a quadrupole ion trap to illustrate the potential utility for random noise as part of an ion trap experiment.

EXPERIMENTAL SECTION Apparatus and Procedure. All experiments involving electron ionization and positive ion chemical ionization were carried out with an ion trap mass spectrometer (ITMS) (r, = 1.0 cm, Q/27r= 1.1MHz) manufactured by Finnigan-MAT,San Jose, CA. All negative ion and electrospray experiments were performed with an ITMS modified for ion inje~tion.~3J5-~~ In each case, the experiment began with a period of ion accumulation, either via in situ ionization (EI, CI) or via ion injection (NCI, electrospray). Following ion accumulation,parent ions of interest were isolated by a series of two resonance ejection ramps to eject ions of lower and higher mass/charge values. Parent ions were then subjected either to conventional single-frequencyCA or to random noise CA under conditions described in further detail below. For MS/MS experiments, CA was followed by a final data acquistion mass scan using either axial modulationz7or, in the case of electrospray data, resonance ejection to extend the mass/charge range.11J5 For MS3 experiments, a second singlefrequency CA step was incorporated; a second ion isolation step was not employed. The mass scan then followed the second CA period. White noise was provided by a solid-state noise generator (Model 6103, NOISE/COM, Paramus, NJ) which provides a relatively flat power distribution from 10 Hz to 500 kHz and was coupled with the ITMS as indicated in the block diagram of Figure 1. A pulse from the scan acquisition processor circuit board in the ITMS electronics, which is actuated via the ITMS software version 4.1, was used to trigger a variable-length pulse generator (BerkeleyNucleonicsModel 6010). The output of the pulse generator provided a TTL pulse of 20-150 ms to the noise generator which then output a noise signal for the duration of the pulse. The noise signaland the output of the ITMS frequency synthesizer were fed into the inputs of a differential amplifier (Tektronix Model 7A22). This amplifier is equipped with a high pass and a low pass filter. Experiments were typically performed with a low frequency 3 dB point of 10 kHz and a high frequency 3 dB point of 1MHz for the summed output of the differential amplifier. This combined signal was then fed into a fixed gain (50 dB) rf amplifier (EN1Model 2100L) the output of which was connectedto the rf input of the ITMS “Balun box”,which provides equivalent signals to the end-cap electrodes phase-shifted 180” (23) Marshall, A. G.; Wang, T . 4 . L.; Ricca, T. L. J.Am. Chem. SOC. 1985,107,7893. (24) Ijames, C. F.; Wilkins, C. L. Chem. Phys. Lett. 1984,108, 58. (25) Marshall, A. G.; Wang, T.-C. L.; Ricca, T. L. Chem. Phys. Lett. -1984. _ _,_-708. - -, 62. - -.

(26) McLuckey, S. A.; Van Berkel, G. J.; Glish, G. L.; Huang, E. C.; Henion, J. D. Anal. Chem. 1991,63, 375. (27) Tucker, D.B.; Hameister, C. H.; Bradshaw, S. C.; Hoekman, D. J.; Weber-Grabau, M. In Proceedings of the 36th Conjerence on Mass Spectrometry and Allied Topics; San Francisco, CA, June 1988, p 628.

I

.

ITMS .

Electronics

Trig

Pulse Generator

Axial Mod

Differential Amplifier

Noise Generator

Figure 1. Block diagram of the electronlcs used to couple random noise to the Ion trap end-cap electrodes for noise CA experiments.

