Anal. Chem. 1993, 65, 704-700
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Investigation of a “Screened” Electrostatic Ion Trap for Analysis of High Mass Molecules by Fourier Transform Mass Spectrometry John A. Castoro, Claw Koster, and Charles L. Wilkins’ Department of Chemistry, Uniuersity of California, Riuerside, Riuerside, California 92521
With the recent adaptation of matrix-asslsted laser desorption/ ionization (MALDI ) of large biomolecules and polymers to Fourier transform mass spectrometric analysis, it is of interest to explore the possible advantages of using a “screened” electrostatic ion trapping technique. It is demonstrated that the observed rate of change of ion cyclotron resonance frequency with trapping potential is in excellent agreement with theory, being independent of mass and charge, in the mass range from 1185 to Just under 17 000 Da. When a screened cubic trap is used in a ca. 7-T magnetic field, the electrical potential within the trap is effectively reduced, and ion cyclotron resonance frequency shifts as a function of trapping potential are decreased by a factor of 25. There is no evidence of significant improvement in m a s resolution with this trap design, except when higher trapping potentials are compared. I n a 9 . 7 4 screened trap measurement, a bovine insulin spectrum with an average resolution of 10 000 is obtained. Comparable mass resolution under conventional trapping conditions could not be obtainedfor trapping potentials greater than 1.2 V. It is evident from this study that use of the screened trap technique could offer advantages for MALDI-Fourier transform mass spectrometry.
INTRODUCTION Recently, there has been encouraging progress in adapting the method of matrix-assisted laser desorption/ionization (MALDI)l-*for high-resolution mass spectrometric analysis of biomolecules by Fourier transform mass spectrometry (FTMW5-’ As a consequence, it is of interest to examine whether use of the “screened”electrostatic ion trap cell design? introduced by Wang and Marshall a few years ago, can offer analytical advantages in high mass MALDI-FTMS applications. It is well-known that one can calculate a theoretical upper mass limit, called the “critical”mass, above which ions cannot be trapped under FTMS conditions, regardless of the dimensions of the trapping cell.9 However, when typical cell dimensionsand geometries (e.g. 2.54-cm or 5.08-cm cubic cells) are used and when ions are not at thermal equilibrium, (1) Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1987, 78, 53. (2) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299. (3) Tanaka,K.; Waki,H.;Ido,Y.; Akita,S.;Yoshida,Y.RapidCommun. Mass Spectrom. 1988, 2, 151. (4) Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T. Anal. Chem. 1991,63, 1193A. ( 5 ) Castoro, J. A.; Koster, C.; Wilkins, C. Rapid. Commun. Mass Spectrom. 1992, 6, 239. (6)Koster, C.: Castoro, J. A.: Wilkins. C. L. J. Am. Chem. SOC. 1992. 114, 7572. (7) Castoro, J. A.; Chiu, R. W.; Monnig, C. A.; Wilkins, C. L. J. Am. Chem. SOC.1992, 114,7571. (8) Wang, M.; Marshall, A. G. Anal. Chem. 1989, 61, 1288. (9) Ledford, E. B., Jr.; Rempel, D. L.; Gross, M. L. Anal. Chem. 1984, 56. 2744. 0003-2700/93/0365-0784$04.00/0
theoretical upper mass limits which are much lower than the ideal critical mass are calculated.10 Fortunately, by use of a variety of experimental strategies, several of which are mentionedas possibilities in ref 10,the detectable mass range for a cubic trappihg cell operated in a ca. 7-T magnetic field can be extended 1 to values significantly higher” than the 12 300-Da trapping limit (which corresponds to a detection limit of about 24dO Da) calculated by Wood and co-workers for a 2.54 cm cubic cell.lo Because mass analysis in FTMS depends upon accurate measurement of ion cyclotron frequencies