Anal. Chem. 1989, 61, 375-382
Nevertheless, it is important to recall the severe restrictions on information incorporated into this predictive model; one might naturally expect more reliable predictions to be a result of either (a) using a larger number of characteristic solvents (and solutes) 01 (b) dowing ternary solvents to act 89 members of the basis set. Current work is under way to extend the predictive method to other column/solvent systems with the inclusion of more comdicated and varied solute classes. LITERATURE CITED Glajch, J. L.; Klrkland, J. J.; Squire, K. M., Minor, J. M. J. Chromatogr. 1980, 16,57. Snyder, L. R.; Dolan, J. W.; Gant, J. R J. Chromatogr. 1979, 765, 3. Sny&r, L. R.; Dolan, J. W.; Gant, J. R. J. Chromatogr. 1979, 165, 31. Quarry, M. A.; Grob, R. L.; Snyder, L. R. Anal. Chem. 1988,5 8 , 907. Koopmans, R. E.; Rekker, R. F. J. Chromatogr. 1984,285, 267. Jlnno, K.; Kawasaki, K. Chromatographia 1984, 18, 90. Bylina, A.; Gluzinskl, L.; Radwanskl, B. Chromatographia 1983, 77, 132. Wise, s.; Sander, L. C . HRC CC, J . High Resolut. Chromatogr. Chromatogr. Commun. 1985,8 , 248. Hanai, T.; Tran, C.; Hubert, J. HRC CC, J . High Resolut. Chromatogr. Chromatogr. Comrnun. 1981,4 , 454. Kaliszan, Roman Crit. Rev. Anal. Chern. 1986, 16, 323. Schoenmakers, P. J.; Billlet, A. H.; de Galan, L. J. Chromatogr. 1981, 218, 261. Schoenmakers. P. J.: Bllliet, A. H.; de Galan, L. J. Chromatogr. 1982, 282. 107.
375
(13) Schoenmakers, P. J.; Billlet, A. H.; de Galan, L. Chromatographia 1982, 15, 205. (14) Lo~muller, c, H.; Hamzavi-Abedi, M. Chu-Xiang, o, J , chromtogr. 1988,387, 105. (15) Malinowski, E. R.; Howery, D. G. factor Analysis in Chemistfy;Wlley: New York, 1980. (16) Malinowski, E. R. Anal. Chem. 1977,4 9 , 606. (17) Wold, S. Technometrics 1987,2 0 , 397.
!i:i
J;
~ ~ ~ ~ ~ , w ~ k ~ Lg8i: , ~ . R " lg87, ~ " 1, , 221, ~ ~ ~ ~ ~ , (20) HOWeN. D. G.: Soroka. J. M. J. Chemom. 1987. 1. 91. i21j weine;,'~. H.;'Howe&'D. G. i n e l . Chem. 1972;4 4 , 1189. (22) Kindsvater, J. H.; Weiner, P. H.; Klingen, T. J. Anal. Chem. 1974,46, 982. (23) Weiner, P. H.; Parcher, J. F. Anal. Chem. 1973,4 5 , 302. (24) Weiner, P. H.; Malinowski, E. R.; Levinstone, A. J. Phys. Chem. 1970, 7 4 , 4537. (25) Malinowski, E. R.; Howery, D. G.; Welner, P. H.; Soroka, J. R.; Funke, P. T.; Seizer, R. S.; Levinstone, A. FACTANAL, Program 320, Quantum Chem. Program Exchange, Indiana University, Bloomington, IN, 1976. (26) Martin, A. J. P. Biochem. SOC. Symp. 1949,3 , 4. (27) Katz, E. D.; Lochmuller, C. H.; Scott, R. P. W., submitted for publication In J. Chromatogr.
RECEIVED for review August 16, 1988. Accepted November 15, 1988. This work was supported, in part, by a grant from the National Science Foundation, Grant No. CHE-8500658 (to C.H.L.).
Influence of Sample Concentration and Adsorption Time on the Yield of Biomolecule Ions' in Plasma Desorption Mass Spectrometry A. Grey Craig* a n d Hans Bennich
Department of Immunology, Box 582, Uppsala University, S- 751 23 Uppsala, Sweden The yield of intact ions formed In 252Cfplasma desorptlon mass spectrometry has been investigated by analyzing melittin and bovine trypsln at dlfferent concentrations on a nitrocellulose surface. The yield of trypsin ions Is shown-to vary wlth the protein concentration and adsorption time. The singly charged ion of trypsin Is observed when concentrated solutlons ( I O pM to 1 mM) of bovine trypsin are applied for sufficient time.