from one another. A delay period in the scan function to coincide with the period of the noise signal was programmed into the scan function via the ITMS scan editor software. This scheme was found to be a simple method to allow noise CA to be incorporated into ITMS experiments without compromising the capability for resonance ejection using a single frequency. A helium bath gas pressure of 1mTorr was maintained for all studies. Single-frequencyCA was effected with qz values of 0.20.3, supplementary rf amplitudes of 500 mV-1.5 V p-p, and duration of 20-40 ms. The frequency and amplitude in each case was tuned to optimize the fraction of parent ions converted to detectable product ions. Noise CA experiments were also performed with parent ion qz values within the range of 0.2-0.4. The amplitude of the noise signaltypically optimized in the range of 10-25 V p-p and the time of activation ranged from 20 to 150 ms. As expected, the activation time and the amplitude of the noise signal tend to be inversely related such that, beyond about 20 ms, similar spectra were observed at constant fluence. Materials. N,N-Dimethylaniline,obtained commercially(Aldrich) and used without further purification, was admitted into the unmodified ITMS to a pressure of roughly 1 X 104 Torr and subjected to electron ionization. Cocaine, also obtained commercially (Sigma),was admitted via a solidsprobe and was ionized by in situ methane chemical ionization.28 Molecular anions of 2,4,6-trinitrotoluene (TNT) were formed in an atmospheric samplingglow dischargeionization(ASGDI)source29andinjected into an ion trap fitted with this type of external ion 80urce.l~ Vapors of TNT were sampled directly into the ASGDI source from the headspace in a 25-mL vial containing about 1 mg of TNT crystals. Neuromedin U-8(Sigma) was dissolved in highperformance liquid chromatography grade water, methanol, and glacial acetic acid in relative approximate proportions of 20%, 75 % , and 5 % , respectively, to give a solution concentration of 5 pmol/pL. The solution was directly infused at 1pL/min through a 120-wm-i.d.dome-tipped needle held at 3-4 kV. Ions resulting from electrospray were drawn into an interface based on the ASGDI hardware and injected into the ion trap.15~26

RESULTS AND DISCUSSION Collisional activation in ion trapping instruments, as it is currently effected, is unique among instruments capable of tandem mass spectrometry in that ion acceleration is mass/ charge dependent. In beam-type instruments, such as multiple quadrupole, hybrid, and multiple sector instruments, the laboratory collision energy of all ions is established by the potential difference between the ion source and collision (28) Kelley, P.E.; Stafford, G. C., Jr.; Syka, J. E. P.; Reynolds, W. E.; Louris, J. N.; Todd, J. F. J. In Advances in Mass Spectrometry 1985; Todd, J. F. J., Ed.; John Wiley and Sons: Chichester, 1986; p 869. (29) McLuckey, S.A.; Glish, G. L.; Asano, K. G.; Grant, B. C. Anal. Chem. 1988,60, 2220.