to determine the mass to charge ratio,12-15it is desirable to minimize any effects which shift those frequencies. One of the most important of such effects is that of the electric trapping potentials required to prevent loss of ions from the trap in the axial (or z-axis) parallel to the magnetic field direction.lO For any particular trapping voltage and field strength, the magnitude of the expected frequency shift (which is mass independent) can be calculated16 and is simply the difference between the magnetron frequency (w,) and the cyclotron frequency (w,) in the absence of trapping field. Thus, the effective frequency, weffective, is readily calculated. For a cubic cell, the geometry factor, a = 1.387, a is the distance between the trap plates (meters), and VT is the trapping voltage relative to ground potential, VO.B is the magnetic field strength (Tesla), q is the ionic charge (Coulombs),and m the ionic mass (kilograms). Ueffective
wc
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(3) Obviously, from eqs 1-3, the effect of the frequency shift becomes increasingly unimportant as magnetic field strength increases. However, even with magnetic field values near 7 T, as in the present study, trapping shifts of about 30 Hz/V are predicted. More important, for high-resolution applications, are the deleterious effects of space-charge interactions produced with higher trapping voltages, which can preclude trapping analyte ions for the periods requisite for highresolution mass measurements. (10) Wood, T. D.; Schweikhard, L.; Marshall, A. G. Anal. Chem. 1992, 64, 1461. (11) In unpublished experiments in our laboratory, dimer ions of carbonic anhydrase (miz 58 000) have been successfully trapped and
detected. (12) Comisarow, M. B.; Marshall, A. G. J. Chem. Phys. 1975,62,293. (13) Marshall, A. G.; Comisarow, M. B. Anal. Chem. 1975,47, 491A. (14) White, R. L.; Ledford, E. B., Jr.; Ghaderi, S.; Wilkins, C. L.; Gross, M. L. Anal. Chem. 1980.52. 1525. (15) Alleman, M.; Lellerhals, Hp.; Wanczek, K. P. I n t . J . Mass Spectrom. Ion Processes 1983,46, 139. (16) Dunbar,R. C.;Chen, J. H.; Hays, J. D. Intern. J.MassSpectrom. Ion Processes 1984, 57, 39. @ 1993 Amerlcan Chemical Society
ANALYTICAL CHEMISTRY, VOL. 65, NO. 6, MARCH 15, 1993
a b
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Figure 1. Screened electrostatic cublc cell diagram: (a)source trap plate: (b) source screen: (c) conductance limit screen: (d) conductance limit trap plate: (e) analyzer trap plate.
Thus, a variety of electrically compensated trapping cells have been designed and tested.17-19 One of the simplest such designs is the screened trap introduced by Wang and Marshalls which is constructed by interposing screen electrodes inside the trapping cell between the ion trapping region and the trap plates. In their original demonstration of the technique, Wang and Marshall used an orthorhombic cell (2.5 in. X 2.0 in. X 2.0 in.), with screen electrodes constructed from 0.0016in.-diameter tungsten wires and a mesh spacing of 16/in. Use of that design resulted in reduction of the frequency shift for benzene molecular ion, observed at 3 T, by a factor of about 100. In the present study, a l’/s-in. cubic cell of similardesign was used, with a somewhat higher screen density of 40wires/in., fabricatedfrom0.001-in. tungsten wires (Figure 1). Because conditions for MALDI-FTMS are so much different from the electron ionization studies of benzene and perfluorotri-n-butylaminediscussed in the original report, it is of interest to investigate whether theoretical predictions are accurate even for high-mass laser-desorbed species. Furthermore, it is also of interest to investigate whether any of the predicted analytical advantages, such as higher resolution, could be realized for molecules with masses above lo00 Da.