INTRODUCTION Plasma desorption mass spectrometry (PDMS) ( 1 ) utilizes 262Cffission fragments as primary ions to bombard solid samples, producing secondary ions of the sample. The secondary ions are mass analyzed by their time of flight. Soon after its discovery, PDMS was recognized to enable intact desorption and ionization of labile molecules (2). The measurement of insulin, and thereafter a series of higher mass proteins including porcine trypsin, illustrated the potential for mass determination of biomolecules (3-5). Recently the measurement of porcine pepsin has demonstrated the ability of PDMS to ionize intact molecules above 30 kdaltons (6). Early measurements with this technique used the electrospray sample preparation method (3,where a solution of the sample is sprayed from a capillary, set at a high potential,
* Author to whom correspondence
should be addressed. This term refers to a charged molecule, but the resolution and mass accuracy of the instrument used are not sufficient to determine whether the single (or multiple) positive charge is due to the loss of one (or more) electron(s) or to the addition of one (or more) proton($. The same caveat holds for the notations M', Mz+,etc., in the figures.
onto a thin, grounded aluminum or aluminized Mylar foil. Alternative sample preparation methods have been developed to enhance the molecule ion yield. These include the use of insoluble matrices such as Ndion (8)and nitrocellulose ( l o ) , or coelectrospraying the anal* with a glutathione matrix (9). The (electrosprayed) nitrocellulose film is suitable for adsorbing large peptide and protein samples (10). A distinct feature of the spectra obtained when using a glutathione matrix or a nitrocellulose film is the presence of intense multiply charged molecule ions (9,lO). In the case of nitrocellulose this enhancement has been suggested as resulting from the formation of preformed ions ( l o ) ,while glutathione was proposed to decrease the electrostatic attraction of preformed ions, or alternatively enable desorption of neutral ion pairs which subsequently dissociate (9). The similarity between the spectra observed when using a glutathione matrix and a nitrocellulose film has been noted (9). The yield of intact ions formed by using PDMS together with the nitrocellulose sample preparation method has been investigated (10-13). Previously, the yield of doubly charged ions of bovine insulin was found to be less dependent on the amount of sample deposited on nitrocellulose, as compared to the yield of singly charged ions (13). In this paper the effect of sample concentration on the behavior of the singly and multiply charged ions of melittin and trypsin is investigated. MATERIALS AND METHODS Measurements were made with a BIOION 20 mass spectrometer (Bio-Ion Nordic AB, Uppsala, Sweden). The instrument was operated at +20 kV accelerating potential; spectra were accumulated for lo6 primary ions for melittin and 2 X lo6 primary ions for bovine trypsin. The nitrocellulose foils were prepared as previously described (13). Spectra were calibrated by using
0003-2700/89/0361-0375$01.50/00 1989 American Chemical Society
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H+ and CH3+since no Na+ peak was observed for trypsin samples prepared with ethanol, as described below. Comparison of spectra calibrated with H+/CH3+and H+/Na+for several peptides including melittin results in a comparable accuracy of mass measurement (14).The measured masses correspond to the centroid at half peak height. The mass spectrometer used in this study has a resolution below 500 and mass accuracy of 0.1% (13). The instrument is therefore only able to determine the mass of the species observed within these limits. As a result we use the expression “molecule ion” in this presentation rather than a more descriptive term, such as “protonated molecule ion”. The term “molecular ion” is considered unsuitable since it may be confused with the M+*species. Molecular ions (M+’)of peptides have thus far not been observed by using za2Cf. Melittin (p.A.) was purchased from Serva Feinbiochemica, Heidelberg. Bovine trypsin was a gift from Novo Industri A/S, Denmark. The stock solution of melittin (1mM) and the dilution series from 0.1 mM to 1 pM were prepared in 0.1% aqueous trifluoroacetic acid (aqTFA). Stock solutions of bovine trypsin (2 mM) were also prepared in 0.1% aqTFA. An aliquot was diluted with an equal volume of 99.5% ethanol. Subsequent dilutions (0.1 mM to 0.1 pM) were made by mixing 1part of the solution to 9 parts of a 1:1 mixture of 0.1% aqTFA and ethanol. A separate trypsin solution that did not contain ethanol was prepared from the 2 mM stock solution by mixing an aliquot with an equal volume of 0.1% aqTFA. Aliquots (10 pL) varying from 1 mM to 0.1 pM were deposited onto the nitrocellulose foils. Since spectra of trypsin measured from solutions that had been stored for 24 h or longer did not show intense molecule ions and sometimes contained interference peaks, fresh stock solutions of trypsin were made for each set of measurements (Figures 6, 7, and 8). The reproducibility of the concentrations of the stock solutions used was estimated to be within 3%. Adsorption Time. The protein solution was left on the nitrocellulose surface for a specified time which will be referred to as the “adsorption time”. The nitrocellulose foil was then rinsed with water and dried with nitrogen. During the adsorption period, the foil was kept in an atmosphere of water and ethanol to avoid drying of the sample. Analysis of Sample Rinse. A variation of the above procedure, applied to one series of experiments with trypsin, was undertaken to analyze the sample which was normally