ANALYTICAL CHEMISTRY, VOL. 64, NO. 13, JULY 1, 1002

regi~n.~O,~l In ion trapping instruments, parent ions are accelerated selectively based on their characteristic frequencies of motion. In the quadrupole ion trap, frequencies of ion motions are dependent upon the frequency and amplitude of the rf trapping signalapplied to the ring electrode,the physical dimensions of the ion trap, the masslcharge ratio of the ion, and any dc field that might be present.32 Most ion trap CA experiments involvethe excitationof the “fundamental secular motion” of the parent ions in the z-dimension, the interend-cap dimension. Typically, no dc field is intentionally established during ion acceleration in ion trap MSIMS experiments. However, the presence of other ions of like charge can establish a dc field strong enough to shift significantlythe frequenciesof motion of ions from their values with no dc field.33 This so-called “space charge” effect is well-known and can lead to frequency shifts large enough to cause ions experiencing space charge to appear to fall into resonance with the supplementary rf signal at one mass unit or so higher than expected based on the resonance frequency measured with minimal space charge.21 The characteristics of ion acceleration in the ion trap described above have important analytical ramifications. First, the fact that ion acceleration is mass selective relaxes ion isolation requirements in the MSIMS experiment. Indeed, the first demonstrations of MS/MS with an ion trap eliminated only the ions of masslcharge lower than that of the parent ion prior to CA.28 Second, the parent ion masslcharge must be known before it can be collisionallyactivated. Of course, the parent ion masslcharge must be known before it is isolated in an MSIMS experiment, but a first step of mass selection may not be necessary in cases in which only one compound is admitted into the mass spectrometer, such as with on-line separations, and a soft ionizationmethod is employed. Third, a significant amount of tuning is required to optimize CA in the ion trap. Much of this tuning is associated with finding the optimum supplementary excitation frequency. This is particularly troublesome in conjunction with on-line separationP which limit the time for tuning and typically present a continually changing analyte quantity. In the absence of measures to adjust ionization conditions to keep the number of ions constant during analyte elution, significant frequency shifts might occur across the the chromatographic peak. Fourth, only ions of one masslcharge ratio are accelerated a t a time when only a single supplementary frequency is used. In many applications, this is a desirable feature. However, in some MSIMS applications, such as the targeted product ion mode of analysis,34 it is desirable to activate ions over a wide range of masslcharge values. Fifth, due to the relatively slow nature of parent ion internal energy deposition and the fact that product ions are not subsequently kineticallyexcited, ion trap CA typically leads to the formation of only one or a few different ions. This is particularly so when one dissociationchannel requires significantlyless internal energy than any others. Therefore, structural information inherent in the MSIMS spectrum may be limited. Fortunately, the ion trap is capable of multiple stages of CA, but it is often undesirable to be forced to resort to an MS” experiment with its added complexity. (30) McLafferty,F. W., Ed. Tandem Mass Spectrometry; John Wiley and Sons: New York, 1983. (31) Busch, K.L.; Glish, G. L.; McLuckey, S. A. Mass Spectrometry/ Mass Spectrometry: Techniques and Applications in Tandem Mass Spectrometry; VCH Publishers: New York, 1988. (32) March, R. E.; Hughes, R. J. Quadrupole Storage Mass Spectrometry: John Wiley and Sons: New York, 1989. (33) Todd, J. F. J.; Waldren, R. M.; Freer, D. A.; Turner, R. B. Int. J . Mass Spectrom. Ion Proc. 1980, 35, 107. (34) McLuckey,S.A.;Glish, G. L.; Grant, B. C. Anal. Chem. 1990,62, 56.

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loo%, M’

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Flguro 2. Spectra obtained In various stages of a single-frequency CA MS3 experlment beginning with the molecular Ion of N,MImethylanlline. The spectrum after lsolatlon of the parent Ion and prior to CA (a). The MS/MS spectrum obtained after slngle-frequency CA of the molecular ion (b). The MS3spectrum after sequential single-frequency CA of flrst the molecular Ion, and then the (M W)+ product Ion (c). The relatively large signal at m/z 122 Is due both to the ’3c contalnlng parent Ion and an bn/molecule reaction product.36

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A wide variety of ions has been subjected to collisional activation with random noise in our laboratory to determine the feasibility of noise CA for analytical MSIMS applications. These include radical cations, singly protonated molecules, multiply protonated molecules, and radical anions. Data describedhere were drawn from each category and were chosen to illustrate the general observations noted thus far in the use of noise for dissociating ions in an ion trap MS/MS experiment vis-a-visconventional single-frequencycollisional activation. Radical Cation. Figure 2 shows spectra resulting from various stages of an MS3 experiment using single-frequency CA. Figure 2a shows the signal due to the molecular ion of NJV-dimethylaniline (mlz 121) after ion isolation but before any CA. Figure 2b shows the MSIMS spectrum that results from single-frequency collisional activation of the molecular ion. Note that the molceular ion fragments exclusively, and with essentially 100% efficiency, by loss of an atom of hydrogen. This represents a case in which structural information in the MS/MS spectrum is almost absent. Such an uninformative MSIMS spectrum is not the norm with the ion

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C-H

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1

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Figure 3. MS/MS spectra of ionized N,Ndimethyianilineobtained using noise CA. The spectrum normalized to the base peak (a). The spectrum shown on the same scale as that of Figure 2c (b).

