Aerosol MALDI with a Reflectron Time-of-Flight Mass Spectrometer

directly into a reflectron time-of-flight mass spectrometer, and the aerosol particles were irradiated and ionized with a frequency-tripled Nd:YAG las...
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Anal. Chem. 1996, 68, 1143-1147

Aerosol MALDI with a Reflectron Time-of-Flight Mass Spectrometer Xun Fei, Gang Wei, and Kermit K. Murray*

Department of Chemistry, Emory University, Atlanta, Georgia 30322

Aerosol matrix-assisted laser desorption/ionization (MALDI) has been combined with a reflectron time-of-flight mass spectrometer for improved mass resolution. A methanol solution of matrix and analyte was sprayed directly into a reflectron time-of-flight mass spectrometer, and the aerosol particles were irradiated and ionized with a frequency-tripled Nd:YAG laser at 355 nm. Mass resolution of over 300 was observed for the peptides bradykinin, angiotensin II, and gramicidin D and for vitamin B12. This represents a resolution enhancement of approximately 10-fold over that previously reported for aerosol MALDI with a linear time-of-flight instrument. Over the past decade, matrix-assisted laser desorption/ionization (MALDI) has developed into a powerful method for mass spectrometric analysis of biomolecules.1,2 MALDI is particularly well suited to mass analysis of peptides and proteins. For example, proteins as large as 106 Da have been mass analyzed by MALDI mass spectrometry.3 MALDI has also been demonstrated for peptide sequencing, either by mass analysis of products of post-ion-source decay (PSD)4 or fragments produced by chemical ladder sequencing.5 MALDI has been demonstrated for DNA mass analysis for oligonucleotides up to 500 base pairs.6 Although MALDI is typically used with time-of-flight (TOF) mass spectrometers,1 Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers have been used with MALDI for both highresolution7 and high-mass work.8 A major limitation of MALDI mass spectrometry is the difficulty in coupling it to direct liquid sample introduction. Direct liquid introduction is useful for coupling liquid separations such as liquid chromatography (LC) and capillary electrophoresis (CE), monitoring reactions in solution, and automated sample delivery. In a typical MALDI mass analysis, the sample is deposited on a metal solids probe, dried, and inserted into vacuum for solid surface desorption and ionization.1 Only a few direct liquid introduction methods have been used with MALDI. A continuous-flow probe has been developed that uses the liquid matrix 3-nitrobenzyl (1) Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T. Anal. Chem. 1991, 63, 1193A-1203A. (2) Burlingame, A. L.; Boyd, R. K.; Gaskell, S. J. Anal. Chem. 1994, 66, 634R683R. (3) Nelson, R. W.; Dogruel, D.; Williams, P. Rapid Commun. Mass Spectrom. 1995, 9, 625. (4) Kaufmann, R.; Kirsch, D.; Spengler, B. Int. J. Mass Spectrom. Ion Processes 1994, 131, 355-385. (5) Chait, B. T.; Wang, R.; Beavis, R. C.; Kent, S. B. H. Science 1993, 262, 89-92. (6) Tang, K.; Taranenko, N. I.; Allman, S. L.; Cha´ng, L. Y.; Chen, C. H. Rapid Commun. Mass Spectrom. 1994, 8, 727-730. (7) Ko¨ster, C.; Castoro, J. A.; Wilkins, C. L. J. Am. Chem. Soc. 1992, 114, 75727574. (8) Solouki, T.; Gillig, K. J.; Russell, D. H. Anal. Chem. 1994, 66, 1583-1587. 0003-2700/96/0368-1143$12.00/0

