Anal. Chem. 1995, 67, 1981-1 986
Aerosol Matrix-Assisted Laser Desorption Ionization: Effects of Analyte Concentration and Matrix-to-Analyte Ratio Michelle D. Beeson, Kennit K. Murray,t and David H. Russell* Department of Chemistty, Texas A& M Univetsity, College Station, Texas 77843-3255
We have recently developed an aerosol-liquid introduction interface for matrix-assistedlaser desorption ionization (MALDI) mass spectrometry. In this study, we examine the effect of matrix-to-analyteratio and analyte concentration on analyte ion yield. These studies were performed using bradykinin, gramicidin S, bovine insulin, and myoglobin as analytes and a-cyano-4-hydroxycinnamic acid and 4-nitroanilineas matrices. The optimum matrix-to-analyte molar ratio for aerosol MALDI was determined to be 10-100:1, which is lower than that typically used for conventional surface MALDI (10010 0OO:l). The ion yield was found to be a nonlinear function of analyte concentration. Possible explanations for these observations are discussed. The ability to introduce solutions directly into a mass spectrometer affords many opportunities for the study of solution phase species. Applications of mass spectrometric liquid introduction include coupling high-performanceliquid chromatography to mass spectrometry (LC/MS) investigating biomolecule associations in s o l u t i ~ n , ~and - ~ probing protein behavior in s~lution.~ The primary difticulty in developing liquid interfaces for mass spectrometry is the incompatibility of solvent with the vacuum requirements of the mass spectrometer. This is particularly true in the case of LC/MS, where typical column effluent flows are in the range of 1-2 mWmin. Various techniques have been developed to decrease the amount of chromatographic solvent introduced into the mass spectrometer by either splitting off some of the eluent before it enters the spectrometefl or removing the eluent before i~nization.~ A continuous liquid beam method has also been reported.8 Two common methods of liquid sample introduction use aerosols formed either in a corona discharge (e1ectro~pray)~J~ or by pneumatic or ultrasonic nebulization followed by chemical or ' Present address: Department of Chemism, Emory University, Atlanta, GA 30322. (1) Arpino, P. J.; Guiochon, G. Anal. Chem. 1979,51, 682A-701A (2) Ganem, B.; Li,Y. T.; Henion, J. D. J. Am. Chem. Soc. 1991,113, 62946296. (3)Ganem, B.; Henion, J. D. Chemtracts 1993,6,1-22 and references therein. (4) Smith, R. D.; Light-Wahl, K. J. Bid. Mass Spectrom. 1993,22, 493-501 and references therein. (5) Miranker, A; Robinson, C. V.; Radford, S. E.; Aplin, R T.; Dobson, C. M. Science 1993,262, 896-900. (6) Emary, W. B.; Lys, I.; Cotter, R J.; Simpson, R; Hoffman, A. Anal. Chem. 1990,62, 1319-1324. (7) McFadden, W. H.; Schwarz, H. L.; Evans, S. J. Chromutogr. 1976,122, 389-396. (8) Mafune, F.; Takeda, Y.; Nagata, T.; Kondow, T. Chem. Phys. Lett. 1992, 199. 615-620. 0003-2700/95/0367-1981$9.00/0 0 1995 American Chemical Society
electron impact ionization.11J2Of these two methods, electrospray ionization @SI) is the best suited for ionization of large biomolecules. The multiply-charged ions formed in the electrospray process reduce the massto-charge ratio to less than 2000, and this enables routine mass measurement accuracy of 0.01%using quadrupole mass analyzer^.'^ Mass resolution of lo6 has been obtained for proteins with molecular masses up to 30 kDa using Fourier transform ion cyclotron resonance (FTICR) instrument^.'