Optical Detection and Charge-State Analysis of ... - ACS Publications

Y. Cai, W.-P. Peng, S.-J. Kuo, S. Sabu, C.-C. Han, and H.-C. Chang*. Institute of Atomic and Molecular Sciences, Academia Sinica, P.O. Box 23-166, Tai...
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Anal. Chem. 2002, 74, 4434-4440

Optical Detection and Charge-State Analysis of MALDI-Generated Particles with Molecular Masses Larger Than 5 MDa Y. Cai, W.-P. Peng, S.-J. Kuo, S. Sabu, C.-C. Han, and H.-C. Chang*

Institute of Atomic and Molecular Sciences, Academia Sinica, P.O. Box 23-166, Taipei, Taiwan 106

Charged polystyrene nanoparticles are generated by matrixassisted laser desorption/ionization (MALDI) and detected by laser-induced fluorescence (LIF) in a quadrupole ion trap. Employing the LIF technique, observations of individual fluorescent nanospheres (27 nm in diameter and containing 180 fluorescein dye equivalents) have been achieved with an average signal-to-noise ratio of ∼10. With the trap operating at a frequency around 5 kHz, charge state analysis of the particles reveals that the number of charges carried by the spheres is between 1 and 10. It suggests a mass-to-charge ratio (m/z) in the range of 105-106 for the MALDI-generated particles. To effectively trap such large particles (m > 5 MDa), damping of the particles’ motions by using ∼50 mTorr He buffer gas is absolutely required. Similar findings are obtained for particles with a nominal size of 1 µm in diameter, demonstrating that production of charged particles with a molecular mass as high as 1012 Da is possible using the MALDI technique. Matrix-assisted laser desorption/ionization (MALDI), first described by Karas, Bachmann, and Hillenkamp in 1985,1 has now evolved into one of the most powerful tools in mass analysis of nonvolatile and high-molecular-weight compounds. It is a soft ionization method and produces little fragmentation in the desorption/ionization process. The method has allowed investigation of DNA fragments up to a size of 2180 nucleotides, or a molecular mass exceeding 600 kDa.2 Using all-trans-retinoic acid as the matrix, Schriemer and Li3 demonstrated further that MALDI mass spectra of polystyrenes with a molecular mass of 1.5 MDa can be attained. This mass analysis range (up to 1.5 MDa) is comparable to what has been achieved using electrospray ionization (ESI), first illustrated by Fenn and co-workers4 for multiply charged poly(ethylene glycol) ions. However, compared to ESI, MALDI may have an upper mass-analysis limit, depending on the nature of the analyte and how the analyte interacts with the matrix molecules.3 In an earlier publication,5 we reported detection of single submicrometer-sized particles produced by ESI and pre(1) Karas, M.; Bachmann, D.; Hillenkamp, F. Anal. Chem. 1985, 57, 29352939. Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (2) Berkenkamp, S.; Kirpekar, F.; Hillenkamp, F. Science 1998, 281, 260262. (3) Schriemer, D. C.; Li, L. Anal. Chem. 1996, 68, 2721-2725. (4) Nohmi, T.; Fenn, J. B. J. Am. Chem. Soc. 1992, 114, 3241-3246. (5) Cai, Y.; Peng, W.-P.; Kuo, S.-J.; Lee, Y. T.; Chang, H.-C. Anal. Chem. 2002, 74, 232-238.

