Anal. Chem. 1996, 68, 3595-3601
Matrix-Assisted Laser Desorption/Ionization of Size- and Composition-Selected Aerosol Particles Bashir A. Mansoori and Murray V. Johnston*
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716 Anthony S. Wexler
Department of Mechanical Engineering, University of Delaware, Newark, Delaware 19716
Matrix-assisted laser desorption/ionization (MALDI) was performed on individual, size-selected aerosol particles in the 2-8 µm diameter range. Monodisperse aerosol droplets containing matrix, analyte, and solvent were generated and entrained in a dry stream of air. The dried particles were drawn from atmospheric pressure directly into the source region of a reflecting field time-of-flight mass spectrometer. Individual particles were then detected by light scattering and analyzed on-the-fly by MALDI. The particle size and composition were systematically varied, and both liquid and solid matrices were studied. As the analyte-to-matrix mole ratio increased, the analyte signal intensity first increased and then leveled off. Quenching effects were observed when two different peptides were present in the same particle. These dependences were interpreted on the basis of analyte surface activity and adsorption isotherms. With a liquid matrix, the analyte is thought to partition between the particle surface and bulk. The signal intensity increases with analyte surface coverage until a monolayer is formed. With a solid matrix, the analyte is thought to adsorb on the surface of the original droplet containing matrix, analyte, and solvent. When the solvent evaporates, the analyte deposits on the dry particle surface. Again, the signal intensity increases with analyte surface coverage until a monolayer is formed. With either a solid or liquid matrix, signal quenching is observed when multiple analytes compete for surface adsorption. Matrix-assisted laser desorption/ionization (MALDI) is widely used for molecular weight determination of natural and synthetic macromolecules.1-3 In most cases, molecular ions are produced with little or no fragmentation. Quantitation of the analyte based on the MALDI signal intensity, however, is impeded by poor reproducibility. The signal intensity is strongly affected by sample morphology4 and laser irradiance5 as well as analyte concentration. We discuss here a method to control many of the variables (1) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299. (2) Hillenkamp, F.; Karas, M. Anal. Chem. 1991, 63, 1193A. (3) Vertes, A.; Gijbels, R. In Laser Ionization Mass Analysis; Vertes, A., Gijbels, R., Adams, F., Eds.; John Wiley & Sons: New York, 1993; Chapter 3a. (4) Doktycz, S. J.; Savickas, P. J.; Krueger, D. A. Rapid Commun. Mass Spectrom. 1991, 5, 145. (5) Ens, W.; Mao, Y.; Mayer, F.; Standing, K. G. Rapid Commun. Mass Spectrom. 1991, 5, 117. S0003-2700(96)00338-1 CCC: $12.00
© 1996 American Chemical Society
associated with sample morphology so that the analyte dependence of signal intensity can be quantitatively assessed. Sample preparation for MALDI normally involves applying a solution containing matrix and analyte to a probe tip and evaporating the solvent. The analyte is incorporated into matrix crystals as they grow.6,7 After drying, the sample is transferred to the mass spectrometer and irradiated with a pulsed laser beam, and then both the matrix and the analyte are ejected into the vapor phase. The analyte signal intensity is dependent on the size, shape, and distribution of matrix crystals that are formed and the degree of analyte incorporation. Since crystals are inhomogeneously distributed across the probe tip, the signal intensity varies greatly with location. Recently, techniques have been developed to produce homogeneous polycrystalline films which are less subject to signal intensity variations.8,9 The method chosen to prepare matrix crystals may also affect how the analyte is incorporated.10 Several researchers have explored the quantitative relationship between signal intensity and concentration.9,11-15 In most cases, sample inhomogeneities on the probe tip preclude a direct correlation between the absolute signal intensity and analyte concentration. Therefore, the signal intensity is usually measured relative to that of an internal standard. This approach works best if the internal standard possesses chemical properties similar to those of the analyte, presumably because both are incorporated into the matrix crystal and subsequently ionized in a similar fashion. Other factors to be considered are the laser irradiance and total number of shots incident on a given location.8 The laser irradiance is normally kept above threshold to minimize the effect of pulse-to-pulse variations. At threshold, the signal intensity varies with the fourth to sixth power of the laser irradiance, which amplifies pulse-to-pulse variations. The total number of shots must (6) Strupat, K.; Karas, M.