^ Although great progress has been made with ESI, it can be dacult to interpret ESI mass spectra of mixtures,I3and the presence of salts and buffers reduces the sensitivity and complicates quantifi~ati0n.l~ We recently developed a method for liquid sample introduction that is compatible with matrix-assisted laser desorption ionization (MALDI),1'97 MALDI is potentially useful for mass spectrometric analysis of liquid samples because the predominant ions produced correspond to the protonated m01ecule~~J~ and the abundance of 6ragment ions can be varied by judicious matrix selection.20-22 Most MALDI experiments are performed by applying a solution containing the matrix and analyte to a metal surface. The solvent is evaporated before the sample is inserted into the mass spectrometer, where the matrix and analyte are then ablated and ionized using a pulsed laser. The typical matrix used for MALDI is a low molecular weight organic compound which absorbs at the wavelength of the pulsed UV laser. For optimum analyte ion signal, the matrix is present in large molar excess over the analyte, usually by 2-4 orders of m a g n i t ~ d e . ' ~One ~ ~ ~limitation for (9) Fenn, J. B.; Mann, M.; Meng, C. IC;Wong, S. F.; Whitehouse, C. M. Mass Spectrom. Rev. 1990,9, 37-70. (10) Bruins, A P.; Covey, T. R; Henion, J. D. Anal. Chem. 1987,59, 26422646. (11) Browner, R F. Microchem. J. 1989,40, 4-29. (12) Willoughby, R C.; Browner, R F. Anal. Chem. 1984,56, 2626-2631. (13) Bieman, K. Annu. Reu. Biochem. 1992,61, 977-1010. (14) Senko, M. W.; McLafferty, F. W. Annu. Rev. Biophys. Biomol. Struct. 1994, 23, 763-785. (15) Moseley, M. A; Jorgenson, J. W.; Shabanowitz, J.; Hunt, D. F.; Tomer, K. B. J. Am. Sot. Mass. Spectrom. 1992,3, 289-300. (16) Murray, K. IC; Russell, D. H. Anal. Chem. 1993,65, 2534-2537. (17) Murray, K. IC; Russell, D. H. J Am. SOC.Mass Spectrom. 1994,5, 1-9. (18) Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T. Anal. Chem. 1991, 63, 1193A-1202A (19) Karas, M.; Hillenkamp, F. Anal. Chem. 1988,60, 2299-2301. (20) Solouki, T.; Oriedo, J. V. B.; Russell, D. H. Proceedings of the 40th ASMS Conference on Mass Spectromehy and Allied Topics, Washington, DC, May, 1992; pp 89-90. (21) Preston Schaffter, L. M. Ph.D. Thesis, Texas A&M University, College Station, TX,1994. (22) Spengler, B.; Kirsch, D.; Kaufmann, R. J Phys. Chem. 1992,96, 96789684. (23) Stmpat, K; Karas, M.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1991,111, 89-102.
Analytical Chemistry, Vol. 67, No. 13, July 1, 1995 1981
developing MALDI LC/MS is the incompatibility of the mass spectrometer with high flow rate liquid sample introduction. To overcome this limitation, Li and co-workers developed an interface for MALDI LC/MS similar to flow FAEL2* In our laboratory we developed a pneumatic nebulizer to form aerosol particles that are sprayed into the vacuum. The aerosol particles are ablated by irradiation from the 355 nm output of a Nd:YAG laser, and ionization of the molecules that constitute the aerosol is enhanced by the presence of matrix and s01vent.I~ For LC/aerosol MALDIMS, the matrix is mixed postcolumn with the LC effluent using a mixing tee.25 The utility of aerosol MALDI for LC/MS and for investigating the association behavior of proteins in solution25has been demonstrated. Our current work on aerosol MALDI is focused on developing a better understanding of the experimental parameters that effect the ionization process. The objective of this work is to determine the parameters that most strongly affect ion yield and limit our ability to make quantitative measurements under a range of chromatographic conditions, including solvents systems and the type and presence of buffers. The experimental parameters of laser intensity, sample preparation, matrix, and molar ratio of the matrix and analyte influence the ion yield in the aerosol MALDI experimentz6 The work described in this paper focuses on two of these parameters: (1) analyte concentration and (2) matrixto-analyte ratio. In addition, we address the differences observed in optimum matrix-to-analyte ratio between aerosol MALDI and surface MALDI. EXPERIMENTAL SECTION The aerosol MALDI apparatus has been described in detail.17 A solution of analyte and matrix is delivered by a syringe pump to a pneumatic nebulizer where nitrogen gas sprays the aerosol into a vacuum chamber. The chamber is evacuated by a 300 L/s roots blower and