rinsed from the foil. After an adsorption time of 30 min, 20 pL of a 1:l mixture of 0.1% aqTFA and ethanol was added to the initial sample on the nitrocellulose foil. Immediately thereafter 20 p L was transferred to a new foil (first ”rinse-deposited”)while the initial foil was rinsed with water and dried with nitrogen. After an adsorption time of 30 min 20 pL of 0.1%aqTFA and ethanol (1:l) was added to the first “rinse-deposited” foil and 20 pL of the diluted solution was transferred to a new foil (second “rinsedeposited”),while the first “rinse-deposited” foil was rinsed and dried as above. After 30 min the second “rinse-deposited“ foil was rinsed and dried. This procedure was carried out with trypsin solutions having initial concentrations from 1pM up to 1 mM. Thus a total of three foils from each of the four “initial solution concentration” (isc) samples were produced. Reproducibility of Measurements. Eight nitrocellulose f o l were each deposited with 10 p L of 0.1 mM bovine trypsin solution (0.1% aqTFA and ethanol (1:l)) and rinsed after 10 min of adsorption time. The eight foils were introduced into the instrument at the same time and each foil waa analyzed 3 times in succession. The yield of the doubly charged molecule ion for the repeated measurement of the same sample had an average standard deviation of *2.7% (standard deviations for individual samples ranged between &2 and &6%). The standard deviation between the average yield for the eight samples was A l l % . The yields were measured above the peak base of background subtracted spectra. In the experiments presented the minimum yield that is measured is approximately 500 counts. RESULTS Figure 1 shows the PDMS spectrum of melittin (2847.5 daltons) where 10 p L of a 0.1 mM solution (2.8 rg) was adsorbed for 5 min and rinsed. Two intense peaks are observed a t m / z 2849 and 2876.3 corresponding to the singly charged
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molecule ions of melittin and its N-formyl analogue (15), respectively. Also present are two less intense peaks a t m/z 1425.6 and 1438.5 corresponding to the doubly charged molecule ions of the above species. A small peak corresponding to the mass of a singly charged dimer ion is also present. Dimers and higher oligomer ions have previously been observed in PDMS from peptides (16). Peptides with mass below 2.5 kdaltons have not been observed to form multiply charged ions (17, 18). Singly and doubly charged molecule ions have been observed for several peptides between 3 and 5 kdaltons (18-20). Above this mass, peptides or small proteins can form even higher charged species up to and including sextuply charged molecule ions (IO). The relative intensity of the singly and doubly charged molecule ions of melittin are dependent on the concentration of the solution deposited on the nitrocellulose as shown in Figure 2. The solutions were rinsed after an adsorption time of 5 min. Figure 3 shows the PDMS spectrum of bovine trypsin (23 293.2 daltons) (a) without baekground subtraction (low mass region inset) and (b) background subtracted. In this case a 1mM solution (0.23 mg) in 0.1% aqTFA and ethanol was adsorbed for 30 min and rinsed. Intense peaks at m/z 23 332, 11652,7780, and 5841 are observed corresponding to singly, doubly, triply, and quadruply charged molecule ions. Figure 4 shows the PDMS spectrum (without background subtraction) of trypsin without ethanol. For this measurement the same 2 mM stock solution of trypsin, diluted with 0.1% aqTFA, was used as in Figure 3. In the spectrum shown in Figure 4 peaks are observed at m/z 5166,5997, and 8961 which to not appear to correspond to multiply charged protonated or sodium cationized molecule ions of trypsin. These peaks may reflect impurities present in the trypsin preparation or fragments produced from autocleavage of P-trypsin (21). The
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intensities of the Na+ peaks observed in Figures 3 and 4 (inset) were both significantly lower than those observed for samples prepared from the same solutions without the rinsing step. Figure 5 shows the spectrum of trypsin when at 1pM solution (0.2 pg) in 0.1% aqTFA and ethanol was adsorbed for 30 min and rinsed. Intense peaks at m/z 11690,7821, and 5853 were present corresponding to the doubly, triply, and quadruply charged molecule ions. When the adsorption time for the 1mM solution was extended longer than 30 min, the yields, of the singly and doubly charged ions were further enhanced, relative to the triply and quadruply charged ions. When the adsorption time of the 1 pM solution was extended longer than 30 min, the yield of the triply charged ion increased relative to the doubly charged ion, while the singly charged ion remained absent. Measurement of 10 p L of a 0.1 pM solution (23 ng) after an adsorption time of 30 min or longer, produced a spectrum where only the triply and quadruply charged species were present. Figures 6,7, and 8 show (a) the singly and doubly and (b) the triply and quadruply charged molecule ion yields for the different concentrations of trypsin after adsorption times of 1, 4, and 30 min, respectively. After an adsorption time of 1 min the singly charged species is observed with the most concentrated solution (1 mM) (Figure 6a). For longer ad-
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sorption times, the singly charged molecule ion is observed from the 10 pM and 0.1 mM solutions. In Figures 7a and 8a
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Flgure 8. Plot of the yield (a) singly and doubly and (b) triply and quadruply charged molecub ions versus the concentration of the trypsin solution (adsorption time 30 rnin).