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1991, 141, 23. (7) Beavis, R. C.; Bridson, J. N. J. Phys. D 1993, 26, 442. (8) Vorm, O.; Roepstorff, P.; Mann, M. Anal. Chem. 1994, 66, 3281. (9) Gusev, A. I.; Willkinson, W. R.; Poctor, A.; Hercules, D. M. Anal. Chem. 1995, 67, 1034. (10) Cohen, S. L.; Chait, B. T. Anal. Chem. 1996, 68, 31. (11) Gusev, A. I.; Wilkinson, W. R.; Proctor, A.; Hercules, D. M. Appl. Spectrosc. 1993, 47, 1091. (12) Duncan, M. W.; Matanovic, G.; Cerpa-Poljak, A. Rapid Commun. Mass Spectrom. 1993, 7, 1090. (13) Harvey, D. J. Rapid Commun. Mass Spectrom. 1993, 7, 614. (14) Tang, K.; Allman, S. L.; Jones. R. B.; Chen, C. H. Anal. Chem. 1993, 65, 2164. (15) Nelson, R. W.; McLean, M. A.; Hutchens, T. W. Anal. Chem. 1994, 66, 1408.
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be controlled, since the signal intensity generally decreases with increasing exposure to the laser radiation. It is not known whether this dependence reflects an inhomogeneous distribution of analyte in the matrix crystals or a laser-induced change in the crystal morphology. A direct correlation between signal intensity and concentration is not universally observed. However, a linear relationship over 1-2 orders of magnitude in concentration may be obtained for specific combinations of matrix, analyte, and internal standard.9,11,14,15 With so many interrelated factors influencing the MALDI signal intensity, it is difficult to ascribe changes in the signal intensity to specific phenomena. This situation may be simplified by studying aerosol particles. One version of an “aerosol-MALDI” experiment has been studied as a means of coupling MALDI to liquid chromatography.16-18 The aerosol generator used in these experiments, however, was chosen primarily for its applicability to liquid chromatography and produced a polydisperse aerosol having ill-defined particle size and composition. In the work described here, we extend the aerosol-MALDI approach to monodisperse aerosols. The methodology is analogous to our previous work involving trace detection of inorganic species in liquid droplets.19 A commercial vibrating-orifice aerosol generator is used to produce well-defined particles whose size and composition are systematically varied. The aerosol enters the mass spectrometer through a differentially pumped inlet. Individual particles are detected by light scattering from a continuous laser beam, and the scatter pulse triggers an excimer laser, which ablates the particle in-flight.20-22 Thus, each mass spectrum arises from a single particle ablated by a single laser pulse. If desired, mass spectra can be averaged over many particles. This method is used to study particle size and composition dependences of the MALDI signal intensity. EXPERIMENTAL SECTION Particle Generation. Particles containing matrix and analyte were produced by drying aerosol droplets composed of matrix, analyte, and solvent. Two matrices were studied: a liquid, 3-nitrobenzyl alcohol (NBA), dissolved in a 45:55 (v/v) mixture of water and methanol at a concentration 0.1% by volume, and a solid, 2,5-dihydroxybenzoic acid (DHB), dissolved in a 50:50 (v/ v) mixture of water and methanol at a concentration 0.06% by weight. Monodisperse aerosol droplets were produced by flowing the respective solutions through a vibrating-orifice aerosol generator (Model 3450; TSI, St. Paul, MN). These droplets were mixed with dry air to remove the solvent. Particle size measurements showed that drying was complete within 2-3 s. This time scale is comparable to that of the “fast evaporation” methods used to prepare conventional MALDI samples.8,10 The dry particles then passed through a flow tube connecting the aerosol generator to the mass spectrometer inlet. The residence time of dry particles in the flow tube was ∼10 s. Thus, the total transit time between droplet generation and particle analysis was ∼12 s. The size and (16) Murray, K. K.; Russell, D. H. Anal. Chem. 1993, 65, 2534. (17) Murray, K. K.; Russell, D. H. J. Am. Soc. Mass Spectrom. 1994, 5, 1. (18) Beeson, M. D.; Murray, K. K.; Russell, D. H. Anal. Chem. 1995, 67, 1981. (19) Mansoori, B. A.; Johnston, M. V.; Wexler, A. S. Anal. Chem. 1994, 66, 3681. (20) Johnston, M. V.; Wexler, A. S. Anal. Chem. 1995, 67, 721A. (21) Carson, P. G.; Neubauer, K. R.; Johnston, M. V.; Wexler, A. S. J. Aerosol Sci. 1995, 26, 535. (22) McKeown, P. J.; Johnston, M. V.; Murphy, D. M. Anal. Chem. 1991, 63, 2069.