rotary piston backing pump. The aerosol beam is skimmed as it enters a 25 cm long, 4 mm i.d. drying tube heated to 500 "C. The dried aerosol particles enter the ion formation region and are irradiated by the pulsed 355 nm output of a frequency tripled Nd:YAG. The laser beam is focused using a cylindrical lens to 7 mm x 0.2 mm, with the long axis parallel to the aerosol beam. For the experiments described here, the laser energy was 13 mJ/pulse (160 MW/cm2). Ions formed by the laser are extracted by a repeller plate held at 10 kV with the extraction grid at ground. Mass separation is achieved in a differentially pumped 1.1 m flight tube, and ions are detected with a microchannel plate particle multiplier. The ion formation chamber is evacuated by a 2400 L/s, liquid nitrogen-trapped 6 in. diffusion pump, and the fight tube is evacuated by a 1200W s ,4 in. diffusion pump, Mass spectra are acquired and averaged with a digital oscilloscope and transferred to a microcomputer for analysis. All mass spectra presented here were an average of 10o0 laser shots, containing 40000 data points at 5 ns/data point. The spectra were averaged and smoothed using procedures described p r e v i o ~ s l y . ~The ~ ~ *integrated ~ peak area was determined by summing the ion signal in the region of the protonated molecule and subtracting an estimated baseline signal. Because the peaks (24) Li, L.; Wang, A. P. L.; Coulson, L. D.Anal. Chem. 1993,65, 493-495. (25) Murray, IC IC; Lewis, T. M.; Beeson, M. D.; Russell, D. H. Anal. Chem. 1994,66,1601-1609. (26) Murray, K IC;Russell, D. H. In LaserAblation: Mechanisms andApplications It Miller, J. C., Geohegan, D. B.. Eds.; American Institute of Physics: New York. 1994; pp 459-464.
1982 Analytical Chemistry, Vol. 67, No. 13, July 1, 1995
resulting from adducts with the analyte molecule, for example [M Nalt, were not completely resolved in the bovine insulin and myoglobin spectra, adduct signals are included in the peak area integration for these analytes. For consistency, adduct signals are also included in integrations of spectra in which the peaks are resolved. The integrated peak areas are taken to be indicative of the analyte ion [M HI+ yield. Mass calibration was performed as described previ0us1y.l~ Briefly, this method uses solvent clusters of the type (ROH),*H+ and (ROH),.Na+, where ROH denotes the alcohol solvent used and n = 1-6, to calibrate the mass spectra to approximately m/z 200. The calibration is then extended to high m/z ratios using reference compounds such as bradykinin or gramicidin S. For example, extrapolation of the low mass calibration data to include peptide standards typically results in mass measurement accuracy of &2 m/z units up to approximately m/z 1500. Using reference compounds, extrapolation of the mass calibration to m/z values of 5000-10 000 typically yields mass measurement accuracy of h20 m/z units. The mass measurement accuracy can be as good as f2 m/z units if internal standards are used. The analyte and matrix solutions were prepared in a methanol solvent containing 6% (v/v) trifluoroacetic acid. For the studies that examined the effects of matrix-to-analyte molar ratios, the matrix concentrations were 6 mg/mL, and the analyte concentrations were varied. The limited solubility of bovine insulin and myoglobin prevented studies of these analytes at matrix-to-analyte molar ratios lower than 1OO:l. The bradykinin acetate salt, bovine insulin, horse heart myoglobin (95-100%, Sigma Chemical Co., St. Louis, MO), and gramicidin S hydrochloride from Bacillus brevis (Fluka Chemika-BioChemika, Ronkonkoma, NY) were used without further pwiiication. The solvents used were trinuoroacetic acid (Sigma) and anhydrous 99.8% spectrophotometric grade methanol (Mallinckrodt, Paris, KY). The compounds a-cyano-4 hydroxycinnamic acid (97%)and 4nitroaniline (99+%, both from Aldrich Chemical Co., Milwaukee, WI) were used as matrices without further purification.