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sented the spectra of these particles with mass-to-charge ratios (m/z) in the range of 108-1010. This paper explores the possibility of using MALDI as an alternative method to produce charged particles with molecular masses higher than 1.5 MDa, or m/z g 106. In analysis of MALDI-generated ions, conventional time-of-flight (TOF) mass spectrometers are often used. For large (or macro) molecular ions,6 this requires the use of high acceleration voltages (up to 30 kV), which inadvertently leads to electric discharge of residual gas in the ion source. With currently available detector designs (such as microchannel plates and electron multipliers), there remains a need to enhance the detection sensitivity for macro-ions because of the low impact velocity of such ions on detector elements.7 A combination of these two factors has restricted routine analysis of large biomolecular ions (m/z > 106) with the conventional detection schemes. Radically different approaches have thus been adopted to solve this problem.6,8-12 Using a trapping plate, Nelson et al.11 collected laser-ablated materials from frozen aqueous solutions containing high-molecular-weight DNA molecules and verified the presence of 410 kDa DNA fragments in a follow-up gel electrophoresis (GE). With a similar two-step approach, Handschuh et al.12 collected laserdesorbed poly(ethylene glycol) particles and analyzed the particle size distribution with an atomic force microscope (AFM). They concluded that the polymers can be deposited on the trapping plate either as individual molecules or as small clusters. These two detection methods (GE and AFM), however, do not provide relevant charge state information of the incipiently desorbed species. Laser-induced fluorescence (LIF) has long been utilized as a method in the analysis of bioparticles labeled with dye molecules.13 (6) Fuerstenau, S. D.; Benner, W. H. Rapid Commun. Mass Spectrom. 1995, 9, 1528-1538. Fuerstenau, S. D.; Benner, W. H.; Thomas, J. J.; Brugidou, C.; Bothner, B.; Siuzdak, G. Angew. Chem., Int. Ed. 2001, 40, 541-544. (7) Glimore, I. S.; Seah, M. P. Int. J. Mass Spectrom. 2000, 202, 217-229. (8) Park, M. A.; Callahan, J. H. Rapid Commun. Mass Spectrom. 1994, 8, 317322. (9) Imrie, D. C.; Pentney, J. M.; Cottrell, J. S. Rapid Commun. Mass Spectrom. 1995, 9, 1293-1296. (10) Bahr, U.; Ro¨hling, U.; Lautz, C.; Strupat, K.; Schu ¨ renberg, M.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1996, 153, 9-21. (11) Nelson, R. W.; Rainbow, M. J.; Lohr, D. E.; Williams, P. Science 1989, 246, 1585-1587. (12) Handschuh, M.; Nettessheim, S.; Zenobi, R. Appl. Surf. Sci. 1999, 137, 125-135. (13) See, for example, Taylor, J. R.; Fang, M. M.; Nie, S. Anal. Chem. 2000, 72, 1979-1986. Ma, Y. F.; Shortreed, M. R.; Yeung, E. S. Anal. Chem. 2000, 72, 4640-4645. Kolodny, L. A.; Willard, D. M.; Carillo, L. L.; Nelson, M. W.; Orden, A. V. Anal. Chem. 2001, 73, 1959-1966. 10.1021/ac020205l CCC: $22.00

© 2002 American Chemical Society Published on Web 07/26/2002

EXPERIMENTAL SECTION The experimental setup is depicted schematically in Figure 1. The quadrupole ion trap (Jordan) has electrodes with parabolic