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composition of the dry particles were determined by the matrix and analyte concentrations in the original solution. Matrix-toanalyte mole ratios were typically in the 102-104 range. The size distribution of the dry particles was independently monitored with an aerodynamic particle sizer (Model 3310; TSI, St. Paul, MN). The experimentally measured distribution was typically log-normal with a standard deviation of 0.3 µm. The actual standard deviation was probably smaller since this value is close to the limit of resolution of the particle sizer. Particle diameters given in the text represent the mean diameters measured with the particle sizer during an experiment. Mass Spectrometer. The aerosol mass spectrometer used in this work has already been described in detail elsewhere.22 Aerosols were sampled into the mass spectrometer through a twostage momentum separator. The resulting particle beam passed through the center of the source region, where it intercepted a continuous helium/cadmium laser beam. The transit time between the inlet and source region was ∼1 ms. The scattered radiation from each particle triggered an excimer laser (Model PSX-100; MPB Technologies, Droval, QC, Canada) which ablated the particle in-flight. Ions produced by laser desorption/ionization were characterized with a reflecting field time-of-flight mass analyzer. Thus, each mass spectrum corresponded to a single particle ablated by a single laser pulse. MALDI spectra were obtained at the 248 nm operating wavelength of the excimer laser. Although the ionization yield is generally lower at this wavelength than in the near-ultraviolet,23 reasonable spectra can be obtained.4 The mean laser pulse energy was periodically measured with an energy meter (Model J25LP2; Molectron, Portland, OR). Pulse energies between 0.2 and 2 mJ were studied. Since the focal spot of the excimer beam in the source region was 250 µm × 200 µm, a laser pulse energy of 1 mJ corresponded to a fluence of ∼2 J/cm2. The irradiance of the excimer laser was essentially flat across the beam, so particles located at different positions in the beam nominally experienced the same irradiance. However, it is likely that the beam contained “hot” spots whose location and intensity varied from shot to shot. Chemicals. 3-Nitrobenzyl alcohol (98%) and 2,5-dihydroxybenzoic acid (99%) were obtained from Aldrich. Leucine enkephalin (acetate salt) and gramicidin S (hydrochloride) were obtained from Sigma. All were used as received. RESULTS AND DISCUSSION Conventional MALDI samples have been limited by a heterogeneous distribution of particle sizes and shapes and an ill-defined matrix-to-analyte mole ratio at specific locations across the sample probe. In contrast, the aerosol particles studied here had uniform size, shape, and composition. The particle size and composition were determined by the amounts of matrix and analyte in the original droplets, while solvent surface tension maintained a spherical morphology during drying. Particles composed of a liquid matrix remained spherical after solvent evaporation, while those composed of a solid matrix were polycrystalline. Both liquid (NBA) and solid (DHB) matrices were considered in this study. With the liquid matrix (NBA), the 10 s time period in the transfer line between the aerosol generator and mass spectrometer was sufficient for the matrix-analyte solution to come to equilibrium. Thus, the distribution of analyte on the particle surface and in the bulk solution reflected its surface (23) Ehring, H.; Karas, M.; Hillenkamp, F. Org. Mass Spectrom. 1992, 27, 472.
