Anal. Chem. 1994,66, 3102-3108
Effect of Solution Ionic Strength on Analyte Charge State Distributions in Positive and Negative Ion Electrospray Mass Spectrometry Guangdi Wang and Richard B. Cole' Department of Chemistryl University of New Orleans, Lakefront, New Orleans, Louisiana 70 148 The variability of ESMS charge state distributions was investigated for a number of analytes when the ionic strength of their solutions was altered over a wide range. Solution ionic strength was adjusted by adding varying amounts of electrolytes (NHdOAc, CsCl, or (mCgH9)4N+Cl-) to protein solutions of myoglobin and lysozyme. Measured solution pH values were found to be nearly constant over the entire range of CsCl and (+CJ49)4N+Cl- addition, and a slight increase in pH was observed upon NH40Ac addition. Analyte peak intensities in positive ion ESMS reproducibly decreased as the solution ionic strength increased. However, analyte charge state distributions remained quite constant despite 4 orders of magnitudevariation in electrolyte concentration. In negative ion ESMS of phenolphthalein diphosphate (free acid form) and eosin Y, analyte charge state distributions also exhibited only minor variations while signal intensity decreased dramatically with increasingsolutionionic strength upon addition of CsCl. These observations in positive and negative ion ESMS provide evidence that "spectator" electrolytes present in solution have no significant effect upon the degree of multiple charging. The factors which determine the distribution of multiply charged analyte ions in the gas phase are thus independent of certain others which influence the efficiency of production of gasphase analyte ions. In the context of theoretical descriptions of the electrospray process, the rate constants for desorption of multiply charged ions into the gas phase described in ion evaporation theory are likely to be invariant with spectator electrolyte concentration. Alternatively, the presence of added ion pairs in the ultimate droplet produced according to single ion in a droplet theory does not alter the level of charge attachment/charge separation of analyte molecules. Charge state distributions of analytes in electrospray ionization mass spectrometry (ESMS) have been the subject of fundamental studies which seek to elucidate the multiprocess electrospray ionization mechanism.1-12 Protein molecules acquiring several to more than 100 charges (via proton
attachment) are found to exhibit various profiles of charge state distributions as a function of protein conformation,'** solution P H , solvent ~ compo~ition,~ instrumental condition^,^ temperature: presence of detergent,' and limitingly, the number of basic or acidic sites.8 These previous studies have revealed some important clues to guide ESMS applications which probe the structures of biopolymers, as well as clues to the understanding of fundamental processes inherent to electrospray ionization. Many aspects of the electrospray process, however, remain unresolved. For example, the ES mass spectrum is believed to be representative of the ions already present in the solution subjected to electrospray condition^.^ Yet in both positive and negative ion ESMS, the solution pH of proteins was not determinant to the formation of multiply charged proteins,1° indicating more complexity to the process including the possibility of acidity enhancement in the charged dr0p1ets.l~It was also found that, for smaller peptides, analyte charge state distributions in ESMS do not reflect proportionally the equilibrium distributions of protonation in the solution phase." For analytes forming only singly charged ions in solution, the dependence of ion abundance on electrolyte concentration has been studied by Kebarle and co-workers.14-18 It was proposed16 that, for a two-electrolyte system, the mass spectrometrically detected ion abundance may be expressed as
where I A + , is~ the ~ ion current of analyte A+ detected by the mass spectrometer, p is a constant expressing the efficiency of the mass spectrometer for detecting gas-phase ions produced in the ES ion source,fis the fraction of droplet charge converted to gas-phase ions, [A+] and [B+] are the electrolyte concentrations initially present in the electrosprayed solution, kA+ and k ~ are + the rate constants expressing the rate of transfer
(1) Katta, V.; Chait, B. T. Rapid Commun. Mass Spectrom. 1991, 5, 214-217.
(2) Loo, J. A.; Loo, Ogorzalek, R.R.; Udseth, H. R.; Edmonds, C. G.; Smith, R. D. Rapid Commun. Mass Spectrom. 1991, 5, 101-105. (3) Chowdhury, S. K.; Katta, V.;Chait, B. T., J. Am. Chem. SOC.1990, 112, 9012-901 3. (4) Edmonds, C. G.; Loo, J. A,; Barinaga, C. J.; Udseth, H. R.; Smith, R. D. J. Chromatogr. 1989, 474, 21-39. ( 5 ) Ashton, D. S.; Beddel, C. R.; Cooper, D. J.; Green, B. N.; Oliver, R. W. A. Org. Moss Spectrom. 1993, 28, 579-583. (6) Mirza, U. A.; Cohen, S. L.;Chait, B. T. Anal. Chem. 1993, 65, 1-6. (7) Vorm,O.;Chait, B.T.;Rocpstorff,P.Proceedingsofthe4IstASMSConference on MassSnectrometrv and Allied Tonics. San Francisco. CA, 1993; DD .. 621a62 1b. ( 8 ) Smith, R. D.; Loo, J. A.; Ogorzalek Loo, R. R.; Busman, M. Mass Specrrom. Rev. 1991, 10, 359-451.