surfaces and standard geometries of z0 ) 7.07 mm and r0 ) 10 mm, as described in detail elsewhere.5 Two holes (with a diameter of 3.1 mm) were made on the end-cap electrodes for sample introduction and entry of the MALDI laser beam into the ion trap, and four holes (3.8 mm in diameter) were made symmetrically on the ring electrode for entry/exit of the probe laser and fluorescence collection. A Roots mechanical pump evacuated the ion trap chamber to a base pressure of 1 mTorr. In conducting the MALDI measurement, a light beam from a frequency-tripled Nd:YAG laser (Continuum Surelite) entered the ion trap through the hole on the lower end-cap. It was focused through an f ) 0.5 m lens, yielding a spot size 1 mm in diameter on the sample substrate. The MALDI-generated particles, as dispersed in the gas phase, were trapped in the ac field provided by a home-built power amplifier.17 The trapping frequency (Ω/2π) was variable from 100 Hz to 20 kHz at a constant peak-to-peak voltage of 2Vac ) 400 V. To effectively trap the MALDI-generated particles, helium gas was introduced into the chamber and regulated by a leak valve at a pressure p ≈ 50 mTorr. The particles were thereby damped to the trap center after collisions with the buffer gas atoms. They were typically stored in the ion trap for 60 s before fluorescence measurements. A polarized Ar ion laser (Coherent Innova 90C) operated at 488 nm probed the desorbed polymeric ions. The probe laser was focused with an f ) 1 m lens through one of the ring-electrode holes to produce a beam waist of ∼150 µm in the trap center. The estimated power density of the laser beam at the point of excitation was F ≈ 400 W/cm2. Laser-induced fluorescence was collected in a 90° configuration with an f/3 lens system (cf. Figure 1). It passed through a spatial aperture and then a holographic notch filter (Kaiser) to reject the scattered 488-nm light. A filter stack, composed of one short-pass (540 nm) and two long-pass (500 and 520 nm) filters, was further used to reduce the background signals. A thermoelectrically cooled photomultiplier tube (PMT, Hamamatsu R943-02) collected the emission, and the signal after preamplification was analyzed with a photon counter (Stanford Research SR400). The typical integration time was 60 ms, with a dwell time of 2 ms, in the photon counting measurements. The sample (FluoSpheres), consisting of 27 ( 4 nm yellowgreen fluorescent polystyrene beads in water, was obtained from Molecular Probes.18 Each particle contained on average 180 fluorescein dye equivalents, which absorb strongly at 488 nm and emit photons with a spectrum peaking at 515 nm. They were used in this experiment without further purification. To prepare the sample for MALDI, the yellow suspension was diluted with deionized water to a concentration in the range of 1011-1014 particles/cm3. Equal volumes of the sample and the matrix, a saturated solution of 3-hydroxypicolinic acid (3HPA) in the solvent mixture (70% acetonitrile and 30% water), were combined on a glass slide.19 After brief mixing of these two solutions, 3 µL of the mixture was deposited on a stainless steel MALDI sample

(14) Shera, E. B.; Seitzinger, N. K.; Davis, L. M.; Keller, R. A.; Soper, S. A. Chem. Phys. Lett. 1990, 174, 553-557. (15) Weston, K. D.; Carson, P. J.; Metiu, H.; Buratto, S. K. J. Chem. Phys. 1998, 109, 7474-7485. (16) Whitten, W. B.; Ramsey, J. M.; Arnold, S.; Bronk, B. V. Anal. Chem. 1991, 63, 1027-1031. Barnes, M. D.; Ng, K. C.; Whitten, W. B.; Ramsey, J. M. Anal. Chem. 1993, 65, 2360-2365.

(17) Ting, J. EDN, 2001, April 26, 136-138. (18) Haugland, R. P. Handbook of Fluorescent Probes and Research Chemicals, 6th ed.; Molecular Probes: Eugene, OR;1996. (19) See, for example, Zhu, L.; Parr, G. R.; Fitzgerald, M. C.; Nelson, C. M.; Smith, L. M. J. Am. Chem. Soc. 1995, 117, 6048-6056. Zhu, Y. F.; Lee, K. L.; Tang, K.; Allman, S. L.; Taranenko, N. I.; Chen, C. H. Rapid Comm. Mass Spectrom. 1995, 9, 1315-1320.

Figure 1. Schematic diagram of the experimental setup. An ac voltage of Vac ) 200 V is applied across the two end caps and the grounded ring electrode of the quadrupole ion trap. The enclosed cross at the trap center denotes the Ar ion laser beam (λ ) 488 nm) directed into the ion trap through two holes in the ring electrode and on the axis perpendicular to this cross-sectional view.