© 1996 American Chemical Society

alcohol,9 and a flowing aqueous matrix probe has also been reported.10 One potential drawback to the continuous-flow probe is that it is limited to flow rates of less than 5 µL/min. A method that uses aerosols for direct liquid introduction has also been reported.11 In the aerosol MALDI method, a solution of matrix and analyte is sprayed into the mass spectrometer at a liquid flow rate of up to 1 mL/min. A high-speed pumping system and a heated drying tube are used to remove solvent and carrier gas. The dried aerosol particles are irradiated with pulsed laser radiation to form protonated analyte ions that are mass analyzed in a TOF mass spectrometer. Aerosol MALDI has been coupled with liquid chromatography for on-line LC/MALDI-MS12 and has been used to elucidate fundamental aspects of MALDI and aerosol ionization processes.13-16 Aerosol MALDI has two drawbacks that limit its utility as an analytical method: sample consumption and mass resolution. Sample consumption is high because the aerosol beam is continuous whereas the laser is pulsed; a typical nanosecond pulsed laser has a duty cycle of at best a few parts in 108. An aerosol MALDI mass spectrum requires the consumption of hundreds of nanomoles of analyte, although femtomole quantities of material are irradiated.13 Sample consumption can be reduced using a higher repetition rate laser or by improving the efficiency of sample delivery, perhaps as single aerosol particles.17 The mass resolution reported for aerosol MALDI is in the range of 10-100 fwhm11-15 and is limited in a linear TOF by the initial ion spatial spread. Additional contributions to the observed peak widths result from unresolved adducts, metastable decay, and ion/ molecule collisions during acceleration.13 TOF mass resolution can often be improved by employing a reflectron mass spectrometer.18 Reflectron mass spectrometers have been used in conjunction with MALDI for mass resolution of m/∆m > 3000 for peptides and m/∆m > 1000 for proteins up to 12 kDa.1 A reflectron mass spectrometer compensates for initial (9) Li, L.; Wang, A. P. L.; Coulson, L. D. Anal. Chem. 1993, 65, 493-495. (10) Williams, E. R.; Jones Jr., G. C.; Fang, L.; Nagata, N.; Zare, R. N. In Applied Spectroscopy in Materials Science II; Golden, W. G., Ed.; Proc. SPIE 1636, Los Angeles, CA, 1992; pp 172-181. (11) Murray, K. K.; Russell, D. H. Anal. Chem. 1993, 65, 2534-2537. (12) Murray, K. K.; Lewis, T. M.; Beeson, M. D.; Russell, D. H. Anal. Chem. 1994, 66, 1601-1609. (13) Murray, K. K.; Russell, D. H. J. Am. Soc. Mass Spectrom. 1994, 5, 1-9. (14) Murray, K. K.; Russell, D. H. In Laser Ablation: Mechanisms and Applications - II; Miller, J. C., Geohegan, D. B., Eds.; American Institute of Physics: New York, 1994; pp 459-464. (15) Beeson, M. D.; Murray, K. K.; Russell, D. H. Anal. Chem. 1995, 67, 19811986. (16) Russell, D. H.; Beeson, M. D. J. Mass Spectrom., submitted. (17) McKeown, P. J.; Johnston, M. V.; Murphy, D. M. Anal. Chem. 1991, 63, 2069-2073. (18) Mamyrin, B. A.; Karataev, V. I.; Shmikk, D. V.; Zagulin, V. A. Sov. Phys. JETP 1973, 37, 45-48.

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Figure 1. Aerosol MALDI reflectron TOF mass spectrometer. Aerosol particles formed in vacuum pass through a heated drying tube into the ion source chamber. The 355 nm pulsed Nd:YAG laser, perpendicular to both the aerosol and laser beams, irradiates the particles to form ions that are accelerated into the reflectron TOF mass spectrometer.