Table I. Yields of Singly and Multiply Charged Ions Measured from a 0.1 mM Trypsin Solution, after Varying Adsorption Times
Table 11. Yields of Singly and Multiply Charged Ions Measured from a 10 pM Trypsin Solution, after Varying Adsorption Times
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the yield of singly charged ions is observed to increase linearly with logarithmic increase in concentration to 1mM. The yield of the doubly charged molecule ion, is appreciably higher than that of the singly charged species. The yield of doubly charged ions increases with the increased concentration from 1to 10 pM but shows indications of saturation at higher concentrations. The yield dependence of the triply charged molecule ions also shows a different behavior. The maximum yield is not observed at 1 mM but rather at the 10 pM or 0.1 mM concentrations. In addition, the drop in the yield of the triply charged ions between 10 and 1 pM is not as significant as for the singly and doubly charged ion yields. The yield measurements of the quadruply charged ion show some trends which suggest a behavior similar to that of the triply charged ions; however the yields are significantly lower. The effect of the adsorption time between application and rinsing was further investigated. The yields of molecule ions, shown in Tables I and 11, were measured when 0.1 mM and 10 pM solutions were deposited onto the nitrocellulose foil. These samples were rinsed after either 1,5,10,or 20 min. The
yields of the singly and multiply charged molecule ions (Tables I and 11) are generally observed to increase with longer adsorption time. As a result, the total yield increases with adsorption time. The variation in the concentration of trypsin remaining on the nitrocellulose after an adsorption time was also investigated. This was undertaken by analyzing the remaining solution that was normally rinsed from the nitrocellulose surface. As outlined in the Experimental Section the adsorption time was 30 min for the initial and “rinse-deposited” samples. Parts a-d of Figure 9 show the yield of the singly, doubly, triply, and quadruply charged molecule ions, respectively, measured from the initial and “rinse-deposited” samples. Four important features can be discerned from these measurements. Firstly, although of reduced intensity, spectra were observed for all of the “rinse-deposited” samples with the exception of the 1p M initial solution concentration (isc) sample. Secondly, a significant difference was observed between the spectra measured from the first and second rinses of the 1 0 pM (isc) sample; the latter was observed to produce a less intense
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spectrum than the initial 1 pM sample. Thirdly, the yields of the singly, doubly, and triply charged ions measured from the first and second rinse samples of the 0.1 mM (isc) sample were more intense than the first rinse of the 10 pM (isc) sample. Fourthly, the first rinse of the 1mM (isc) produced yields of singly and doubly charged ions of similar relative intensity to the initial 0.1 mM sample.
DISCUSSION The measurements of melittin indicate that the yield of singly charged molecule ions is more dependent on concentration than that of doubly charged ions. This behavior is consistent with the results reported for bovine insulin (13) and other polypeptides such as nisin, glucagon, and thioredoxin (IS). The yield of the singly charged species increases linearly with the logarithmic increase of concentration. In contrast the yield of doubly charged molecule ions shows a much weaker dependence with the concentration of the solution deposited, particularly above 10 rM. The spectra shown in Figure 3 and 4 illustrate the effect of the addition of ethanol to trypsin. The ethanol results in a significant increase in the yield of the singly and multiply charged molecule ions. Interestingly, this enhancement is at the expense of the species observed when no ethanol is added. The reasons for this dramatic change in the spectrum of trypsin with the addition of ethanol are not completely understood. The removal of salt contaminants has previously been noted as a possible reason for the increased molecule ion yields when using the nitrocellulose method (10). Since the Na+ ion intensity is further reduced by the addition of ethanol, the increased molecule ion yield may be the result of a lower salt concentration.
The enhancement may also be due to the effect of the addition of alcohol to the protein solution; three different modes of action are worthwhile pointing out. Firstly, an increased binding of nitrocellulose when alcohol is present (22) may reflect swelling of the nitrocellulose pores or a wetting effect which makes the surface more accessible for the protein. Alternatively the effect of alcohol on the protein may result in the enhanced interaction. The denaturing effect of ethanol (23) may be compared with the unfolding of a protein that occurs concomitant with adsorption (24). The probability that interaction between the denatured protein and the nitrocellulose surface results in binding is thereby markedly increased. Some protein flocculation may even be considered superimposed on adsorption (24). Thirdly, it may be that the denatured molecules once adsorbed are more readily desorbed in the ionization process. Ethanol molecules surrounding the denatured protein, rather than an aqueous hydration sphere, may be involved in the protein interaction with the nitrocellulose. Removal of the ethanol molecules under vacuum would remove the ethanol-mediated binding interaction and thereby help overcome the barrier for desorption of the protein. In comparison an aqueous hydration sphere may be less easily removed under vacuum. The variations in the yield measurements of bovine trypsin with concentration show a more complicated dependence than observed for melittin and other peptides (13, 18). The dependence of the singly charged ions of melittin can be characterized as positive, that is the yield increases with concentration. This is also the case for the singly charged ion of trypsin. The trypsin doubly charged ion shows a positive dependence at low concentrations, but at higher concentra-
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 4, FEBRUARY 15, 1989