Exceedingly high sensitivity can be achieved using this method. For example, Shera et al.14 observed single rhodamine-6G molecules in solution in a flow cell, whereas Weston et al.15 detected fluorescence from single molecules adsorbed on a glass surface at room temperature. Detection of a single dye molecule embedded in a single microdroplet levitated electrodynamically in a quadrupole ion trap has also been demonstrated by Ramsey and co-workers.16 A signal-to-noise ratio (S/N) approaching 10 is readily attained for a single rhodamine-6G molecule. The high detection sensitivity of this optical method has made LIF a potentially useful approach for investigation of single dye-labeled particles as well as fluorescent nanometer-sized materials to be transported from condensed phases to the gas phase for detailed analysis using the MALDI technique. We report herein on the use of laser-induced fluorescence in combination with a quadrupole ion trap as a method to detect and analyze charged particles generated by MALDI. The method is proposed to surmount, in part, the difficulties that the conventional TOF mass spectrometry has encountered in conducting detection and mass analysis of particulate and polymeric molecules. In this study, fluorescent polystyrene spherical particles are employed as the sample and high-pressure buffer gas is used to damp the particles toward the center of the quadrupole ion trap for spatial confinement. With the trap operating at a frequency around 5 kHz, the electrodynamically confined particles have m/z values on the order of 106. Charge-state analysis of the MALDIgenerated particles is possible using this ion-trapping device along with theoretical calculations.

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holder, dried in air, and then left in a vacuum overnight. The sample was mounted on a surface located close to the upper endcap hole (Figure 1) and irradiated with ∼5 mJ pulses from the frequency-tripled YAG laser. The estimated power density on the sample substrate was ∼1 × 107 W/cm2. RESULTS AND DISCUSSION Generation and Trapping of Charged Nanoparticles. One key feature for trapping charged macroscopic particles in this experiment is utilization of high-pressure buffer gas. For molecular ions, typically 1 mTorr of He gas is employed in commercial ion trap mass spectrometers for effective trapping and, at the same time, establishing good mass resolution. The buffer gas clearly may play an even more important role here, because the MALDIproduced ions are heavy and thus possess enormous amounts of kinetic energy.20,21 In conventional ion trap mass spectrometry, maintaining a steady-state buffer gas pressure above 1 mTorr is incompatible with the use of ion detectors, such as the electron multipliers. In an effort to trap and analyze high-mass ions generated by MALDI, Schlunegger et al.22 employed pulsed buffer gas to provide a peak pressure of 10 mTorr (with a duration of 1-10 ms) in the ion trap region. Although this design has made possible detection of singly charged molecular ions of IgG (Mw ∼ 150 000), the average buffer gas pressure inside the chamber already rises up to 10-4 Torr. In our experiment, the nanoparticle has a mass on the order of 104 kDa, and an even higher pressure is required to damp the particle’s motions effectively for trapping. The fluorescence detection scheme proposed in this work, which avoids the use of high voltages inside the chamber, may offer a solution to circumvent the difficulty when high buffer gas pressure is needed in operation. Figure 2 displays the time course of the emission collected for MADLI-generated gas-phase particles from the samples prepared at various concentrations (1.6 × 1011 to 4.8 × 1014 particles/cm3). Typically, three laser shots are used in particle generation, and the particles are being trapped under the same conditions, p ) 50 mTorr, Ω/2π ) 5 kHz, and Vac ) 200 V, in each fluorescence measurement. Essentially, no signal is detected at the lowest concentration (Figure 2d), except that derived from the scattered laser light, which stays constant over time as the background signal. Increasing the sample concentration by 1-2 orders of magnitude noticeably enhances the intensity of the emission at 500-540 nm. The emission shows a quick decay, a behavior distinctly different from that of the background signal. The typical number of the photons collected for the emission is ∼5000/60 ms in the first 1 s of the excitation, as shown in Figure 2b,c. Further increase of the sample concentration, however, does not increase the S/N level accordingly. The emission becomes significantly weaker when the concentration exceeds 4.8 × 1014 particles/cm3 (Figure 2a). This is ascribable to reduction of the MALDI efficiency at higher sample concentrations. Such a concentration dependence of the signals (cf. Figure 2) clearly indicates that the observed emission is derived primarily from the MALDI-generated polystyrene particles, rather than from the laser-ablated matrix molecules or clusters, which are known to absorb poorly at 488 nm.23 The optimum condition for generation (20) Beavis, R. C.; Chait, B. T. Chem. Phys. Lett. 1991, 181, 479-484. (21) Gluckmann, M.; Karas, M. J. Mass Spectrom. 1999, 34, 467-477. (22) Schlunegger, U. P.; Stoeckli, M.; Caprioli, R. M. Rapid. Commun. Mass Spectrom. 1999, 13, 1792-1796.