ion energy spread because higher energy ions take a longer path in the ion reflector. Under the proper conditions, ions of the same mass but different energies will have the same flight times.4 A reflectron can also compensate for an initial ion spatial spread. Ions formed at different positions in an electric field gradient have different kinetic energies; thus the initial spatial spread translates into an energy spread that can be compensated for by the reflectron. In this article, results for aerosol MALDI with a reflectron mass spectrometer are reported. Mass spectra for the peptides bradykinin, angiotensin II, and gramicidin D and for vitamin B12 are reported. The aerosol MALDI reflectron TOF mass spectra show a 10-fold resolution improvement over aerosol MALDI with a linear TOF. Prospects for further improvement of mass resolution as well as detection limit are discussed. EXPERIMENTAL SECTION The aerosol MALDI apparatus used in this work is similar to one that was described in detail previously.13 Two differences are noteworthy: first, the linear TOF mass analyzer has been replaced with a two-stage reflectron mass analyzer,18 and second, the maximum laser pulse energy is a factor of 5 larger than that of the previously described apparatus. A brief description of the aerosol MALDI reflectron TOF apparatus is given below. A schematic diagram of the aerosol MALDI reflectron TOF mass spectrometer is shown in Figure 1. Aerosols are produced in vacuum with a pneumatic nebulizer that consists of a plastic disposable pipet tip affixed to a metal compression fitting with vacuum epoxy adhesive. Note that the view in the diagram is from above. A 1.6 mm o.d., 0.25 mm i.d. PEEK plastic tube inside the nebulizer delivers the liquid to the tip where it is entrained by the nitrogen carrier gas. Matrix and analyte solutions are delivered to the nebulizer using a syringe pump at a flow rate of 0.5 mL/min; the nitrogen carrier gas flow rate is 1.5 L/min. The aerosol formation chamber is evacuated by a 330 L/s roots blower and rotary piston backing pump, resulting in a pressure of 0.1 Torr in the aerosol formation chamber. Solvent is removed from the aerosol particles as they pass through a 27 cm long 3 mm i.d. heated drying tube that forms a differential pumping aperture between the aerosol formation and ionization chambers. The drying tube is heated to 500 °C by a cylindrical resistance heater. The temperature of the aerosol 1144

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particles themselves is unknown but is the result of a competition between evaporative cooling and heat transfer in the drying tube. The ionization chamber is evacuated to 10-4 Torr with a 2400 L/s liquid nitrogen trapped diffusion pump. After exiting the drying tube, the aerosol particles intersect the pulsed laser beam at a 90° angle. The UV source is a 10 Hz pulsed Nd:YAG laser (Powerlite 8000, Continuum, Santa Clara, CA) frequency tripled to 355 nm. The pulse width of the laser is 6 ns, measured with a 200 ps response time Si photodiode (Electro-Optics Technology, Traverse City, MI). A 30 cm focal length cylindrical lens is used to focus the laser to a spot size of 0.1 mm by 5 mm at the laser and ion beam intersection. This spot size represents the contour of 50% of the maximum irradiance, measured using the method of Huth-Fehre et al.19 Ions formed by laser irradiation are extracted perpendicularly to both the laser and ion beams with a continuous voltage of 10 kV applied to the acceleration plate. The opposite plate is held at ground and the central 1 cm hole is covered by a 2 mm, 90% transmission grid. Deflector plates direct the ion packet through a 9 cm long, 1 cm i.d. differential pumping aperture into the flight tube. A pressure of 5 × 10-7 Torr in the flight tube is maintained by a 1200 L/s liquid nitrogen-trapped 4 in. diffusion pump. A commercially available two-stage reflectron (R. M. Jordan, Grass Valley, CA) is used to mass separate the ions. Ions are postaccelerated an additional 2 kV onto a 40 mm dual-microchannel plate particle multiplier. The detector signal is sent to the 50 Ω input of a 500 MHz digital oscilloscope (9350M, LeCroy, Chestnut Ridge, NY) which is triggered by the laser Q-switch output signal. The measured jitter in the Q-switch output is less than 500 ps, and hence triggering from the photodiode was deemed unnecessary. At the maximum temporal resolution of the oscilloscope of 1 ns, the data can be acquired at just under 2 Hz. At 10 ns data point spacing the data can be acquired at 10 Hz. The mass spectra shown in this article result from an average of 100-1000 single mass spectra with a 1 ns data point spacing. Averaged mass spectra are transferred from the oscilloscope to a Power Macintosh microcomputer (Apple Computer, Cupertino, CA) for further analysis. Mass spectra were boxcar averaged for display and plotting. An n point boxcar average is given by