EXPERIMENTAL SECTION Instrumentation. Experiments were performed using a Millipore-Extrel (Madison, WI) FTMS-2000 dual-cell Fourier transform mass spectrometer equipped with a 7-T superconducting magnet and a modified source cell screened cubic trap geometry, 47.6 mm (screen to screen separation) X 47.6 mm X 47.6 mm, with both trap plates 2 mm from the screens, or 51.6 mm apart (Figure 1). The screens were constructed from 0.001in.-diameter tungsten 40 x 40 mesh (Unique Wire Weaving Co., Inc., Hillside, NJ) spot welded on a 3-mm stainless steel frame and electricallyisolated from the source cell trap plates by Teflon washers and from the conductance limit by a Teflon gasket (to allow maintenance of a pressure differential between source and analyzer cells). The wires in the center of the screens were displaced outward to create a 2-mm-diameter opening to avoid interaction with the UV laser beam. For laser desorption, a Lambda Physik EMG-201MSC excimer laser (operating at 308 nm, 180 mJ/28 ns pulse) was used to pump a Lambda Physik FL-2001dye laser. Ultraviolet radiation (355 nm) was produced by pumping the dye laser cell containing a 0.60 g/L dioxane solution of 2,2”’-dimethyl-p-quaterphenyl (BMQ, Lambda Physik), resulting in a maximum output energy of 5 mJlpulse. A 355-nm laser light attenuated by an iris enters the mass spectrometer through a fused silica window and is focused to a 500-pm-diameter beam impinging upon a probe tip (17) Yang, S. S.; Rempel, D. L.; Gross, M. L. 36th Am. SOC.Mass Spectrom. Annu. Conf. Mass Spectrom. Allied Topics 1988, 586. (18)Naito, Y.; Inoue, M. 36th Am. SOC.Mass Spectrom. Annu. Conf. Mass Spectrom. Allied Topics 1988, 608. (19) Hanson, C. D.; Castro, M. E.; Kerley, E. L.; Russell, D. H. Anal. Chem. 1990,62, 520.
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(incidence angle 0’) by a 12.5-cm focal length optical glass lens. This lens is mounted on a rotating lens assembly attached to the analyzer flange, allowing the lens to be rotated out of the way when electron ionization or chemical ionization measurements are made. The sample probe is positioned exterior to the source cell, approximately 1-2 mm from the front trap plate. The power is adjusted to obtain a power density of 106-107 W/cm2,which can be fine-tuned by attenuating the laser beam. Sample Preparation. 2,bDihydroxybenzoic acid, DHB (FlukaChemical Co., Buchs, Switzerland), was used as the matrix for the MALDI insulin (from bovine pancreas, Sigma Chemical Co., St. Louis, MO) and myoglobin (from equine skeletal muscle, type I, Sigma Chemical Co.) spectra. Samples were prepared by mixing a suitable quantity of a 0.5 mmol/L analyte solution in 0.1 % aqueous trifluoroacetic acid, TFA (Mallinckrodt, Inc., St. Louis, MO), with a 50 mmol/L matrix solution (0.1% TFA in methanol). For high-resolution insulin spectra, the matrix was modified by addition of a 50 mmol/L D-fructose (AldrichChemical Co., Milwaukee, WI) comatrix methanol solution. The volumes used were adjusted to give an analyte:matrix:comatrix molar ratio of 1:5000:5000 for insulin spectra measurements. For the lowresolution myoglobin measurements, no comatrix was used and the ratio of myoglobinto DHB was 1:3000. Poly(ethy1ene glycol) 1000 was dissolved in methanol and diluted to a 1:1000 molar ratio (PEG/DHB) solution and used as external calibrant. MALDI solutions were sprayed as aerosols onto a rotating stainless steel probe tip for homogeneous deposition. Electron ionization mass spectra were obtained by depositing 2,4,6-tris(perfluoroheptyl)-1,3,5-triazine, TpT (Fulka Chemical, Buchs, Switzerland), on a probe tip which was then inserted into the mass spectrometer, resulting in a source cell pressure of ca. 10-7-10-* Torr. Spectral Measurements. To measure MALDI spectra, the front trap plate and front screen are set to ground potential while the rear screen is set to 9 V, prior to firing the desorption/ ionization laser pulse. The laser is then triggered and, for both screen and nonscreen (or normal) trapping experiments, the appropriate trapping potential is applied followinga 9-V retarding potential delay of 160 p s for insulin and 255 p s for myoglobin. For screened trap measurements, the screens are gounded and trapping voltages applied to the trap plates in the conventional manner. In the normal measurements, the trap plates are grounded and the screensserve as trap plates. Electron ionization spectral measurements of TpT employed electron energies of 20 eV and emission currents of 200-400 nA in order to minimize the number of ions formed and space charge effects, which may influence the frequency and/or peak shape of the resulting spectrum. The screens/plates were held at either 0.2 or 0.5 V during the ionization event and then set to the appropriate trap voltages as described