Figure 1. MALDI spectrum of LE in NBA. Average of 25 single particle spectra. Particle diameter, 6.3 µm; LE/NBA mole ratio, 4.3 × 10-2; laser pulse energy, 1.1 mJ.
activity. With the solid matrix (DHB), diffusion did not occur in the transfer line. Instead, the surface versus bulk distribution of analyte reflected the dynamics of crystal formation during the 2-3 s during the drying step. With either matrix, the total amount of analyte in a given particle was well-defined since individual droplets/particles remained isolated in the gas stream. The aerosol samples considered in this study also had a few disadvantges for MALDI. First, it was not possible to wash the sample to remove salts and other unwanted species. Thus, alkali salt contamination was significant. Second, the mass resolutions of the aerosol-MALDI spectra were somewhat lower than those obtained by conventional MALDI. In aerosol MALDI, ionization occurs in the center of the source region, and a relatively low electric field gradient must be employed to maintain a reasonable total acceleration voltage. The lower gradient is more sensitive to the initial distribution of ion kinetic energies produced by the desorption event than the high gradients normally used in conventional MALDI. Liquid Matrix. Figure 1 shows a MALDI spectrum of leucine enkephalin (LE) in NBA. The particle diameter is 6.3 µm, and the mole ratio of LE to NBA is 4.3 × 10-3. In the LE molecular ion region, ions corresponding to (M + H)+, (M + Na)+, (M + K)+, and (M - H + 2Na)+ are observed. The high relative intensities of cationized species in combination with strong signals at m/z 23 (Na+) and 39 (K+) show that salt contamination in the chemicals, or more likely in the aerosol generator, is high. In the low-mass region, matrix molecular ions (M•+, (M + H)+, (M + Na)+, and (M + K)+) and fragmentation products of matrix monomer and clusters are observed. Detailed analysis of this region gives no direct evidence of solvent cluster formation. This result contrasts previous aerosol-MALDI work18-20 and suggests that solvent evaporation in the current system is complete. Complete evaporation is also supported by particle size measurements, which match the size predicted on the basis of the operating conditions of the aerosol generator. The mass resolution (R ) m/∆m, where ∆m is the full width at half-maximum) in Figure 1 is 260 in the LE molecular ion region. This value is somewhat lower than that in conventional MALDI and reflects the limited electric field gradient of the aerosol mass spectrometer. Figure 2 shows a plot of (M + Na)+ peak area for LE as a function of laser pulse energy. In each case, the particle diameter is 4.7 µm, and the LE to NBA mole ratio is maintained at a relatively high level of 10-2. Each data point is the average of 25 single particle spectra, and the error bars represent the standard
Figure 2. (M + Na)+ peak area of LE vs laser pulse energy. Each point is the average of 25 single particle spectra. The error bars show the standard error of the mean. Particle diameter, 4.7 µm; LE/NBA mole ratio, 10-2.