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Figure 2. Temporal profiles of the fluorescence signals from the trapped nanoparticles irradiated by an Ar ion laser. The fluorescence was collected for the particles produced by MALDI with an analyte concentration of (a) 4.8 × 1014, (b) 9.6 × 1013, (c) 1.6 × 1012, and (d) 1.6 × 1011 particles/cm3. The buffer gas pressure used in the measurements was 50 mTorr, and the laser power density at the point of excitation was 400 W/cm2. Note the change of scale in the vertical axis in (a) and (d).

of the 27 nm particles by MALDI, as conducted in this experiment, is in the concentration range of 1012 to 1013 particles/cm3. In Figure 3, we compare results of the measurements for particles trapped under three different conditions, p ) 100, 70, and 35 mTorr, with the sample concentration fixed at 1.6 × 1012 particles/cm3. As seen, the signal lasts longer as the buffer gas pressure increases from 35 to 100 mTorr. It has a typical decay time of roughly 8 s at p g 100 mTorr. This temporal decay is most likely associated with photobleaching of the dye molecules, rather than loss of the particles from the trap. We rule out the possibility of the latter from repeated measurements of the signal, the intensity of which varies quite independently with the particle storage time before the laser excitation. Further support of the first interpretation comes from the measurements of Hamann et al.,24 who reported a photobleaching time of ∼500 s for the same (23) Puretzky, A. A.; Geohegan, D. B. Chem. Phys. Lett. 1998, 286, 425-432. (24) Hamann, H. F.; Kuno, M.; Gallagher, A.; Nesbitt, D. J. J. Chem. Phys. 2001, 114, 8596-8609.

Figure 4. Temporal profiles of the fluorescence signals from the trapped nanoparticles produced by MALDI and interrogated by using an Ar ion laser. The analyte concentration of the sample was 1.6 × 1012 particles/cm3; the trapping conditions were p ) 50 mTorr, Ω/2π ) 5 kHz, and Vac ) 200 V; and the laser power density at the point of excitation was 400 W/cm2. Note the weak and sporadic features appearing at later times of the excitation. Inset: Enlarged view of the well-separated peaks, which are characteristic of fluorescence detection in the single-particle limit.

Figure 3. Temporal profiles of the fluorescence signals from the trapped nanoparticles as a function of He buffer gas pressure of (a) 100, (b) 70, and (c) 35 mTorr. The fluorescence was collected for the particles produced by MALDI with an analyte concentration of 1.6 × 1012 particles/cm3.

fluorescent nanoparticles trapped on a fused-silica surface with use of a power density of 5 W/cm2 from an Ar ion laser. In our experiment, the particles are excited with a power density of ∼400 W/cm2. Assuming that the photobleaching rate is linearly proportional to the excitation photon flux, this suggests a decay time of ∼6 s for such trapped particles at F ≈ 400 W/cm2. The estimate is in good agreement with our observations (∼8 s). With this interpretation, the faster decays observed at the lower buffer gas pressure regions (cf. Figure 3b,c) can then be understood as a result of lessening of the collisional cooling effect.25 Single Particle Detection. Interesting features may emerge when a large number of particles are confined in the trap. Figure 4 displays a histogram obtained with the sample prepared at a concentration of 1.6 × 1012 particles/cm3 and trapped at a buffer gas pressure of 50 mTorr. Weak and sporadic peaks are found in the spectrum, as highlighted in the inset in Figure 4. The peaks are well-separated and become evident after illumination of the particles for more than 10 s. They appear presumably because the trapped particles are all in oscillatory motion and not all of (25) March, R. E.; Hughes, R. J. Quadrupole Storage Mass Spectrometer; Wiley: New York, 1989; Chapter 4.