Yi )

1

n-1

n

k)0

∑y

in+k

where yi are the initial ion signal data points and Yi are the boxcaraveraged data points. Note that a boxcar-averaged data set is reduced in size by a factor of n. For example, a 200 000 point mass spectrum boxcar averaged with n ) 4 will result in a new data set of 50 000 data points. Reduction of the number of data points is necessary for plotting and printing the data. A 600 dot/ in. laser printer, for example, can depict fewer than 10 000 x axis data points on a full sheet of standard size paper. Sample solutions were made from matrix and analyte dissolved in methanol (99.8%; EM Science, Gibbstown, NJ) with 5% trifluoroacetic acid (J. T. Baker, Pittsburgh, NJ) by volume. The matrix compound 2,5-dihydroxybenzoic acid (Sigma, St. Louis, MO) and the analytes bradykinin (83%, B-3259; Sigma), angiotensin II (90%, (19) Huth-Fehre, T.; Westman, A.; Sundqvist, B. U. R. Rev. Sci. Instrum. 1994, 65, 511-512.

Figure 2. Aerosol MALDI reflectron TOF spectrum of angiotensin II with a DHB matrix. The mass resolution is 300 fwhm.

A-9525; Sigma), gramicidin D (G-5002; Sigma), and vitamin B12 (99%, V-2876; Sigma) were used without further purification. For all of the mass spectra reported, the solutions contained 1 mg/ mL analyte and 4 mg/mL matrix. RESULTS AND DISCUSSION An aerosol MALDI reflectron TOF spectrum of angiotensin II is shown in Figure 2. The spectrum is an average of 100 laser shots at 210 mJ/pulse. A 20 point boxcar average was performed on the data shown in the plot. The irradiance within the 0.1 × 5 mm focused laser beam is greater than 700 MW/cm2, nearly 3 orders of magnitude larger than the ion production threshold for solids probe MALDI.1 The compatibility of aerosol MALDI samples with these relatively high irradiance values has been noted previously and is ascribed to excess energy boiling off residual solvent from the aerosol particles.13,14 The peak at 79 µs in Figure 2 is associated with protonated angiotensin [M + H]+ ion. The mass resolution for the [M + H]+ peak is 300 as given by t/2∆t, where t is the flight time and ∆t is the fwhm of the peak as determined by a fit to a Gaussian functional form (note that t/2∆t ) m/∆m). The ion signal between 10 and 50 µs results from matrix fragments and protonated methanol clusters of the form H+‚(CH3OH)n. The presence of protonated methanol clusters in aerosol MALDI mass spectra was noted previously.13 The broad peak at 57 µs is associated with unimolecular or collision-induced postsource decay that is often observed in solids probe MALDI reflectron mass spectra.4 Experiments were performed at constant-acceleration voltage and different reflectron voltages to confirm the assignment of the PSD peak. Aerosol MALDI spectra of angiotensin II taken at a range of reflectron voltages are shown in Figure 3. For each spectrum, the acceleration voltage was held constant at 5 kV. The voltage to the back plate of the reflectron was changed from 5.3 to 5.9 kV in 200 V steps. The voltage drop across the second stage of the reflectron was 70% of the voltage applied to the back of the reflectron, and the front of the reflectron was at ground. As the reflectron voltage was increased, the position of the [M + H]+ peak decreased from 79.7 µs at 5.3 kV to 74.7 µs at 5.9 kV. The broad PSD peak remained at 58 µs in this reflectron voltage range. PSD fragmentation in the flight tube results in ions that do not have sufficient energy to penetrate the second stage of the reflectron. These ions are reflected back from the first reflectron stage with no mass separation and give rise to the broad peak at 58 µs. The flight time of the [M + H]+ ions that penetrate

Figure 3. Angiotensin II aerosol MALDI spectra at various reflectron voltages. The ion kinetic energy is 5 kV.