error of the mean. Between 0.15 and 0.20 mJ, the signal intensity rises very quickly: no analyte ions are observed at or below a 0.15 mJ pulse energy. This corresponds to an apparent threshold for ion production of 0.4 J/cm2, which is significantly higher than that in conventional MALDI5,24 but comparable to a previous aerosol-MALDI experiment.19 Above threshold, Figure 2 shows that the signal intensity is relatively invariant with laser pulse energy. In conventional MALDI, the threshold for ionization increases as the laser spot size decreases, and the signal intensity above threshold becomes less dependent on laser fluence.24 For example, the threshold laser fluence is over an order of magnitude smaller with a 200 µm beam diameter than with a 10 µm beam diameter. Also, the signal intensity with a 200 µm beam increases as the fifth power of the laser fluence just above threshold, while the signal intensity with a 10 µm beam increases only linearly with laser fluence. In Figure 2, the effective spot size is given by the particle diameter, 4.7 µm. The threshold fluence is somewhat higher than the 10 µm beam diameter data in ref 24, and the signal intensity barely changes with increasing fluence. Thus, the dependence in Figure 2 is consistent with the trends in ref 24. Figure 2 is also consistent with a previous aerosol-MALDI study that showed a relatively weak dependence of the signal intensity on laser fluence.19 Figure 3 shows plots of the absolute (M + Na)+ peak area for LE versus the LE-to-NBA mole ratio. Data from two different particle diameters are shown, 2.6 and 4.7 µm. Figure 3a shows the entire range, while Figure 3b shows an expanded view of the low mole ratio region. Initially, the signal intensity increases linearly with mole ratio (Figure 3b), but a negative deviation is observed at higher mole ratios, and the signal intensity eventually reaches a plateau (Figure 3a). This dependence is commonly observed in fast atom bombardment and liquid secondary ion mass spectrometry experiments25-28 and has been attributed to the surface activity of the analyte. Mole ratios above 0.025 were not studied owing to analyte solubility limitations. (24) Dreisewerd, K.; Schurenberg, M.; Karas, M.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1995, 141, 127. (25) Ligon, W. V.; Dorn. S. B. Int. J. Mass Spectrom. Ion Processes 1984, 57, 75. (26) Allmaier, G. M. Rapid. Commun. Mass Spectrom. 1988, 2, 76. (27) Heine, C. E.; Holland, J. F.; Watson, J. T. Anal. Chem. 1989, 61, 2647. (28) Huang, Z.; Cole, R. B. Int. J. Mass Spectrom. Ion Processes 1993, 127, 169.
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Figure 4. (M + Na)+ peak area of LE vs particle diameter. Each point is the average of 25 single particle spectra. The error bars show the standard error of the mean. LE/NBA mole ratio, 4.3 × 10-3; laser pulse energy, 0.32 mJ.
Figure 3. (M + Na)+ peak area of LE vs LE/NBA mole ratio. Each point is the average of 25 single particle spectra. The error bars show the standard error of the mean. Particle diameters are 2.6 (b) and 4.7 µm (9), respectively. The plots in part a are fit according to eq 2. The plots in part b are expanded portions of part a and are fit by linear regression. Laser pulse energy, 0.32 mJ.
The plots in Figure 3 can be interpreted on the basis of the adsorption isotherm equation:29
θ)
bc 1 + bc
(1)
where θ is the fraction of the particle surface covered with analyte, c is the bulk concentration of analyte in the particle, and b ) Kγ/ as, where K is the equilibrium constant for surface adsorption, γ is the activity coefficient of the solute in the bulk solution, and as is the activity of the solvent in the bulk solution. If we assume that the MALDI signal intensity arises primarily from LE molecules adsorbed on the particle surface and that the signal intensity is directly proportional to surface coverage, then eq 1 can be rewritten as
b′m I ) Imax 1 + b′m
(2)
where I is the analyte signal intensity in the MALDI spectrum, Imax is the analyte signal intensity with unit surface coverage, m (29) Adamson, A. W. Physical Chemistry of Surfaces, 3rd ed.; J. Wiley and Sons: New York, 1976; Chapter IX.