trap, but for radical cations hydrogen loss is often the dominant decomposition path. Much more structural information is available in an MS3experiment in which the (M - H*)+product ion (mlz 120) is subjected to single-frequency CA (Figure 2c). From a separate MS3 study involving ions containing one l3C at0m,3~ we have established that the major second generation product ions apparent in Figure 2c are CsHsN+ (mlz 118), CsH7+ (mlz 103), C,Hg+ (mlz 93), C7H7+ (mlz 91), CsH7+ (mlz 7 9 , and C,&+ (mlz 77). Figure 3 shows MS/MS spectraacquired at the same parent ion qz value and for the same collisional activation duration but with noise used as the supplementary signal applied to the end caps (approximately 15 V p-p). Figure 3a shows the spectrum with full scale at 36% that of Figure 2A. The base peak in this spectrum is also due to loss of a hydrogen atom, but more extensive fragmentation, absent in Figure 2b, is also clearly apparent. Figure 3b shows the MSIMS spectrum, obtained using noise CA, on the same scale as that of the MS3 spectrum shown in Figure 2c. Many of the same ions are apparent in both spectra including ions at mlz 118,103,91, and 77. Ions at mlz 93 and 79 are diminished in intensity relative to those at mlz 91 and 77 in Figure 3b due either to further fragmentation, e.g. by Hz loss, or to loss from the ion trap. A relatively small signal at mlz 65 due to C5H5+appears in the noise CA experiment, whereas it is not present under the conditions used in the MS3 experiment. This ion results from dissociation of C7H7+ by loss of CZHZ. Several analytically significant observations can be made from this comparison. First, the single-frequency MS3 study provides slightly greater efficiencies. For example, the total product ion signal in the MS3 spectrum, which includes all first and second generation product ions, constitutes about 80% of the parent ion signal recorded in the absence of CA. All product ion signal recorded in the noise CA MS/MS spectrum constitutes roughly 60% of the parent ion signal prior to CA. Note, however, that a significant signal due to (35) Glish, G. L.; Asano, K. G.; McLuckey, S. A. J. Am. Soc. Mass Spectrom. 1990, I , 166.

80

120

160

200

m/z

-

-

Figure 4. MS/MS and MS3 spectra obtained from single-frequency CA of protonated cocaine. The MSIMS spectrum (a). The MS3 spectrum following the sequence m/z 304 m/z 182 second generation product ions (b).

the parent ion remains. If this signal is included, slightly over 80% of the parent ion signal prior to CA appears as ions in the noise MSIMS spectrum. Another significant difference in the spectra is the much greater degree of sequential fragmentation observed with the noise CA experiment. This almost surely arises from the fact that product ions once formed can also undergo kinetic excitation in the noise CA experiment. In light of the fact that essentially equivalent structural information is inherent in the noise MSIMS and single-frequency MS3 spectra, such a relatively small loss in efficiency (60% vs 80%) would appear to be acceptable, since two stages of frequency tuning and two stages of amplitude adjustment are required to obtain the latter whereas only one amplitude adjustment is required to obtain the former. Protonated Molecule. Most analytical applications of MSIMS employ an ionization method that induces very little fragmentation. Chemical ionization can be a gentle means for ionization, and it is widely used in conjunction with MSI MS. Typically, acidlbase chemistry is used to provide, in the positive ion mode, the protonated analyte molecule. Data for protonated cocaine are described here obtained both with single-frequency CA and noise CA. Figure 4 shows the MSI MS spectrum of protonated cocaine and the MS3 spectrum, following the sequence mlz 304 mlz 182 second generation products, both obtained with single-frequencyCA. The product ion at mlz 182, formed by loss of benzoic acid from the protonated molecule, is the only product ion observed in the MSIMS spectrum. Roughly 50% of the parent ion signal prior to CA appears as product ion signal in the MS/ MS spectrum. Collisional activation of the (MH - CsH5COOH)+ ion yields the second generation product ions due to methanol loss (mlz 150), loss of methyl acetate (mlz 108), and formation of C5HsN+(mlz 821,among other less abundant ions. Almost all of the (MH - CsH&OOH)+ signal prior to the second stage of CA can be accounted for by the sum of the second generation product ion signals yielding an overall efficiency of the MS3 experiment of nearly 50 % . Figure 5 shows the noise CA MS/MS spectrum of proto-

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ANALYTICAL CHEMISTRY, VOL. 64, NO. 13, JULY 1, 1992 15%.