into the second stage of the reflectron shows a stronger dependence on reflectron voltage than the small PSD fragment ions that experience only the steep electric field gradient in the first stage of the reflectron. Figure 4 shows calibrated mass spectra of bradykinin, angiotensin II, gramicidin D, and vitamin B12. The mass spectra are an average of 1000 laser shots, and a boxcar average of order 10 has been performed on the data. The laser pulse energies were 210 mJ for bradykinin, 210 mJ for angiotensin II, 240 mJ for gramicidin D, and 12 mJ for vitamin B12. For all analytes except vitamin B12, the largest ion signal was obtained at the maximum laser pulse energy. The spectra show the same general features as the spectrum in Figure 2. The [M + H]+ peak is the prominent high mass peak for all the mass spectra with the exception of vitamin B12 in Figure 4c, which has a prominent peak corresponding to the protonated molecule that has lost a CN fragment. The [M - CN + H]+ peak is typically larger than the [M + H]+ peak in the solids probe MALDI TOF mass spectrum of vitamin B12 as well.20 Mass resolutions of 270 for bradykinin, 330 for angiotensin II, 310 for gramicidin D, and 400 for vitamin B12 were obtained. The broad peak corresponding to postsource decay is labeled PSD in the mass spectra (Note that the mass calibration in Figure 4 applies to ions formed in the source and does not apply to PSD formed ions.) The ratios of the area of the PSD peak to the [M + H]+ peak are 1.2 for bradykinin, 1.4 for angiotensin II, 9.1 for gramicidin D, and 1.6 for vitamin B12. The peak areas were obtained from uncalibrated mass spectra. These area ratios cannot be directly compared because the irradiance levels for the mass spectra are different; however, the high level of PSD for gramicidin D may help to explain why, for example, the [M + H]+ ion signal for this analyte is 20 times lower than that of angiotensin II. (20) Kinsel, G. R.; Preston, L. M.; Russell, D. H. Biol. Mass Spectrom. 1994, 23, 205-211.

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Figure 5. Mass resolution, m/∆m, for the [M + H]+ ion of bradykinin plotted as a function of laser pulse energy.

Figure 4. Aerosol MALDI reflectron TOF mass spectra of (a) bradykinin, (b) angiotensin II, (c) gramicidin D, and (d) vitamin B12 with DHB matrix. The mass resolution values are 270, 330, 310, and 400, respectively.

The mass measurement accuracy for the analytes in Figure 4 is 0.1% using the protonated methanol cluster peaks as an internal calibrant. Accuracy values of 0.01% can be achieved with solids probe MALDI when a peptide or protein internal calibrant is used.21 Mass measurement accuracy can be improved by increasing mass resolution and signal-to-noise ratio. Differences in spatial, temporal, or energy distributions between the analyte and calibrant ions can lead to a nonlinear mass scale and thus limit mass measurement accuracy. An internal calibrant with properties similar to the analyte will thus yield the highest mass measurement accuracy. Calibration using the protonated methanol cluster peaks, however, is much simpler and will be sufficient in many cases. A plot of the mass resolution for the [M + H]+ ion of bradykinin in a DHB matrix is shown in Figure 5. Below 10 mJ, no ion signal is observed. At 10 mJ, the mass resolution is the greatest at m/∆m ) 440. Mass resolution decreases as the laser pulse energy increases, to just below 300 at the maximum available (21) Beavis, R. C.; Chait, B. T. Anal. Chem. 1990, 62, 1836-1840.