tions the yield saturates. The triply charged ions also show a positive dependence at lower concentrations, but at higher concentrations a negative dependence is observed. The quadruply charged ion shows similar trends to the triply charged ion; however, the variations are not significant compared with the absolute yields and the expected reproducibility (see Materials and Methods). Therefore the quadruply charged ion can best be regarded as independent of the concentration and adsorption time measurements presented in Figures 6, 7, and 8 and Tables I and 11. This behavior is similar to the melittin doubly charged ion. The spectrum of bovine trypsin is shown to vary with the adsorption time. An increased adsorption time results in an enhanced molecule ion yield for the singly and multiply charged species. In particular the singly charged molecule ions are observed at lower concentrations. As a consequence the total molecule ion yield increases with adsorption time. Spectra measured of samples prepared at both high and low concentrations with adsorption times longer than 30 min are consistent with trends observed in both Figures 6,7, and 8 and Tables I and 11. This includes the enhancement of both singly and doubly charged ions relative to the triply charged when a 1 mM solution is adsorbed. The reduced intensity of the triply and quadruply charged ions at this concentration and their enhancement when a 0.1 pM solution was measured suggest a general trend for these ions at extreme conditions which is not clearly observed for the quadruply charged ion in the figures and tables. Although the trends of both concentration and adsorption time discussed are not observed uniformly throughout the tables and figures presented this may reflect the limitations of preparing identical electrosprayed nitrocellulose foils. An increase in the yield of insulin molecule ions with the thickness of the nitrocellulose film has been observed ( I I , I 2 ) . Variation in the amount of nitrocellulose electrosprayed may occur as a result of evaporation of acetone from the nitrocellulose solution or fine details of the electrospraying conditions. The contribution of this variation should, however, be encompassed within the standard deviation for separate samples discussed in Materials and Methods. The consistency between the yield measurements presented in Figures 6,7, and 8 may also suffer from differences in the concentration of the stock and accordingly the dilution solutions. It is suggested that the observed dependence on concentration and adsorption time show a general trend. That is, the singly and to a lesser extent doubly charged ions are enhanced with both increasing concentration and adsorption time. In contrast, the triply charged ion shows a positive dependence with both concentration and adsorption time at low solution concentrations, which changes to a negative dependence at high solution concentrations. The dependence of the quadruply charged ion at extreme conditions is similar to the triply charged ion. On this basis the singly and doubly charged ion behavior will be categorized together as “lower charge” species and the triply and quadruply ions behavior will be categorized as “higher charge” species. On the basis of the measurements of the initial and “rinse-deposited” samples, it is possible to obtain a rough approximation of how much sample binds from different concentrations. This discussion is intended to note that there is a variation in the amount of sample binding from the different solutions. The absence of a discernible specrum from the 1pM (isc) “rinse-deposited” samples is in contrast to the measurement of the 0.1 pM sample with the same adsorption time. This would be consistent with the major portion of the 1 pM sample being bound to the initial foil. For the purpose of establishing that more sample binds with increasing concentration, con-
sider that 10 pmol (i.e. the maximum that can bind to the 1 pM initial foil) binds to the 10 pM initial foil. Of the remaining 90 pmol on the initial foil two-thirds are transferred, that is a 3 pM solution. Although it is to be expected that less than 10 pmol would bind from this solution, we can at least say that 10 pmol would be the maximum, given only 10 pmol binds to the initial foil. This would leave 50 pmol on the first “rinse-deposited” foil, of which half would be transferred resulting in a 1.25 gM solution on the second “rinse-deposited” foil. However, in the case of the 10 pM (isc) sample, the concentration of the solution transferred to the second “rinsed-desposited” foil produces significantly lower yields of both the doubly and triply charged ions than the 1 pM solution. It is concluded that more sample binds to the initial foils from the 10 pM than the 1pM concentration. The same argument can be applied to the amount of sample binding from the 0.1 mM concentration. Since the second “rinsedeposited” sample produces less intense yields of the singly and doubly charged ions than the 10 pM initial sample, we can conclude that the amount of sample binding from the 0.1 mM is significantly more than from the 10 pM. In the case of the 1 mM first “rinse-deposited” sample, the yield is not significantly below the initial 0.1 mM concentration. This, it is argued, is indicative of a lower proportion of the total molecules in the initial solution binding to the nitrocellulose, from the 1 mM compared to the 0.1 mM solution. Although more protein probably binds from the 1mM than the 0.1 mM concentrations, the nitrocellulose is reaching a saturation level. The variation of the yield of multiply charged molecule ions with the amount of sample deposited is difficult to reconcile with the suggestion of preformed ions (IO). While the charge of trypsin in solution will be a function of the pH and the dielectric constant of the solution, these parameters should not vary with the different dilutions. The addition of alcohol to an acidic solution will raise the protonic activity (paH)of the solution (25) and significantly lower the dielectric constant (26). In aqueous-organic mixtures the proton activity (aH) is defined relating to the conventional scale in water (25). However, these effects should be uniform for all the concentrations employed. Even more difficult to reconcile if the ions are presumed to be preformed is the dependence of the distribution of multiply charged molecule ions on adsorption time. The results presented indicate that the charge state of the trypsin molecule in solution cannot be the main parameter which determines the observed ion charge. The variation of the relative intensities of ions of different charge is proposed to result from the different amounts of sample adsorbed to the nitrocellulose surface. The increase of the yield of the singly and doubly charged molecule ions of bovine trypsin with concentration, reflects the increased amount of sample that is