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Flgure 5. The MS/MS spectrum of protonated cocaine acquired using

Flguro 6. The MS/MS spectrum of the molecular anion of 2,4,6-TNT acquired using noise CA.

nated cocaine normalized to the signal at mlz 182. There are several noteworthy observations in comparing the noise MS/ MS spectrum with the MSIMS and MS3 data acquired with single-frequency CA. First, all of the first and second generation product ions observed in the MS3 experiment with single-frequencyCA are observed as product ions in the noise CA MSIMS spectrum. Once again, two stages of frequency tuning and amplitude adjustment were required to obtain the MS3 data whereas only one amplitude adjustment was required for the noise CA data. Second, the fraction of parent ions converted to product ions in the noise experiment was about 20% as opposed to a 50% conversion in the MS3 experiment. Once again, the efficiency of the noise CA experiment is somewhat lower than the single-frequency CA approach. This is a general observation. However, the relative efficiencies of the two experiments tend to become more nearly equal at higher qz values and when there is a relatively low threshold dissociation. In the case of the cocaine experiment, the single-frequencyand noise MSIMS data were acquired with qz value of 0.2 whereas the qz value for the second CA step in the MS3 experiment was 0.26. Third, noise CA conditions which maximize product ion abundances do not result in as large a depletion in parent ion signal as observed in the single-frequencyCA experiment. This is also a general observation. More detailed study is needed to determine the underlying reasons for the latter two observations. For now, we speculate that efficiencies are lower in the noise CA experiment due to a greater chance for product ion loss. Some product ions are likely to be formed with positions and velocities such that kinetic excitation by a frequency component in the noise signal can eject them. Thus, efficiencies tend to increase with the strength of the trapping field, which increases with qz over the range of interest here, and when relatively low amplitudes are required for dissociation. Parent ion diminution is also smaller with the noise experiment due to less power at the parent ion frequency being present in the noise signal. Longer CA times or greater amplitudes are effective at ejecting parent ions, but product ion signals are typically not enhanced beyond some optimum value. Radical Anion. Electron capture or negative chemical ionization are the preferred means of ionization for detection and identification of some analytes. This is the case for many organic high explosives, such as TNT.36837 Single-frequency CA of the molecular anion of TNT (mlz 227) yields almost exclusively the product ions (M - OH')- (mlz 210) and (M NO')- (mlz 197) with an efficiency of 30-40%.38 Figure 6 shows the MSIMS spectrum obtained with noise CA. This

spectrum shows much more extensive fragmentation than is observed with single-frequencyCA presumably due to further fragmentation of the (M - OH*)-and (M - NO*)-anions. The conversion of molecular anions to product ions in this case is 25 96. It is also noteworthy that the absolute abundances of the ions in this spectrum changein proportion to the number of parent ions admitted but not relative to one another nor to the parent ion signal, even under conditions in which significant changes in the parent ion frequencies occur due to space charge. Thus, MSIMS spectra acquired with noise CA are essentially independent of the number of ions in the ion trap, as expected due to the broad band nature of the technique. Multiply Protonated Molecule. Multiply charged ions have recently become important in analytical mass spectrometry due to the advent of electrospray (ES).3w1 Peptides and proteins have proved to be particularly well suited to ionization by electrospray. An important application in this area is the on-line separation of protein tryptic digests with subsequent analysis by electrospray coupled with MS/MS.42-u Such an application poses difficulties with single-frequency CA in the ion trap due to possible shifts in frequency during analyte elution and, more significantly, problems with tuning CA conditions prior to the chromatographic run. In the work reported with ion trap MSIMS and MS3 applied to tryptic digest components in an on-line separation followed by ion spray,26 preliminary tuning was performed by infusing the tryptic digest and reducing the signal at the mlz value of the anticipated parent ions. Because chemical noise present in the ion spray mass spectrum of the unseparated digest was too great to determine if product ions were formed, the tuning was probably not optimal. One of the major conclusions from that work was the tuning limitation of single-frequency CA in the ion trap for on-line LC/MS/MS of tryptic digests. We have examined several multiply charged peptides, primarily doubly protonated species which are frequently formed in ES of tryptic digests, using both single-frequency CA and noise CA. Data for neuromedin U-8, an octapeptide of molecular mass 1111, are presented here and illustrate the general observations made for all of the doubly protonated peptides that we have subjected to noise CA. Figure 7 compares MSIMS spectra of doubly protonated neuromedin

noise CA.