3598 Analytical Chemistry, Vol. 68, No. 20, October 15, 1996
is the mole ratio of LE to NBA, and b′ ) Kγ/γs, where γs is the solvent activity coefficient. The assumption that the MALDI signal intensity arises primarily from surface-adsorbed molecules is reasonable since previous laser desorption/ionization studies have shown that organic molecular ions are produced only from the outer 10 nm of a sample.30 For the range of analyte mole ratios and surface coverages considered here, most (>70%) of the analyte molecules in the outer 10 nm region of the particle reside on the surface. The solid lines in Figure 3a represent the best fit to the data using eq 2. When m is small, the b′m term in the denominator of eq 2 becomes small, and I becomes linearly dependent on m. Hence, a linear plot is observed in Figure 3b. The linear range extends over 1 order of magnitude in mole ratio, which is comparable to conventional MALDI experiments.14,15 It should be noted that the signal intensity dependence in Figure 3 is not caused by detector saturation. Inorganic ions dissolved in glycerol droplets show a linear relationship between signal intensity and concentration across a similar range.19 Figure 3 shows that the LE signal intensity is greater from 4.7 µm particles than that from 2.6 µm particles. This observation may reflect the greater surface area of the former and is reflected by a corresponding increase in Imax in eq 2. However, the signal intensity does not scale linearly with particle surface area. For the plots in Figure 3a, the surface area increases by a factor of 3.3, while Imax from eq 2 increases by only a factor of 2.0 as the particle diameter increases from 2.6 to 4.7 µm. Figure 4 shows a plot of the (M + Na)+ peak area for LE versus particle diameter for an LE-to-NBA mole ratio of 4.3 × 10-3. Although the signal intensity increases between 2 and 5 µm, it is constant or slightly decreasing above 5 µm. The plot in Figure 4 cannot be rationalized on the basis of a simple surface adsorption model. Instead, this plot may reflect varying ion formation and/or ion collection efficiencies with particle size, or, as discussed below, solution characteristics that are unique to small particles. In large droplets, only a small fraction of the total amount of analyte is needed to form a monolayer. Thus, the analyte activity in the bulk solution is not affected by monolayer formation. In small particles, the surface-to-volume ratio becomes much larger, and a significant fraction of the total amount of analyte in the (30) Van Vaeck, L.; Gijbels, R. Fresenius J. Anal. Chem. 1990, 337, 755.
Figure 5. MALDI spectrum of LE and GS in NBA. Average of 25 single particle spectra. Particle diameter, 3.3 µm; LE/NBA mole ratio, 1.2 × 10-3; GS/NBA mole ratio, 2.3 × 10-3; laser pulse energy, 1.1 mJ.
particle may be consumed during monolayer formation. For example, consider a small peptide that occupies 1 nm2/molecule on a particle surface. If the mole ratio of analyte to matrix is 5 × 10-3 and the particle diameter is 2 µm, then formation of a monolayer of analyte on the particle surface requires 1.3 × 107 molecules or 12% of the total amount of analyte present in the particle. In this case, eq 2 would not strictly apply, and the initial increase in signal intensity with concentration would be more gradual. This situation is suggested in Figure 3a. When the data are fit with eq 2, b′ is smaller for 2.6 µm particles (530) than for 4.7 µm particles (1400). Ideally, b′ should be invariant with particle size since the bulk solutions are the same. The smaller value of b′ for the smaller particle reflects a more gradual rise in the signal intensity with concentration and is consistent with analyte depletion from the bulk solution as the surface monolayer forms. Analyte depletion from the bulk solution could also inhibit micelle formation in small particles. In Figure 4, the constant or slightly decreasing signal intensity above 5 µm may reflect the formation of micelles in large particles, which reduces the analyte surface coverage. The weak dependence of the analyte signal intensity on particle size suggests that the detection sensitivity can be increased by decreasing the particle diameter. As the particle diameter decreases, a greater fraction of the analyte resides on the particle surface. In the current experiments, the smallest particle diameter we studied was 2 µm, and a measurable signal was consistently observed for a 5 × 10-4 mole ratio, which corresponds to 20 amol of analyte in a single particle. The plots in Figure 3 are obtained only in the absence of other analytes. Figure 5 shows a spectrum of NBA containing both leucine enkephalin (LE) and gramicidin S (GS). Again, salt contamination causes the (M + Na)+ peaks to be dominant in the respective molecular ion regions. The resolution of the (M + Na)+ peak for GS is 260. Figure 6 shows the effect of mole ratio on signal intensity when these two analytes are in solution. The particle diameter is held constant at 3.3 µm, while the mole ratios of LE to NBA and GS to NBA are both increased. The ratio of LE to GS remains constant at 1.7. The (M + Na)+ peak area of GS at first increases with increasing mole ratio and then levels off. As with LE in Figure 3, the GS plot in Figure 6 follows eq 2. The LE plot in Figure 5, however, is much different than Figure 3 and indicates signal suppression. The (M + Na)+ peak
Figure 6. (M + Na)+ peak area of LE (b) and GS (9), respectively, vs peptide (LE or GS)-to-NBA mole ratio. The absolute amounts of LE and GS increase, while the LE/GS mole ratio remains constant at 1.7. Each point is the average of 25 single particle spectra. The error bars show the standard error of the mean. The GS plot is fit to eq 2, the LE plot is fit to eq 4, and the LE signal intensity is multiplied by a factor of 3. Particle diameter, 3.3 µm; laser pulse energy, 0.37 mJ.