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RESULTS The studies described in this paper focus on two parameters that influence aerosol MALDI ion signal: analyte concentration and matrix-to-analyte molar ratio. The analytes were chosen to provide a range of molecular masses and analyte characteristics. Bradykinin (1060.2 Da) is a linear nonapeptide which contains basic residues on both the N- and Gterminus; gramicidin S (1141.5 Da) is a cyclic decapeptide; bovine insulin (5733.6 Da) is a globular protein containing intramolecular disulfide bonds; and myoglobin (16 890 Da), also a globular protein, contains a heme group with an iron center. a-Cyano4hydroxycinnamicacid and 4nitroaniline were chosen as matrices because in previous studies they provided the largest signals of several matrices studied." Figure 1contains the aerosol MALDI mass spectra of the four analytes. The mass spectrum of bradykinin shows characteristics typical of an aerosol MALDI mass spectrum. The protonated molecule, denoted [M + HI+, is observed at approximately m/z 1050. The sodium adduct ion, [M + Nal+, is also present, and it is only partially resolved from the [M HI+ ion. In spectra where the [M HI+ and [M Nalt ion signals are resolved, the largest signal is due to the protonated molecule.25 We assume that the sodium contribution is constant for all matrix and analyte combinations. The validity of this assumption is supported by
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Figure 1. Aerosol MALDI mass spectra of the analytes used. (a) A 1.5 mM solution of bradykinin with 4-nitroaniline at a ratio of 30:l.(b) A 4.4 mM solution of gramicidin S with a-cyano-4-hydroxycinnamic acid at a 5:l matrix-to-analyte ratio. (c) A 0.3 mM solution of bovine insulin with 4-nitroaniline at a ratio of 1OO:l. (d) A 0.1 mM solution of myoglobin with a-cyano-4-hydroxycinnamic acid at a ratio of 9O:l.
atomic absorption data which indicate that sodium impurities come primarily from the methanol solvent. Adducts of matrix fragments with bradykinin are also resolved. Protonated clusters of bradykinin are also observed, denoted [2M HIt and [3M HI+ for clusters of two and three bradykinin molecules, respectively. The intense peaks below m/z 300 are always observed in the aerosol MALDI mass spectra and result from solvent clusters of the form (ROH),*H+ and (ROH),-Na+,l7where ROH denotes the alcohol solvent used. Figure lb-d contains mass spectra of gramicidin S, bovine insulin, and myoglobin, respectively. These spectra are similar to that noted in Figure la. The sodium adduct, [M Na]+, is partially resolved for gramicidin S but is unresolved for bovine insulin and myoglobin. The presence of [M Nalt in the mass spectrum is indicated by a shift of the analyte peak centroid to m/z values higher than the molecular weight of the singly protonated analyte. The analyte peak in the mass spectra is labeled [M HI+ because this is the largest peak.25 Doubly protonated analytes are also observed for bovine insulin and myoglobin. Although the myoglobin and bovine insulin signals appear smaller than the gramicidin S and bradykinin signals, the integrated peak areas are similar. For example, the peak height of the myoglobin [M HI+ is less than 4% of the bradykinin [M HI+ peak height, whereas the myoglobin peak area is about 50%that of bradykinin. Also note that if the peaks in the bovine insulin spectrum labeled [M + HI+, [2M HI+, and [3M + HI' are integrated together, the signal intensity increases by a factor of 2.
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Figure 2. Log/log plots of the [M + H]+ peak area as a function of analyte concentration at a matrix-to-analyte ratio of 1OO:i for (a) 4-nitroaniline and (b) a-cyano-4-hydroxycinnamic acid matrices. corresponds to bovine insulin, to myoglobin, A to gramicidin S, and 0 to bradykinin. The error bars represent 20% variation.