bound to the nitrocellulose. In the same way the increased yield of the “lower charge” species with longer adsorption times before rinsing also reflects an increased amount of bound sample molecules. The amount of sample adsorbed to the nitrocellulose also determines the yield of the triply and quadruply charged molecule ions of bovine trypsin. A t very low concentrations the yield of these ions is dependent on the amount of sample bound to the nitrocellulose in much the same way as proposed for the singly and doubly charged ions. However, at higher concentrations, the formation of “lower charge” species predominates over the formation of “higher charge” species. The switch from “higher charge” to “lower charge” species can be explained in terms of the amount of sample adsorbed onto the nitrocellulose surface. In the case when concentrated solutions are applied, there is a large reservoir of molecules in solution, and therefore a large number of all available sites on the nitrocellulose will be occupied. As a result a consid-
ANALYTICAL CHEMISTRY, VOL. 61, NO. 4, FEBRUARY 15, 1989
erable number of the sample molecules, adsorbed to the nitrocellulose, are in contact with other sample molecules. A common ,intermediate step has been postulated (27) for all ionization methods whereby a charged interface is formed during the ionization event. The molecules in contact in the region of this charged interface may collectively share a number of charged sites between them, leading to charge competition. The desorbed ion can be envisaged as the most successful molecule to retain a charge and break the adsorption bonds between the surface and the other molecules. Because of this charge competition, it is proposed, the ejected ion is less likely to contain several charge sites and therefore this event produces predominantly “lower charge” molecule ions. In contrast when low concentration solutions are applied, the reservoir of molecules that remain in solution is small. A considerable number of available binding sites on the nitrocellulose may therefore remain unoccupied. As a result a larger portion of the sample is adsorbed in “isolation” (i.e. the adsorbed molecules are not in contact with each other). This isolation reduces or excludes the competition for the charge in the ionization process. Therefore the desorbed molecule ions are able to retain several sites of ionization. A theoretical model by N o r s k ~ vand Lundqvist (28) has been used to describe some features of the plasma desorption ionization event (29). On the basis of this model it has been proposed that the formation of an intact ion occurs as a result of desorption of a cluster of molecules with subsequent ejection of a single intact ion (9,27,29). The proposed charge competition may be envisaged occurring on the desorption surface or alternatively taking place within the desorbing cluster of molecules. The proposal, that the proximity to other charge competing molecules determines the charge state of the ejected ion, can be extended to explain the similar mass spectra observed when a glutathione matrix is electrosprayed with the sample. It has previously been reported that at high matrix-to-sample concentrations, the ratio of the doubly charged to singly charged molecule ion of insulin is at a maximum (9). A t a high glutathione matrix concentration, it is assumed that the insulin molecule is surrounded by glutathione molecules. Therefore, during the ionization event the insulin molecules cannot compete for the charge against each other. Since glutathione does not interfere with the ionization of insulin, this allows a high charge retention on the insulin molecule thereby forming more of the doubly charged ions. This can be compared to the low sample concentrations applied to nitrocellulose, where the samples are isolated and retain higher charges due to the absence of other nearby protein molecules competing for the charge. As the glutathione matrix concentration is reduced, the molecules come in contact and compete with each other for the charge, producing more of the singly charged insulin molecule ions. The maximum number of charges observed can be explained as a consequence of the proposed charge competition. The observed yield dependency can only be explained by charge competition, provided that the total number of charges that the molecules are envisaged to compete for is not proportional to the number of sample molecules which are competing for the charge. If the contrary were the case, then as the number of bound trypsin molecules increased, they would attract more charge, and subsequently share the charge rather than compete for it. Therefore a consequence of the charge competition proposal is that a certain number of chargeproducing events emanate from the impact of a fission fragment, and this number is independent of the sample on the surface. This would create an average number of charge events per unit area. When the surface contains an isolated molecule, the size of the molecule determines the area that the molecule
381
is able (or required) to interact with charge-producing events. In the case of trypsin, the molecule extends over an area which enables it to interact with charge-producing events which ultimately lead to a maximum of sextuply charged species (IO). A smaller molecule such as insulin is restricted to triply charged species (13),while melittin produces only a doubly charged ion. Peptides of below 2.5 kDa do not interact with charge-producing events leading to more than one site of ionization.
CONCLUSION The dependence of the distribution of multiply charged molecule ions with sample concentration and adsorption time has important implications for this technique from both physical and analytic viewpoints. The results presented indicate that the molecule ions observed are not due to ions preformed in solution. The observation of molecule ions with “higher charge”, when the amount of sample deposited is reduced, reflects the reduced amount of sample bound to the nitrocellulose. The increased yield of molecule ions with “lower charge” when more concentrated samples are deposited is due to the increased amount of sample bound to the nitrocellulose, enabling charge competition. These observations may be used to enhance the yield of singly charged molecule ions, assisting molecular mass determinations of biomolecules using PDMS. The increase in yield when samples are exposed for longer periods of time may also provide useful mass spectra from dilute sample solutions. Registry No. Trypsin,9002-07-7;mellitin, 37231-28-0;ethanol, 64-17-5.