area of LE initially increases with increasing mole ratio and then decreases. No LE signal is observed above a GS/NBA mole ratio of 1.2 × 10-2. This dependence suggests a competition between LE and GS for surface adsorption. As the mole ratios increase, GS occupies a greater fraction of the total surface area and displaces adsorbed LE. As the GS coverage approaches one monolayer, the LE surface coverage and signal intensity go to zero. Preferential adsorption of GS on the particle surface is not surprising since the hydrophobicity of GS in aqueous solution, as given by the data of Bull and Breese31 or Nozaki and Tanford,32 is much greater than that of LE. Based on this simple model, the surface coverage of LE should vary according to
θLE )
(
)(
)
bGScGS bLEcLE 11 + bLEcLE 1 + bGScGS
(3)
where θ, b, and c are the same as in eq 1, and the LE and GS subscripts refer to the two peptides. The first term in eq 3 represents the increasing surface coverage with increasing cLE, while the second term represents the decreasing number of available surface sites for LE as cGS increases. As with eq 1, eq 3 can be rewritten in terms of the LE signal intensity:
(
ILE ) ILE,max
)(
)
bGS′cGS bLE′mLE 11 + bLE′mLE 1 + bGS′cGS
(4)
where I, b′, and m are the same as in eq 2. Figure 6 shows that the experimentally derived plot of ILE vs mLE qualitatively follows eq 4. Quantitative differences may reflect experimental artifacts such as activity coefficient variations or impurities. Solid Matrix. The above results were interpreted on the basis of solution chemistry in the liquid NBA matrix. A solid matrix, (31) Bull, H. B.; Breese, K. Arch. Biochem. Biophys. 1974, 161, 665. (32) Nozaki, Y.; Tanford, C. J. Biol. Chem. 1971, 246. 2211.
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2,5-dihydroxybenzoic acid (DHB), was also studied for comparison. The mass spectra of LE and GS in DHB are similar to those shown in Figures 1 and 5. Salt contamination causes cationized species to dominate in the molecular ion region. In addition, ions corresponding to (M + H)+, (M + K)+, (M + 2Na - H)+, and (M + 2K - H)+ are observed. In the low-mass region, three types of ions are observed: atomic ions (Na+, K+), matrix ions (M•+, (M + H)+, (M + Na)+, (M + K)+), and ions derived from fragmentation and/or clustering of the matrix. No peaks corresponding to water and/or methanol clusters are detected, confirming that solvent evaporation is complete. The laser pulse energy dependence of peptide (M + Na)+ ions from DHB is similar to that from NBA in Figure 2. For GS in DHB, the threshold energy is 0.15 mJ (0.3 J/cm2), and the signal intensity is relatively invariant with pulse energy above threshold. When LE or GS alone is in solution, a plot of signal intensity versus mole ratio (not shown) is remarkably similar to Figure 3. That is, the signal intensity initially increases with mole ratio and then levels off. The similarity between the mole ratio dependences in NBA and DHB is at first surprising, since the NBA results were interpreted on the basis of solution chemistry that cannot occur in the (solid) DHB matrix. Our tentative explanation of the DHB data is based on the dynamics of crystal formation during the drying step. In the original droplet containing matrix, analyte, and solvent, the droplet surface is enriched in analyte, and the surface coverage is determined by the bulk analyte concentration. As the droplet dries, matrix crystals begin to form on the interior of the droplet, and analyte molecules in the bulk solution may incorporate into these crystals. The droplet surface, however, remains enriched in analyte. As solvent evaporation proceeds to completion, these adsorbed molecules are deposited on the dry particle surface. This process is similar to that encountered in our studies of inorganic salt cocrystallization in aqueous particles.33 As an aerosol droplet containing several dissolved salts dries, surface tension causes the solid phases formed to migrate toward the center of the droplet and be surrounded by the remaining aqueous phase. The composition of the solid that