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Table 1. Values of n from a Least-Squares Fit to S = A@, Where S is the [M + H]+Signal from Figure 2, c is the Analyte Concentration, and A is a Proportionality Constant
analyte
4-nitroaniline
matrix a-cyano-4hydroxycinnamic acid
bradykinin gramicidin S bovine insulin myoglobin
1.84 2.14 2.78 1.29
1.32 1.61 1.42 1.32
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Figure 2 contains log-log plots of the integrated peak area for each analyte as a function of analyte concentration. The error bars represent the instability of the aerosol generator over time, variations in the laser pulse energy, and the error in estimating the baseline in the area determinations. The error was estimated by repetitive area determinations to be 20%. The lines through the data points are the results of a least-squares fit to the function S = Acn,where S is the integrated peak area, A is a scaling factor, c is the analyte concentration, and n is the power to which the concentration is raised. The parameters A and n were varied in the fit. The n values from the fit are shown in Table 1 and are discussed below. Figure 2a contains a plot of the integrated [M HI+ signal as a function of the analyte concentration using the matrix Cnitroaniline. The signals for gramicidin S and bradykinin are similar in relative abundance throughout the concentration
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Analytical Chemistty, Vol. 67, No. 13, July 1, 1995
1983
range studied. The lines fit to the data points for bovine insulin have a slope similar to those for gramicidin S and bradykinin, although the bovine insulin signal is lower. At low concentrations, the [M HI+ signals for myoglobin indicate greater relative abundance than the [M HI+ signals for the other analytes. Figure 2b contains a plot of the [M HI+ peak area as a function of the analyte concentration using the matrix a-cyano-4-hydroxycinnamic acid. In this plot, the slopes of the lines are similar, and the [M HI- signals show less variance than those in Figure 2a. In both plots, the relative ion yield is myoglobin > gramicidin S x bradykinin > bovine insulin. Comparison of the plots in Figure 2, parts a and b, reveals that the signals are larger with the a-cyano-4-hydroxycinnamic acid matrix than with the 4nitroaniline matrix. The fact that a-cyano-4-hydroxycinnamicacid produces greater ion signal than 4nitroaniline with aerosol MALDI has been observed previously with bovine insulin.17 Table 1 summarizes the results of the least-squares fit to S = Acn for the data in Figure 2. A value of n = 1 indicates that the ion signal is linearly dependent on the analyte concentration. In general, the n values are smaller for acyano4hydroxycinnamic acid than for 4nitroaniline, indicating that the ion signal is less strongly dependent on concentration with a-cyano-4-hydroxycinnamic acid than with Cnitroaniline. However, the n values for myoglobin are nearly identical for both matrices. For all analytes and with both matrices, n is greater than unity, indicating a nonlinear dependence of [M HI+ signal on analyte concentration. Figure 3 contains log-log plots of the integrated [M HIT peak area as a function of the matrix-to-analyte molar ratio. The limited solubility of myoglobin and bovine insulin prohibits measurements of [M HIf signal for these analytes at matrixto-analyte ratios lower than 1001. Figure 3a contains a plot of the integrated [M HI+ signal as a function of the matrix-toanalyte ratio for the matrix 4nitroaniline. Note that the [M+ HI+ ion signal decreases at matrix-to-analyte ratios larger than 1OO:l. This is particularly noticeable for bradykinin, which has an optimum matrix-to-analyte ratio in the range 30-1031. Figure 3b contains a plot of the integrated [M HI+ signal as a function of the matrix-to-analyte ratio for the matrix a-cyano-4-hydroxycinnamic acid. The maximum bradykinin [ M HI signal occurs at matrix-to-analyte ratios between 10 and 30:l. The bovine insulin [M HIt signal is optimum at a matrix-to-analyte ratio of approximately 1OO:1, and the myoglobin [M HI+ signal is optimum for matrix-to-analyte ratios of 1OO:l or less. It appears that for the matrices and analytes used, the optimum matrix-toanalyte ratio lies in the range of 10-1OO:l.
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DISCUSSION
The ionization of samples introduced into the mass spectrometer from a solution is a complicated process, especially if the sample is still in a condensed phase environment. For example, it is known that aerosol particles sampled in the aerosol MALDI experiment retain significant amounts of solvent because clusters of the solvent are observed in the mass spectra.17 Because the aerosol particles are composed of analyte, matrix, residual solvent, and other species in the solution (salts, buffers, trifluoroacetic acid), the formation of gas phase ionic forms of the analyte requires separation of the charged species from the matrix and solvent. The ionization process occurs concurrently with separa1984 Analytical Chernistty, Vol. 67,No. 73,July 7, 7995
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Figure 3. Loghog plots of the [M H]+ peak area as a function of matrix-to-analyte ratio for (a) 4-nitroaniline and (b) a-cyano-4hydroxycinnamic acid matrices. corresponds to bovine insulin, to myoglobin, and 0 to bradykinin. The error bars represent 20% variation.