LITERATURE CITED Sundqvist, 6.; Macfarlane, R. D. Mass Spectrum Rev. 1085, 4 , 421-460. Torgerson, D. F.; Skowronski, R. P.; Macfarlane, R. D. Biophys. Res. Commun. 1074, 60, 616-621. Sundqvist, 6.; Fohlman, J.; Peterson, P.; HAkansson. P.; Kamensky, I.; McNeal, C. J.; Macfarlane, R. D. J. Am. Chem. SOC. 1082, 104, 2948-2949. Sundqvlst, 6.; Mkansson, P.; Kamensky. I.; Kjellberg, J.; Salehpour, M.; Wlddiyasekera. S.; Fohiman, J.; Peterson, P.; Roepstorff, P. Biomed. Mass Spectrom. 1084, 7 7 , 242-257. Sundqvist, 6.; Roepstorff, P.; Fohlman, J.; Hedin, A,; Mkansson, P.; Kamensky, I.; Lindberg, M.; Salehpour, M.; Save, G. Science 1084, 226, 696-698. Craig, A. G.; Engstrom, A.; Bennich, H.; Kamensky, I . Am. Soc.Mass Spectrom. Allied Top. 1987, 35, 528-529. McNeal, C. J.; Macfarlane, R. D.; Thurston, E. L. Anal. Chem. 1979, 57, 2036-2039. Jordan, E. A.; Macfarlane, R. D.; Martin, C. R.; McNeal, C. J. Int. J. Mass Spectrom. Ion Phys. 1083, 53, 345-348. Alai, M.; Demirev, P.; Fenseiau, C.; Cotter, R. J. Anal Chem. 1088, 58, 1303-1307. Jonsson. G. P.; Hedin, A.; Hikansson, P.; Sundqvist, 8.; Save, G.; Nlelsen, P. F.; Roepstorff, P.; Johansson, K.-E.; Kamensky, I.; Lindberg, M. S. L. AnalChem. 1986, 58, 1084-1087. Roepstorrf, P.; Nielsen. P. F.; Sundqvist, B. U. R.; HAkansson, P.; Jonsson, G. Int. J. Mass Specfrom. Ion Processes 1087, 78, 229-236. Nielsen, P. F.; Roepstorff, P., I n Proceedings from the Work on Physics on Smell Systems; Springer Proceedings on Physics; Hilf, H., Wien, K., Eds.; Springer-Verlag: Berlin. 1986. Kamensky, I.; Craig, A. G. Anal. Instrum. 1087, 16, 71-92. Craig, A. G.; Bennich, H., unpublished results. Jentsch, J. 2.Naturforsch., 8 1060, 248, 264-265. Sundqvist, 6.; Hedin, A.; HAkansson. P.; Kamensky, I.; Salehpour, M.; Save, G. Int. J. Mass Spectrom. Ion Processes 1085, 65, 69-69. Undeberg, G.; Engstrom, A; Craig, A. G.; Bennich, H. I n The Andysis of Peptides and Proteins by Mass Specfrometty; McNeal, C. J., Ed.; John Wlley and Sons: New York, 1988; pp 1-14. Craig, A. G.; Engstrom, A.; Lindeberg, G.; Bennich, H., unpublished results. Roepstorff, P.; Nielsen, P. F.; Kamensky. I . ; Craig, A. 0.;Self, R. Biomad. Enviorn. Mass Spectrom. 1088, 15, 305-310. Engstrom, A.; Bennich, H.; Kamensky, I.Biomed. EnviCraig, A. 0.; ron. Mass Spectrom. 1987, 74, 669-673. Inagami. T. I n Proteins Structure and Function; Fuatsu. M., et al., Eds.; Kodansha: Tokyo, and Wiley: New York, 1972; pp 1-83. Gershoni, J. M.; Palade, G. E. Anal. Biochem. 1083, 737, 1-15. Kauzmann, W. A&. Protein Chem. 1950, 74, 1-63. Macritchie, F. Adv. Protein Chem. 1078, 32, 283-326. Hoa. G. H. 6.; Douzou, P. J. Biol. Chem. 1973, 248, 4649. Green, A. A.; Hughes, W. L. I n Methods in Enzymology; Colowick, S. P., Kapian, N. O., Eds.; Academlc Press: New York, 1985; pp 1-02,
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(27) Derrick, P. J. Fresenius' Z . Anal. Chem. 1886, 324, 486-491. (28) Norskav, J. K.; Lundqvist, B. L. Phys. Rev. 8 1979, 79. 5661. (29) Macfarlane, R. D. A m . Chem. Res. 1982, 75, 268-275.
RECEIVED for review January 12,1988. Accepted October 21,
1988. This work was supported by the Swedish National Board for Technical Development and the Swedish Medical Research Council. A.G.C. acknowledges funding support of OE and Edla Johansson Vetenskapliga Stiftelse and Gunnar Hanssons Forskningsstiftelse.