above. A 1-300-ms delay (for both LD and E1 spectral measurements) is imposed, following application of the trapping potential, to allow for desorbed neutrals to pump away and for the ions to relax and equilibrate within the trap. At the end of this delay, spectra are obtained. Data Processing. A typical scan employed a 200-V peakto-peak excitation sweep from 0 to 200 kHz at 200 HzIps sweep rate. TpT spectra were obtained by averaging 100 time domain data sets of 65 536data points and myoglobin spectra by averaging 5 sets of 16 384 points (collectedusing a 1-MHzdata acquisition rate). To obtain frequency domain mass spectra each averaged data set was augmented by an equal number of zeros and baseline corrected prior to magnitude-mode Fourier transformation. Alternatively, for insulin, single scan spectra, collecting 65 536 data points (collected using a 100-kHz data acquisition rate), zero-filling,and performing a Fourier transform were measured. No apodization was used. Resolution in all cases is estimated from the ratio of peak position to peak width at half-height. ICR frequency shift as a function of trapping voltage was determined from the frequency at the centroid of the monoisotopic 1C12peak for TpT, the 3 0 3 isotopic peak for insulin, or the unresolved molecular ion peak for myoglobin. Magnetic field strength was determined by fitting the frequencies (using the standard Millipore-Extrel software) of 10peaks from the known spectrum of laser-desorbed sodium-cationized poly(ethy1ene glycol) 1000 used as calibrant.
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 6, MARCH 15, 1993 88325
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myoglobin (Figure3a), -28.1 Hz/V (r = -0.997 for the doublycharged molecular ion (Figure3b), and-27.2 Hz/V (r = -1.OOO) for the m/z 1185 molecular ion of TpT (Figure 4). Trapping voltage was varied between 0.1 and 1.2 V for insulin, between 0.1 and 6 V for myoglobin, and for TpT, between 0.5 and 9 V. Negative ion spectra of TpT also were measured, verifying that the frequency shift of 28.3 Hz/V (r = 1.OOO) is of the same absolute magnitude as in the positive ion spectral measurements. Thus, the present resulta are consistent with the 3-T benzene molecular ion data presented by Wang and Marshalla and further confirm the validity of the model by demonstrating that the ion cyclotron frequenciesvary in the expected manner, independent of charge or mass, for ions with masses up to ca. 17 OOO Da.
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RESULTS AND DISCUSSION Ion Cyclotron Frequency Variation with Trapping Voltage in the Absence of Screening. As mentioned above, theory predicts that measured ion cyclotron frequencies should shift linearly with change in trapping potential, with the magnitude of that shift being independent of ionic mass or charge and determined only by trapping cell geometry and magnetic field strength. Figures 2-4 are plots of the frequency shift data obtained for the three test compounds for both screened and nonscreened trapping experiments. The expected frequency shift in the absence of screening was calculated to be -28.5 Hz/V at the estimated field strength of 6.83 T determined using the calibration procedure described. This is in excellent agreement with the measured values of -29.0 Hz/V (correlation coefficient, r = -0.981) for the m/z 5734 molecular ion of bovine insulin (Figure 2), -29.0 Hz/V ( r = -0.994) for the mlz 1695120 molecular ion of
grounding the screens while varying the trapping potentials of the trap plates was also investigated. For laser-desorbed molecular ions, the frequency shift is reduced to -1.2 Hz/V ( r = -0,871) for insulin, -1.5 Hz/V (r = -0.621) for myoglobin, and for electron-ionized TpT molecular ion, to -1.2 Hz/V (r = -1.OOO). From the experimental data, the equivalent nonscreened trapping potential can be calculated for the screened trap. Using a least squares fit for the nonscreened trap data for TpT in the absence of screening potential, and the effective ICR frequency measured with a 9-V screened trapping potential, the equivalent nonscreened electrostatic field is 0.33 V. Thus, the use of the screened trap design for these high mass molecules does accomplish the desired objective of minimizing penetration of the trapping potential into the measurement region of the cell. The value of the geometric constants, a, for a screened cubic trap has not previously been derived. It can be estimated by solving for a in eq 3 and using the experimentally determined frequencyshift with trapping voltage. Using the best fit data, the ICR frequency shift for TpT, 0,/2IIAV = -1.224 Hz/V. Magnetic field strength, B, is 6.812 T, and a, the distance between the screens, is 0.0476 m, resulting in a calculated value for a of 0.059 43. This is the expected order of magnitude, because elongatingthe ion trap can dramatically decrease this constant, as demonstrated by Hunter and coworkers.21 Furthermore, screening the applied potential also decreases a. As a consequence, a for a screened cubic trap is lower than for a normal cubic trap of the same dimensions, but slightly larger than for the screened tetragonal trap, where Wang and Marshall determined ita value to be 0.0166.8 (20) Zaia, J.; Annan, R. S.; Biemann, K. Rapid Commun. Mass Spectrom. 1992, 6, 32. (21)Hunter, R.L.;Sherman, M. G.; McIver, R. T.,Jr. Znt. J. Mass Spectrom. Zon Phys. 1983,50, 259.