them are confined in the probe volume of the laser beam. Although most of the particles are quickly photobleached in the first few seconds, those started with large amplitude motions and not in the probe volume are excited only at later times. Excitation of those particles, which are low in density, thus may yield emission as temporally separated peaks (or sharp spikes) in the histogram. It is noted that the time evolution pattern of these photon bursts closely resembles that observed for single rhodamine6G molecules in a flow cell26 and in an electrodynamically focused microdroplet stream27 in fluorescence detection. They are, therefore, likely to derive from single particles with their oscillatory amplitudes somewhat larger than the spot size (∼150 µm) of the probe laser beam. The evidence that the individual spikes in the inset in Figure 4 are derived from one single particle may be inferred from an analysis of the absolute fluorescence intensity. We calculate the number of photons (Is) detected in each photon-counting channel in the single particle limit as

Is ) NσFφf fctd

where σ is the absorption cross section, φf is the fluorescence quantum yield, N is the number of the dye equivalents in the (26) Goodwin, P. M.; Ambrose, W. P.; Keller, R. A. Acc. Chem. Res. 1996, 29, 607-613. (27) Lermer, N.; Barnes, M. D.; Kung, C.-Y.; Whitten, W. B.; Ramsey, J. M. Anal. Chem. 1997, 69, 2115-2121.

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analysis of the trapping potential in combination with a molecular damping model. Figure 5b displays the result of a calculation for the potential well depth (Dz) of an ideal quadrupole ion trap as a function of both trapping frequency (Ω/2π) and charge number (z).29,30 For particles that are being trapped in this electrodynamic device, they must possess a kinetic energy lower than the depth of the potential well, namely mυ2/2 < Dz. Given the average mass m ) 1.08 × 10-20 kg in the present case, this suggests a value of υ < 30 m/s for the particles trapped in the potential well of this device operating at Ω/2π ) 5 kHz and Vac ) 200 V (cf. Figure 5b). Note that the estimated velocity, υ < 30 m/s, is a result of many collisions of the MALDI-generated particles with background He atoms. To evaluate the initial velocity (υ0), we assume that all of the particles experience the same viscous forces after desorption from the surface, and their motions are described by

dυ ) -kυ dt

m

where k is the constant of damping from the buffer gas. The υ0 can then be evaluated from the solution for this equation of motion, υ ) υ0e-kt/m and υ0 ) υ + Sk/m, where S is the distance that the particle travels to reach the final velocity, υ. For a sphere with a diameter much larger than the mean free path of background gas molecules in thermal equilibrium, the damping constant is expressed as31,32 Figure 5. (a) Dependence of the experimentally measured total fluorescence intensity on ion trapping frequency, Ω/2π. The signals were integrated over 30 s of the fluorescence profiles. (b) Variation of the theoretically calculated potential well depths (Dz) of an ideal quadrupole ion trap with the particle charge number, z, at three different trapping frequencies, Ω/2π ) 5, 7, and 10 kHz. Values of the parameters used in the calculations were Vac ) 200 V and m ) 1.08 × 10-20 kg.