(36)Yinon, J. Mass Spectrom. Rev. 1991, 3,179. (37)Yinon, J. Mass Spectrom. Rev. 1982, 1, 257. (38)McLuckey, S.A,; Asano, K.G.; Glish, G. L. In Proceedings of the 38th Conference on Mass Spectrometry and Allied Topics;San Francisco, CA, June 1988,p 1108.

~

(39)Fenn, J. B.;Mann, M.; Meng, C. K.; Wong, S.F.; Whitehouse, C. M. Science 1989, 246, 64. (40)Fenn, J. B.;Mann, M.; Meng, C. K.; Wong, S.F.; Whitehouse, C. M. Mass Spectrom. Rev. 1990, 9,37. (41)Smith, R.D.; Loo, J. A.;Edmonds, C. G.; Barinaga, C. J.; Udseth, H. R. Anal. Chem. 1990, 62, 882. (42)Huang, E. C.; Henion, J. D.J.Am. SOC.Mass Spectrom. Ion Proc. 1990, 1, 158. (43)Huang, E.C.; Henion, J. D.Anal. Chem. 1991, 63,732. (44)Covey, T.R.;Huang, E. C.; Henion, J. D.Anal. Chem. 1991,63, 1193.

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“2

Flgure 7. MS/MS spectra of doubly protonated neuromedin U-8 (TyrPhaLeu-Phe-Arg-Pro-Arg-Asn-NH2) using noise CA (a) and singlefrequency CA (b).

different relative abundances. The major difference appears to be in the abundances of the lower madcharge products, such as the az+and y3+ ions. This is a general observation. That is, the noise CA experiment appears to discriminate against the lower mass/charge product ions from multiply protonated peptides and proteins relative to single-frequency CA. These ions are lost either by further fragmentation or by ejection from the ion trap. Neither possibility can be precluded from these results. A second noteworthy difference in the behavior of these relatively large ions under singlefrequency CA and noise CA conditions is that longer excitation times, up to 150 ms for noise CA vs 20-40 ms for singlefrequency CA, are required for optimum efficiency in the noise CA experiment. This is presumably due to the lower powers present in the noise signal at the parent ion frequency resulting in a somewhat slower rate of ion activation. The results shown here, and data not described for other doubly protonated peptides and for more highly charged peptides and proteins, indicate that noise CA is effective at dissociating such species at some cost in efficiency (a factor of two seems to be typical), and with some discrimination against low mass/charge product ions. However, as with the data described for singly charged ions, rich structural information can be obtained without recourse to MSnstudies, no tedious frequency tuning is required, and the activation method is insensitive to the number of ions in the ion trap. For these reasons, we find that noise CA is a relatively simple and inexpensive means to address the drawbacks of ion trap MS/MS which result from the madcharge and ion number dependence of single-frequency CA.

U-8obtained with noise CA (Figure 7a) and single-frequency CA (Figure 7b). Major product ions in each spectrum are labeled accordingto the nomenclature scheme commonly used for peptide fragments.45 (Note that the (M H)+ion, which is also observed in the ES mass spectrum, was not ejected from the ion trap prior to single-frequencyCA but was ejected from the ion trap prior to the application of noise CA.) Both spectra show many structurally informative product ions; an efficiency of about 70 % was measured for single-frequency CA whereas an efficiency of about 40 % was noted for noise CA. The spectra also share the major product ions but show

+

(45) Roepstorff,

601.

P.;Fohlman, J. Biomed. Mass Spectron.

1984, 11,

ACKNOWLEDGMENT This research was sponsored by the US.Department of Energy Office of Safeguards and Security under Contract DE-AC05-840R21400with Martin Marietta Energy Systems, Inc. RECEIVED for review December 30, 1991. Accepted March 20, 1992. Registry No. N,N-Dimethylaniline, 121-69-7; cocaine, 5036-2;2,4,64rinitrotoluene, 118-96-7;neuromedin U-8,98530-279.