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Table I. Comparison of MALDI-FTMS Mass Resolution for Normal and Screened Trap Spectra of Insulin as a Function of Traming Voltage screened trap normal trap trap voltage (V) resolution" trap voltage (V) resolution" 0.1 10436 3.0 11796 0.2 10221 4.0 11137 9451 5.0 11136 0.3 8838 6.0 11399 0.4 0.5 10878 7.0 12542 0.6 11433 8.0 9582 10246 9.0 9993 0.7 0.8 11788 12433 0.9 1.0 11107 1.2 7910 5670
Ratio of peak position to peak width at half height, for the 3C13 isotopic peak of the molecular ion (M+ H)+. a
Determining the Unperturbed Cyclotron Frequency. The unperturbed cyclotron frequency can be calculatedfrom eq 2but cannot be directlydeterminedexperimentally because the magnetic field confines the ions in the trap only in the x-y direction. An electrostatic trapping potential is needed to prevent ions from escaping along the 2 direction during spectral measurements. This applied potential produces the effective frequency shift, as discussed above. The unperturbed cyclotron frequencycan be estimated by extrapolating the measured frequency vs trapping potential plots to the Y-intercept, which corresponds to an absence of trapping potential, shown graphically in Figures 2-4. For the magnetic field used, the calculated cyclotron frequency for insulin is 18 333.68 Hz and the average experimental intercept is 18333.13 Hz (0.0030% error). For TpT the theoretical frequency is 88247.10 Hz and the average of the four intercepts is 88 300.43 Hz (0.060% error). Also, as seen in Figures 2-4,the intercepts are very nearly equal in magnitude for either screen or nonscreened experimentsand both positive and negative ions. Effect of the Screened T r a p upon Mass Resolution. It is desirable to minimize cyclotron frequency shifts, as demonstrated here, for ease of accurate mass measurement. Wang and Marshall predicted that use of such a cell should result in higher mass resolution and mass range than in the absence of the screen elementa.8 In the present context, highmass applications of MALDI-FTMS measurements, those benefits would be significant. Accordingly, mass resolution as a function of trapping potential was examined for both screened and nonscreened modes of operation. It would be difficult to assess subtle changes in mass resolution for single-shot matrix-assisted laser desorption spectra under specific trapping conditions because it is not possible to ensure identical numbers of ions are produced from measurement to measurement. However, it is possible to detect large changes or a trend in mass resolution over a range of trapping conditions. Table I summarizes a series of high-resolution measurements performed on trapped insulin molecular ions, for various normal and screened trapping potentials. For normal trapping, resolution averages 10 448 for the 11 measurements. However, no dependence of resolution on trapping voltage is seen. Similarly, when the screened trapping mode is used, there appears to be no dependence of resolution on trapping voltage, nor is the resolution (averaging 11 083) significantly greater than for normal mode measurements. Evidence of the efficacy of the screening function is provided by the observation that only spectra with distorted peak shapes could be obtained using trapping voltages greater than 1.2 V in normal trapping experiments,but that high resolution could be obtained even
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Table 11. Comparison of EI-FTMS Mass Resolution for Normal and Screened Trap Spectra of 2,4,6-Tris(perfluoroheptyl)-l,3,5-triazine as a Function of Trapping Voltage resolution" trapping positive negative voltage normal screened normal screened 1.0 8820 12980 10610 2.0 8800 3.0 7989 7410 8600 4.0 6461 8735 8500 5.0 6390 12819 7830 5943 8750 12280 6810 6.0 7.0 5017 10980 8600 8095 8.0 4997 10545 9370 8005 9.0 6324 12755 7952
" Ratio of peak position to peak width at half height, for the all C1*isotopic peak of the molecular ion, M+, for positive ion spectra and M-, for negative ion spectra;100spectra were averaged to obtain each resolutionestimate. For negative ion spectra,trappingvoltage is the opposite sign from that indicated in the first column.