nanospheres, F is the excitation photon flux, fc is the light collection efficiency (including the PMT detection efficiency), and td is the photon detection time. Given N ) 180, σ ≈ 2 × 10-16 cm2/molecule for the fluorescein dye molecule,28 F ≈ 8 × 1020 photons/cm2s, φf ≈ 0.4,18 and fc ≈ 1 × 10-3, the number of photons detected by the photomultiplier tube is Is ≈ 700 for a td ) 60 ms integration time. Notably, this photon number is about three times larger than the background level (Ib ≈ 230 photons) arising from stray laser light as measured in the present experiment. Accordingly, one single particle of the 27-nm sphere should be detected with an estimated signal-to-noise ratio of S/N ≈Is/xIb ≈ 45 using the current setup. Our observed S/N ratios for the wellseparated features in Figure 4 are somewhat lower than the estimate. There are two possible causes for this discrepancy: first, the dye molecules are already partially photobleached in the first few seconds of the excitation, and second, the particles pass through only the weaker part of the laser beam as a result of their large amplitude motions. On the basis of this single particle detection, we estimate that roughly 50 particles are in the laser probe volume when the histogram shown in Figure 4 is taken. Estimation of Particle Velocity. One may estimate the initial velocity of the MALDI-generated particles in the trap from an (28) Brackmann, U. Lambdachrome Laser Dyes; Lambda Physik: Go ¨ttingen, 1986.

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k≈

(8 + 0.9π) pm 4 F0d

x

8M πkBT

where p is the buffer gas pressure, M and T are the molecular mass and temperature of the buffer gas atoms, and υ, F0 and d are the velocity, density and diameter, respectively, of the particle investigated. In the case p ) 50 mTorr, T ) 300 K, F0 ) 1.05 g/cm3, and d ) 27 nm, the mass-weighted damping constant is calculated to be k/m ≈ 2000/s. With S ≈ 10 mm, which is the distance between the sample surface and the trap center, the initial velocity of the trapped particles is, thus, estimated to be υ0 < 50 m/s. The important message conveyed by the above analysis is that a substantial number of slow particles (υ0 < 50 m/s) are generated by MALDI for the polystyrene nanospheres. The result is markedly different from that of organic and biomolecular ions, whose velocities have been determined to center around 700 m/s with a narrow distribution.20,21 One possible origin for this difference is that the particles studied in this experiment are much larger in size than the matrix molecules. For the nanometer-sized particles (d ) 27 nm), one may expect that the energy received for desorption/ionization in the process of MALDI is proportional to the number of matrix molecules surrounding them. Since the energy scales with d2 and the velocity of the particle at a given energy is in inverse proportion to the square root of its mass (or (29) March, R. E.; Hughes, R. J. Quadrupole Storage Mass Spectrometer; Wiley: New York, 1989; Chapter 2. (30) Sudakov, M. Int. J. Mass Spectrom. 2001, 206, 27-43. (31) Dahneke, B. E. Aerosol Sci. 1973, 4, 147-161. (32) Cai, Y.; Peng, W.-P.; Kuo, S.-J.; Chang, H.-C. Int. J. Mass Spectrom. 2002, 214, 63-73.