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tion from the matrix and solvent. Thus, it would be surprising if matrix and solvent effects did not inhence the ionization process. One of the more striking differences between surface MALDI and aerosol MALDI is the optimum matrix-to-analyte ratio. In surface MALDI, the optimum matrix-to-analyte ratio is typically lO2-lO4.l9J3 In a previous study,26we found that the best ion signal for bovine insulin with Cnitroaniline matrix was obtained at a matrix-to-analyte ratio of 50:1, whereas a typical matrix-to-analyte ratio for surface IvlALDI of bovine insulin is 500-1000:1. No other matrix and analyte combinations were studied, and no matrix-toanalyte ratios below 50:l were investigated. We suggested that differences in optimum matrix-to-analyte ratio between surface MALDI and aerosol MALDI are related to the rate of crystal formation.1i,26 Fast crystallization forces the matrix and analyte into contact and favors a low matrix-to-analyte ratio. The lower matrix-to-analyte ratio for aerosol MALDI may also be due to the particle size. For example, we find that optimum matrix-to-analyte ratios for aerosol spray sample deposition of bovine insulin are 100-500:1, which is lower than the matrix-teanalyte ratio required for evaporation The idea of fast crystallization promoting MALDI ion yields is not limited to aerosol MALDI or aerosol sample deposition. Vorm and cc-workers described a sample preparation method for surface MALDI which uses rapid evaporation of solvent to prepare matrix deposits onto which the analyte-containing solution is deposited.
This preparation method results in improved ion yield and mass re~olution.~~ Nuwaysir and Wilkins also reported improved ion yield and better signal reproducibility for surface MALDI samples formed using a matrix-analyte “redeposition”method.28 In this method, acetone is used to redissolve the matrix arid analyte after deposition from a methanol or water solvent. Xiang and Beavis also reported improved ion yield from surface MALDI sample deposits formed by rapid evaporation of the s0lvent.2~ Aerosol spray methods and solvent evaporation to deposit matrix-analyte films produce crystalline particles with a range of sizes. Deposits from slowly evaporating solvents form large matrix crystals and result in segregation of matrix and analyteF3 Thus, the inclusion of analyte into the matrix crystal lattice is diminished. Under these conditions, analyte deposits may be formed at defect sites within the matrix crystal lattice. Matrixanalyte deposits formed from rapidly evaporating solvent systems result in the formation of smaller crystalline particles and more homogeneous matrix-analyte deposits. Thus, matrix-analyte deposits formed during the evaporation of volatile solvents should favor more uniform distribution of matrix and analyte. In the aerosol MALDI experiment, the solvent quickly evaporates from the droplet, possibly causing the remaining solvent to freeze due to evaporative c o ~ l i n g . ~Matrix-analyte ~.~~ crystal formation occurs in a steep concentration gradient as the volume of the solvent is decreased. Although it may be difficult to develop a temperature-dependent quantitative model that describes the crystal growth within an evaporating liquid dr0plet,3~the contrast between slowly evaporating solvents and rapidly evaporating systems appears rather obvious. Specifically, matrix-matrix, matrix-analyte, and analyte-analyte interactions in the solution phase more directly influence the distribution of the matrix and analyte in the solid phase, whereas solid phase matrix-matrix, matrix-analyte, and analyte-analyte interactions are more important for deposits formed via a slow crystallization pro~ess.33,~ The data contained in Figure 2 show that the aerosol MALDI ion signal is nonlinear in analyte concentration. We attribute the nonlinear responses to residual solvent present in the aerosol particle. At a constant matrix-to-analyte ratio, the analyte concentration is proportional to the ratio of solute to solvent. Thus, in Figure 2, the analyte concentration axis reflects the average solvent content of the aerosol particles, yith the amount of solvent decreasing from left to right. Figure 2 shows that as the aerosol solvent content increases, the ion signal decreases nonlinearly. The best signal is obtained with the least solvent, in agreement with earlier observations that drier aerosol particles produce better ion ~igna1.I~ If our interpretationof the data contained in Figure 2 is correct, the effects of residual solvent on the ion yield are not the same for the matrices and analytes studied. One interpretation of the (27) Vorm, 0.;R o e p s t o ~P.; , Mann, M. Anal. Chem. 1994,66, 3281-3287. (28) Nuwaysir, L. M.; Wilkins, C. L. In Applied Spectroscopy in Materials Science; Saperstein, D. D., Ed.; Proceedings of SPIE 1437; The International Society for Optical Engineering: Bellingham, WA, 1991; pp 112-123. (29) Xiang, F.; Beavis, R C. Rapid Commun. Mass Spectrom. 1994,8,199-204. (30) Klots, C. E. J. Chem. Phys. 1985,83, 5854-5860. (31) Engelking, P. C. J Chem. Phys. 1986,85,3103-3110. (32) Boom, A W.; Cresser, M. S.; Browner, R F. Spectrochim. Acta 1980,358, 823-832. (33) Mattiasson, B.; Kaul, R In Molecular Interactions in Bioseparations; Nago, T. T., Ed.; Plenum Publishing: New York, 1993; pp 469-478. (34) Kopperschlager, G.; Birkenmeier, G. In Molecular Interactions in Bioseparations; Nago, T. T.,Ed.; Plenum Publishing: New York, 1993; pp 499509.