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pulse energy of 200 mJ. Similar behavior is observed for the other analytes: mass resolution is greatest at the threshold pulse energy for observed signal and decreases with increasing pulse energy. A decrease in mass resolution with increasing laser pulse energy is observed for solids probe MALDI as well; however, the ion signal tends to decrease with increasing pulse energy.22 For aerosol MALDI of bradykinin, the ion signal is still increasing at a laser pulse energy 30 times greater than the threshold energy. The mass resolution of 300-400 observed for aerosol MALDI with a reflectron represents an approximately 10-fold improvement over the mass resolution obtainable with a linear TOF.11-15 This resolution is comparable to that obtained for averaged aerosol laser desorption ionization mass spectra using a reflectron mass spectrometer.23 However, the mass resolution is a factor of 10 lower than that achievable with reflectron solids probe MALDI for analytes of similar mass.1 Factors that limit aerosol MALDI mass resolution are the time duration of ion formation, initial ion spatial spread, and ion source pressure. It is possible that aerosol particle breakup and ion formation could last 100-200 ns. Ion formation for as long as 1 µs after laser irradiation has been observed in ions formed from liquid beams.24 Initial ion spatial spread is limited by the laser beam width along the flight axis. If a lower bound to the aerosol MALDI ionization threshold irradiance is taken to be 106 W/cm2,1 the estimated 0.4 mm spatial spread will result in a energy spread of approximately 6% of the total beam energy. A typical reflectron can compensate for kinetic energy spreads up to 10-20% of the ion beam energy,4,18,25 suggesting that the ion spatial distribution is not limiting mass resolution. Ion/neutral collisions in the 10-4 Torr ion source region can also increase the energy spread in the ion beam. Improvements in nebulizer efficiency and multiple-stage acceleration are potential solutions to this problem. The dependence of mass resolution on laser pulse energy does not help to distinguish among the possible resolution-limiting mechanisms. The decrease in mass resolution results from an (22) Ingendoh, A.; Karas, M.; Hillenkamp, F.; Giessmann, U. Int. J. Mass Spectrom. Ion Processes 1994, 131, 345-354. (23) Mansoori, B. A.; Johnston, M. V.; Wexler, A. S. Anal. Chem. 1994, 66, 3681-3687. (24) Mafune´, F.; Kohno, J.; Nagata, T.; Kondow, T. Chem. Phys. Lett. 1994, 218, 7-12. (25) Spengler, B.; Kirsch, D.; Kaufman, R. Rapid Commun. Mass Spectrom. 1991, 5, 198-202.

increase in the [M + H]+ peak width. Higher laser pulse energy could lead to increased spatial and temporal distribution of the ions, both of which will lead to peak broadening. Additionally, a higher pulse energy could lead to an increase in material ejected from the aerosol particles and this could in turn lead to a greater number of collisions and peak broadening. Detailed studies of the dependence of mass resolution on laser pulse energy for various matrix and analyte combinations are underway and may shed further light on this phenomenon. CONCLUSIONS The coupling of aerosol MALDI with a reflectron mass spectrometer has resulted in an improvement in mass resolution by a factor of 10 over a linear TOF-MS: mass resolution between 300 and 400 was observed for the analytes angiotensin II, bradykinin, vitamin B12, and gramicidin D. Mass resolution was found to be greatest at the lowest laser pulse energies that produced ion signal and to decrease with increasing laser pulse energy. Resolution may be limited by initial ion spatial and temporal distributions or by collisions during ion acceleration. Further study is necessary to more clearly understand the factors limiting mass resolution and to further improve the resolution. Future work will also be aimed at improving the upper mass limit and the detection limit of aerosol MALDI.

Several aerosol MALDI instrument modifications are planned. Multiple-stage acceleration will limit the contribution of the initial ion spatial spread to the ion energy distribution. The effects of the initial temporal spread of ions can be reduced by applying the acceleration voltage after ion formation. Single aerosol particle production and ionization will improve both the detection limit and the ion source region pressure. A single particle delivered to the point of ionization will reduce sample waste and improve the duty cycle for ionization. Mass resolution may also be improved for single-particle mass spectra because the ion spatial distribution will be limited by the particle size rather than the width of the ionization laser beam. ACKNOWLEDGMENT This research was supported by Emory University and the Emory University Research Committee. The authors thank the American Society for Mass Spectrometry and Finnigan Corp. for an ASMS Research Award and the Society of Analytical Chemists of Pittsburgh for a SACP Starter Grant Award. Received for review September 13, 1995. December 13, 1995.X

Accepted

AC950913P X

Abstract published in Advance ACS Abstracts, February 15, 1996.

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