using trapping voltages as high as 9.7V,when the screens were grounded (Figure 6). As a second test of the effect of screened trapping upon resolution, electron ionization spectra of the relatively volatile compound 2,4,6-tris(perfluoroheptyl)-l,3,5-triazine(TpT) were obtained using both screened and normal trapping conditions. Here too, mass resolution was determined as a function of trapping voltage while maintaining a reasonably constant sample pressure (between 1.2 X 10-7and 9.0 X 10-8 Torr) to minimize effects from pressure differences. Table I1summarizes the data. Comparison of these results, obtained under normal and screened trap conditions, also shows no pronounced resolution advantage of the screened trap, except when higher trapping voltages are used, where there is some evidence that resolution declines under the conventional trapping protocol, while it remains fairly constant using the screened trap technique.
CONCLUSIONS Ion cyclotron frequency shifts of approximately 30 Hz/V of analyte ion frequencies as a function of trapping voltages in a l7J8-in.cubic cell using a 7-Tmagnetic field can be reduced by almost a factor of 25 by using a screened trap cell of the Wang and Marshall design. Experimental shifts for the three test compounds studied, with masses ranging from 1185 to just under 17 OOO Da are in good agreement with theory and
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show the expected mass independence. It is also seen that the frequency shift is unchanged for doubly-charged myoglobin molecular ion and shows the expected sign, but not magnititude change for negative ion spectra. Furthermore, the screened trap is shown to be compatible with matrixassisted laser desorptionlionization, which is important if expected mass range and resolution benefits are to be realized. However, for the three test compounds investigated, there is no evidence of greatly improved mass resolution as a result of using the screened trap methodology. It can be speculated that this might result, in part, from the relatively dense screen used, which could be limiting mass resolution. The normal trapping mode positive ion electron ionization mass spectral measurementsof 2,4,6-tris(perfluoroheptyl)-1,3,5-triazine do show a systematic decline in resolution as trapping potential is increased, while the corresponding screened trapping experiments show no such trend. Similarly, under normal trapping conditions, no high-resolution MALDI spectra of insulin were obtainable for trapping voltages greater than 1.2 V, although high-resolution spectra could be obtained with
trapping voltages as high as 9.7V when the screenedtrapping method was used. Both observations are consistent with reduced space charge effects when the latter method is employed. This might be significant with high-mass MALDI applications, where higher trapping voltages may be required. Thus, the present study indicates that it would be worthwhile to refine and optimize the screened trapping method as a possible adjunct to MALDI-FTMS techniques.
ACKNOWLEDGMENT Support from NIH Grant GM-44606 and NSF Grant CHE92-01277is gratefully acknowledged. J.A.C. is grateful for partial support under an ACS AnalyticalChemistry Division Summer Fellowship sponsored by the Society of Analytical Chemists of Pittsburgh.
RECEIVEDfor review September 11, 1992. Accepted December 22, 1992.