values given in Table 1 is that the sensitivities of matrix and analyte to solvent in the aerosol particle are not constant. For example, Cnitroaniline is more sensitive to solvent than is acyano4hydroxycinnamic acid, because 4nitroaniline has the larger value of n. Bovine insulin seems particularly sensitive and myoglobin particularly insensitive to the presence of solvent. Previous studies of nonlinear signal response implicate mass transport effe~ts.3~ However, we interpret the differencesobserved for matrix and analyte as evidence that some portion of the nonlinear response is related to the solvent content of the aerosol particles. If the nonlinear signal response were due only to mass transport, the effects should not vary with matrix and analyte, unless chemical properties affect particle size. The data in Figure 2 suggest that the [M + HI+ ion yields are analyte dependent; the relative sensitivities of the various analytes are myoglobin > gramicidin S = bradykinin > bovine insulin. Factors that might cause such an effect include analyte surface area, hydrophobicities, and basic residues. The simplest view would be that the surface area of the analyte could be related to the probability for protonation. In the case of peptides and proteins, the surface area increases with increasing molecular weight.36 The molecular weights of the analytes are as follow: bradykinin, 1060.2; gramicidin S, 1141.5; bovine insulin, 5733.6; and myoglobin, 16 890. With the exception of bovine insulin, ion signal increases with increasing molecular weight. It seems unlikely that the surface area of bovine insulin in methanol is less than that of gramicidin S and bradykinin. Although the relative surface area of bovine insulin may be diminished by the formation of oligomers, summing the integrated areas for bovine insulin [M HI+, [2M HI+, and [3M HI+ increases the total signal only by a factor of 2. This does not change the order of the relative sensitivities of the analytes. Analyte hydrophobicities may also affect the matrix-analyte interaction and thus the ion yield and sen~itivities.3~~~~ Average hydrophobicities for bradykinin, gramicidin S, bovine insulin, and myoglobin were calculated from amino acid compositions using tabulated hydrophobicities of individual amino acids.39 The estimated hydrophobicity of gramicidin S is 1980 cal/mol, bradykinin is 1556 cal/mol, myoglobin is 1023 cal/mol, and bovine insulin is 1020 cal/mol. Thus, the hydrophobicity ordering of gramicidin S > bradykinin > myoglobin = bovine insulin does not correlate with the data from Figure 2. This suggests that analyte hydrophobicitiesalone do not determine the ion yield and sensitivities. Another parameter that could affect the analyte ion yield and sensitivity is the number of basic residues. Basic amino acid residues may act as proton acceptor groups for MALDI proton transfer. (Basic residues are defined here as residues that are positively charged at pH 7.0.) Bradykinin is composed of 22%basic residues, myoglobin of 21%,bovine insulin of 8%,and gramicidin S has no basic residues. This ordering does not correlate with the observed order of analyte ion yield and suggests that the tt
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(35) Ho, J. S.; Behymer, T. D.; Budde, W. L.; Bellar, T. A J Am. SOC.Mass Spectrom. 1992,3, 662-671. (36) Janin, J.; Miller, S.; Chothia, C. J. Mol. B i d . 1988,204, 155-164. (37) Naylor, S.; Findeis, A. F.; Gibson, B. W.; Williams, D. H.J Am. Chem. Soc. 1986, 108,6359-6363. (38) Caldwell, K. A; Gross, M. L Anal. Chem. 1989, 61, 494-496. (39) Bigelow, C. C.; Channon, M. In Handbook of8iochemistv and Molecular 8ioloQ. Proteins; Vol. I, 3rd ed.; Fasman, G. D, Ed.; CRC: Cleveland, OH, 1976 pp 209-243.