d3), we have υ0 ∝ d - 1/2. This simple model suggests that the larger the particle is, the lower the velocity will be. Hence, not surprisingly, a large number of the desorbed nanospheres as investigated in this experiment would not achieve the same velocity of molecular ions as studied previously.20,21 Charge State Analysis. An ion trap is capable of capturing particles with m/z falling in the stability region of its operational diagram. Particles with different m/z values would be captured as the trapping frequency and voltage amplitude are varied, according to the Mathieu equation.29 One may utilize this property and deduce the charge state information by determining the number of particles being captured as a function of Ω/2π at a fixed Vac. Figure 5a displays a plot for the variation of the total fluorescence intensity, a quantity directly correlated with the number of the MALDI-produced particles, with the ion trapping frequency. The intensity is integrated over 30 s, with the data taken after averaging 5-10 measurements to obtain a reliable value. As shown, the fluorescence is in general more intense at lower trapping frequencies, with the intensity peaking somewhere between Ω/2π ) 5 and 7 kHz. The photon count is very low at Ω/2π ) 10 kHz, and essentially no particles are being trapped at Ω/2π > 15 kHz. We ignore the data taken in the lower frequency region (Ω/2π < 4 kHz), because contribution of the signals from the nanosphere aggregates would become significant therein (cf. Figure 5b). We deduce the charge state information on the premise that the kinetic energy distribution of the laser-desorbed particles is independent of their charge numbers. This premise has, indeed, been verified by Gluckmann and Karas21 using a TOF mass spectrometer for different classes of analyte, including both organic and biological molecules. Applying the Mathieu equation to our case, we obtain the potential well depths of an ideal ion trap operating at various trapping frequencies for particles having different charge numbers (cf. Figure 5b). Predicted by this calculation, the presently investigated particles should be more favorably confined in the trap at lower frequencies when their charge numbers are 9, trapping would be more efficient at 10 kHz than at lower frequencies. Since the trapping efficiency is directly correlated with the strength of the forces that the particles experience, one may estimate the charge states of the trapped particles from a comparison of the measurements (Figure 5a) with the calculations (Figure 5b). The comparison leads us to the conclusion that the number of charges on the particles is 100 for the MALDI-generated microparticles. For these particles, one may determine directly the charge state distribution from a measurement of their mass-to-charge ratios using the ion trap as a mass spectrometer. The feasibility of this approach has been demonstrated in our previous work,5 in which we detected scattered laser light from submicrometer-sized particles ejected out of the ion trap operating in an axial mass-selective instability mode. Figure 6 displays the mass spectrum of the 1-µm fluorescent polystyrene microspheres captured by using the ion trap under the conditions, p ) 50 mTorr, Ω/2π ) 200 Hz, and Vac ) 420 V. The spectrum was taken, after pumping out the buffer gas to p ≈ 1 mTorr, by ramping the trap ac voltage from Vac ) 420 to 1700 V and, at the same time, collecting the scattered laser light bursts from the ejected particles as they pass across the probe laser beam. The individual peaks observed in the spectrum (cf. Figure 6) are essentially derived from one single particle.5 Assigning an average mass of 7.4 ( 0.6 × 10-16 kg to each particle (d ) 1.1 ( 0.027 µm and F0 ) 1.05 g/cm3), the number of charges on these laserdesorbed microspheres ranges from 80 to 400 charges. The charge numbers, notably, are ∼1 order of magnitude higher than that obtained for the 27-nm spheres and are much larger than any reported in the literature for the MALDI-generated ions. CONCLUSION AND FURTHER STUDIES We have demonstrated that very high mass (from 106 to 1012 Da) polystyrene particles can be generated by the matrix-assisted laser desorption/ionization technique for spectral mass analysis. With use of 3-hydroxypicolinic acid as the matrix for desorption/ ionization of the fluorescent polystyrene particles 27 nm and 1.1 µm in diameter, we determined that the number of charges on these particles is 1-10 and 80-400, respectively. Such MALDIAnalytical Chemistry, Vol. 74, No. 17, September 1, 2002

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generated particles can be effectively captured by using a quadrupole ion trap operating in the audio frequency region with the assistance of He buffer gas at a pressure of ∼50 mTorr. This work has extended our current investigation capabilities from small molecular ions to nanometer-sized charged particles using laser-induced fluorescence as a detection method. One possible application of this method, in conjunction with MALDI or ESI as the ion source, is to bring fluorescent (organic, inorganic, or biological) nanoparticles with a size of 106, a region remaining underexplored in the field of mass spectrometry. ACKNOWLEDGMENT We acknowledge Dr. R. H. Hsu for the use of the 488-nm notch filter and many helpful discussions. We also thank the Academia Sinica and the National Science Council (Grant No. NSC 91-3112P-001-023-Y) of Taiwan for financial support of this work. Received for review April 1, 2002. Accepted June 11, 2002. AC020205L