is initially formed depends on the relative concentrations and solubilities of the various components in the original droplet. As the particle dries, the composition of the aqueous solution surrounding the solid phase moves toward the eutonic composition. Therefore, when drying is complete, the surface layer is also given by the eutonic composition. An important consequence of this process is that minor components in the original droplet preferentially deposit in the surface layer, while major components preferentially deposit in the particle core. As with NBA, competition among analytes for surface adsorption is indicated in the spectra of DHB. Figure 7 shows the results of an experiment where LE acts as an internal standard (LE-toDHB mole ratio remains constant), while the GS-to-DHB mole ratio is varied. The particle diameter is held constant at 3.3 µm. In Figure 7a, the absolute (M + Na)+ peak areas of LE and GS are plotted versus the GS-to-DHB mole ratio. The GS signal intensity at first increases and then levels off with increasing mole ratio. The solid line represents the best fit to eq 2. Although the LE-to-DHB mole ratio remains constant, the LE signal intensity decreases as the GS-to-DHB mole ratio increases, and the LE (33) Ge, Z.; Wexler, A. S.; Johnston, M. V. J. Colloid Interface Sci., in press.
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Figure 7. (a) (M + Na)+ peak area of LE (b) and GS (9), respectively, vs GS/DHB mole ratio. (b) GS to LE peak area ratio vs GS/DHB mole ratio. The LE/DHB mole ratio is held constant at 2 × 10-3. Each point is the average of 25 single particle spectra. The error bars show the standard error of the mean. In part a, the GS plot is fit to eq 2, the LE plot is fit to eq 5, and the LE peak area is multiplied by 2. Particle diameter, 3.3 µm; laser pulse energy, 0.31 mJ.
signal vanishes when the GS-to-DHB mole ratio exceeds 8 × 10-3. Suppression of the LE signal is interpreted as a displacement of LE from the droplet surface as the GS concentration increases. If GS displaces LE from the droplet surface before solvent evaporation, then the particle surface after drying will contain less LE, and the resulting MALDI signal will be lower. By analogy to eqs 3 and 4, the LE signal intensity should exhibit the following relationship:
(
ILE ) ILE,max 1 -
)
bGS′mGS 1 + bGS′mGS
(5)
where the term in parentheses represents the decreasing number of available surface sites for LE as the cGS increases. Figure 7a shows that the experimental data are consistent with eq 5. Figure 7b shows the ratio of the GS-to-LE signal intensity as a function of GS-to-DHB mole ratio. The overall plot is nonlinear and indicates that LE is not an effective internal standard for GS quantitation. As eqs 3-5 show, this nonlinearity is expected if the analyte and internal standard have different surface preferences.
CONCLUSION In aerosol-MALDI, the size and composition of particles containing matrix and analyte can be systematically varied. When the particle size is held constant, analyte signal saturation and/ or suppression can be observed as the composition changes. This behavior is interpreted on the basis of analyte surface activity in the dry particle or in the original droplet containing matrix, analyte, and solvent. Similar composition dependences have been found in conventional MALDI studies,12,13 but an explanation for this phenomenon was not advanced. Although analyte incorporation into the matrix crystals may be similar to aerosol-MALDI, crystal formation in conventional MALDI is more complex since the matrix and analyte are not spatially constrained. In contrast,
aerosol crystal formation assures intimate contact between the matrix and analyte on the micrometer scale. ACKNOWLEDGMENT This work was supported by grants from the National Science Foundation (Grant No. ATM-9422993) and Environmental Protection Agency (Grant No. R823980-01-0). Received for review April 10, 1996. Accepted July 16, 1996.X AC9603385 X
Abstract published in Advance ACS Abstracts, August 15, 1996.
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