TECHNICAL NOTES Fiber-optic Magnestum and Calcium Ion Sensor Based on a Natural Carboxylic Polyether Antibiotic Koji Suzuki,* Koji Tohda, Yasushi Tanda, Hiroshi Ohzora, Shuji Nishihama, Hidenari Inoue, and Tsuneo Shirai* Department of Applied Chemistry, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223, Japan Recently, the development of various types of optical chemical sensors has been actively investigated (1-4). Among these, sensors aiming at ion measurements are mostly H+ (pH) sensors (5-13) and several other ion sensors for K+ (14, 15), Na+ (16),Be2+(19,Mg2+ (18),Zn2+ (18),Cd2+(18), A13+ (18, 19),and halides (20). Particularly, among all the chemical sensors, including optical sensors and many electrochemical sensors, a proper sensor for Mg2+had not been developed. Here we report the preparation and response characteristics of an optical sensor for Mg2+ and Ca2+ using the natural carboxylic polyether antibiotic A23187, whose chemical structure is shown in Figure 1. This molecule selectively f o m a complex with Mg2+ or Ca2+ (21, 22). In this case, the fluorescence intensity around 430 nm decreases because of the following complex formation (23): 2LH
+ M2+
2ML2
+ H+
(1)
(L: A23187; M2+: Mg2+ or Ca2+) not only are A23187 and its complex insoluble in water because of their lipophilicity, but also the complex formation and decomposition can be controlled reversibly according to the pH of the aqueous solution contacting the molecule. These characteristics are advantageous factors for applying this molecule to an optical chemical sensor for Mg2+ or Ca2+.
EXPERIMENTAL SECTION The composition of the ion-sensing membrane is 4 w t % A23187 (Sigma Chemical Co., St. Louis, MO) with 71 wt % dibutyl sebacate (DBS;Tokyo Chemical Industry Co., Ltd., Tokyo) as the membrane solvent and 25 wt % poly(viny1chloride) (PVC; p9526, high molecular weight type, Sigma Chemical Co.). These compounds were dissolved in 10 times their weight tetrahydrofuran (THF) followed by casting the film on a Teflon board and drying overnight at room temperature. The thickness of the prepared membrane was 14 2 rm. In Figure 2 a schematic view of the optical sensor probe, is shown which is composed of two plastic optical fibers (0.d. 3 mm; ESKA, Mitsubishi Rayon Co., Ltd., Tokyo) and the prepared PVC matrix sensing membrane incorporating A23187. The optical fibers were cut obliquely along a 15-mm length from the fiber end. The cut end and fiber end were polished with emery paper (CC-1000-Cw;Sankyo Co., Ltd., Tokyo) and emery cloth. The two fibers were completely joined together with acetone. The ion-sensing PVC matrix membrane was fixed to a PVC tube having an outer diameter of 3.5 mm (i.d. 3.0 mm) with THF and placed on the tip of the two-optical-fiber bundle. To measure fluorescence spectra, one of the two optical fibers was connected to the window for a light source (150-W
*
0003-2700/89/036 1-0382$01.50/0
UV-Xe lamp, Wacom R&D Corp., Tokyo), and the other one to the window as the detector of a spectrofluorometer equipped with double monochromators (FP-550,Japan Spectroscopic Co., Ltd., Tokyo). The slith widths of the monochromators for obtaining the excitation and emission spectra were set 3 and 5 nm, respectively. To avoid influence from surrounding lights, the measurements were performed in a dark box in which the inside temperature was controlled at 25 2 O C . All containers used for ion measurements were dark brown, and the sample solution was stirred constantly during the measurements. All ion sample solutions (30 mL) were cation chlorides prepared with 0.05 M Tris/HCl (pH 7.0) unless otherwise stated. An HC1 solution of pH 3 was used as the washing solution for the sensing membrane.
*
RESULTS AND DISCUSSION Maximum excitation and emission (fluorescence) wavelengths in the UV-vis spectrum of A23187 (solvent: ethanol) were reported to be 380 and 437 nm, respectively (23). The maximum excitation wavelength of the prepared sensor based on plastic fibers and the PVC matrix membrane containing A23187 was 380.0 f 1.5 nm. The fluorescence spectra obtained with the sensor ranging from 350 to 600 nm are shown in Figure 3. In Figure 3, the four spectra correspond to the case where the sample contains 1 x lo*, 1 X lo4, and 1 X M Ca2+and nothing (blank; 0.05 M Tris/HCl (pH 7) only). The spectra decrease with decreasing concentration of Ca2+. In this case, the maximum fluorescence wavelength was 435 f 2.5 nm (427 f 2.5 nm at 1 X M Ca2+),and this wavelength (435 nm) was fixed in subsequent measurements. The pK, of A23187 has been reported to be 6.9 (24) (DMSO; 6.7 (%), 6.4 (26), 65 wt % methanol in water), and the complex formation of A23187 with an alkaline-earth-metal ion such as a Ca2+hardly occurs a t a pH below 3. The variation in fluorescence intensity of the optical sensor due to pH of the sample solution is shown in Figure 4. The maximum fluorescence intensity decreased in the range of pH 6.8-7.5 in the presence of Ca2+ (5 X lo4 M in Figure 4), and the intensity reached a maximum around pH 4. No intensity change was observed in the range of pH 4-2.5. In this region, the complex formation of A23187 with Ca2+apparently did not occur. Thus, the ion measurements have been performed with the solution adjusted to pH 7 (0.05 M Tris/HCl), and in the case of changing samples, the sensor membrane was washed with an HCl solution of pH 3. Alkaline solution over pH 8 has not been tested because of the precipitate formation of the hydroxide of the alkaline-earth-metal ion. In addition, the fluorescence of A23187 is attributable to the protonated ligand (LH; L:A23187). The anion form (L-) also decreases 0 1989 American Chemical Society