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number of basic residues is not a signiticant factor in determining ion yield and sensitivity. The observation that the analyte ion yield depends on the nature of the analyte and the solvent content of the aerosol particle suggests that quantification may require the use of internal standards. This study was limited to methanol, but it seems logical that different solvents may have different effects on individual biomolecules. For example, it may be possible that matrix-analyte interactions are sensitive to solvent type. Solvents affect the rates of crystallization,which strongly influence inclusion of the analyte in the matrix crystal lattice. Both noncovalent and hydrogen bonding interactions between the matrix and analyte would be expected to change as the solvent characteristics change. Thus, solvent would be expected to affect the MALDI ion yield and quantification. It is also important to consider how changing the matrix and analyte concentrations affect the aerosol particle size. Particle size is important for two reasons: (1) aerosol particle transport efficiency depends on particle size40r41 and (2) aerosol MALDI ion yields may depend on particle size. Within the mass spectrometer, large and small aerosol particles are not transported with the same efficiencies. Consequently, large and small aerosol particles may not be sampled identically in the time-of-flight source region. Particle size may also influence the ion yield. Under conditions of complete desolvation, higher concentrations of matrix and analyte produce aerosol particles with larger diarr1eters.4294~ However, because desolvation is incomplete in the aerosol MALDI experiment, it is not clear whether the aerosol particle size and the resulting ion yield are strongly affected by the concentrations of the matrix and analyte. To determine if the matrix and analyte (40) Winkler, P. C.; Perkins, D. D.; Williams, W. K.; Browner, R. F. Anal. Chem. 1988,60,489-493. (41) Browner, R. F.; Boom, A W.; Smith, D. D.Anal. Chem. 1982,54, 14111419. (42) Allen, L. B.; Koropchak, J. A Anal. Chem. 1993,65, 841-844. (43) Charlesworth, J. M. Anal. Chem. 1978,50, 1414-1420.
1986 Analytical Chemistv, Vol. 67,No. 13, July 7, 1995
concentrations influence the size distribution of the aerosol particles requires direct particle size measurements. In summary, three observations concerning aerosol MALDI ion yields can be made from the results presented here. (1) The optimum matrix-to-analyte ratio in aerosol MALDI is in the range 10-1OO:l for the analytes and matrices studied. This optimum matrix-to-analyte ratio is smaller than the optimum matrix-toanalyte ratio observed in surface MALDI. We postulate that the lower optimum matrix-to-analyte ratio is a result of rapid desolvation occurring in aerosol MALDI, forcing the matrix and analyte into contact with each other. Rapid desolvation promotes more efficient matrix-analyte interactions. (2) Desolvation also influences the [M HI+ ion signal. We interpret the observation that the [M HI+ ion signal is a nonlinear function of the analyte concentration as an indication of the importance of aerosol desolvation. Drier aerosol particles produce larger aerosol MALDI ion signal. (3) The relative sensitivities for the different analytes are due to the chemical properties of the analyte. The order of the [M HI ion yields is myoglobin > gramicidin S bradykinin > bovine insulin. After examining the hydrophobicities, number of basic residues, and surface areas of each analyte, we tentatively suggest a correlation between analyte size and ion yield.
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ACKNOWLEDGMENT This research was funded by the Texas Advanced Technology Program and by the Division of Chemical Sciences, Office of Basic Energy Sciences, U S . Department of Energy under Grant No. DEFG05435ER-13434. The Nd:YAG laser and additional equipment were purchased with funds provided by the National Science Foundation under Grant No. CHE-9223629. Received for review September 19, 1994. Accepted April
6,1995.@ AC9409344 @Abstractpublished in Advance ACS Absfracts, May 15, 1995.