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Analyrical Chemlstty, Vol. 66, No. 21, November 1, 1994
(9) Guevremont, R.; Siu, K. W. M.; Le Blanc, C. Y.; Berman, S . S . J. Am. SOC. Mass Spectrom. 1992, 3, 216-224. (10) Kelly, M. A.; Vestling, M. M.; Fenselau, C. C. Org. Mass Spectrom. 1992, 27, 1143-1147. (11) Wang. G.; Cole, R. B. Org. Mass Specrrom. 1994, 29, 419-427. (12) Cole, R. B.; Harrata, A. K. Rapid Commun. Mass Specrrom. 1992,6, 536539. (13) Gatlin, C. L.;Turecek, F. Anal. Chem. 1994, 66, 712-718. (14) Ikonomou, M. G.; Blades, A. T., Kebarle, P. Anal. Chem. 1990,62,957-967. (15) Ikonomou, M. G.; Blades, A. T., Kebarle, P. Anal. Chem. 1991, 63, 19891998. (16) Tang, L.;Kebarle, P. Anal. Chem. 1991, 63, 2709-2715. (17) Kebarle, P.; Tang, L.Anal. Chem. 1993, 65, 972A-986A. (18) Tang, L.; Kebarle, P. Anal. Chem. 1993, 65, 3654-3668. 0003-2700/94/0366-3702$04.50/0
0 1994 American Chemical Soclety
of the respective ions from charged droplets to the gas phase, and l i s the total electrospray current leaving the ES capillary. The expression correlates well with experimental results obtained for a number of coexistinganalytes which form singly charged ions in solution.'* The fact that addition of external electrolytes significantly suppressesthe analyte signal intensity is qualitatively accounted for by eq 1. The situation for analytes that form multiply charged ions in solution may be more complicated. As shown in previous work" involving comparative studies of solution-phase protonation and charge state distributions of detected ES ions, pH-induced shifts in analyte charge state distributions are often accompanied by increases or decreases in total signal intensity (i.e., sum of all detected charge states). Furthermore, electrospray solvent effect studies in both the positive" and negativeI9 ion modes revealed that higher charge state distributions appeared with higher signal intensities (e.g., when thesolvent was methanol), whereas lower charge state distributions were accompanied by decreased total signal intensities (e.g., in butanol). Fundamental solvent properties such as dielectric constant,19 volati1ity,l1J0 and solvent basicity" have been proposed to contribute to the simultaneous variations in the charge state distributions and in the total signal intensity from multiply charged analytes in ESMS. The present report seeks to investigate the variability of analyte charge state distributions in response to varying "spectator" electrolyte concentrations, i.e., solution ions which do not participate in the process of charge attachment to analytes. Raising the concentration of electrolytes leads to higher solution conductivities, and the added ions must contribute to the establishment of the charge excess known to exist in droplets formed during the electrospray process. In addition, at higher total electrolyte concentrations, analyte activities can decrease to a considerable extent when their concentrations are held constant. In the present report, the expression derived for systems employing exclusively singly charged ions (eq 1)16will be extended to the case of multiply charged ions.
EXPERIMENTAL SECTION A Vestec-201quadrupole mass spectrometer (Vestec Corp., Houston, TX) equipped with an electrospray ionization source as described previouslyz1was used for all mass spectrometry experiments. In the positive ion mode, the electrospray needle voltage was held at 2-2.5 kV above ground potential. The voltage at the nozzle and the skimmer4ollimator voltage difference were kept at 300 and 6 V (minimal fragmentation conditions), respectively. In the negative ion mode, the needle and nozzle voltages and the skimmer-collimator voltage difference were held at -1.5 to -2.5 kV, -250 V, and -6 V, respectively. Sample solutions were delivered into the electrospray ion source via a syringe pump (Sage Instruments, Cambridge, MA) at a flow rate of 1.6 pL/min. The temperature in the source block was maintained at 260 f 1 OC, and the temperature in the vicinity of the electrospray needle was indicated by thermocouple to be 47 f 1 OC for each series of runs. Total electrospray ion current, I , was measured at the nozzle counterelectrode. Solution pH (19) Cole, R. B.; Hamata, A. K. J . Am. Soc. Mass Spectrom. 1993.4, 546-556. (20) Fenn, J. B. J. Am. Soc. Mass Spectrom. 1993, 4, 524-535. (21) Allen, M. A.; Vestal, M. L. J. Am. Soc. Mass Spectrom. 1992, 3, 18-26.
measurements were made with a digital ion analyzer (Orion Research, Cambridge, MA). A conductivity bridge (Model RC 16B2, Beckman Instruments Inc., Cedar Grove, NJ) was used to make solution conductance measurements. Myoglobin (horse heart), lysozyme (hen egg), and phenolphthalein diphosphate (free acid form) were purchased from Sigma Chemical Co. (St Louis, MO). Eosin Y and CsCl were from Aldrich (St. Louis, MO). All protein solutions were prepared in 1:l (v/v) MeOH/HZO solvent for positive ion ESMS. Acidic protein solutions were prepared by adding 0.1% (v/v) glacial acetic acid, whereas "neutral" protein solutions contained no added acid. For negative ion ESMS experiments, phenolphthalein diphosphate and eosin Y were dissolved in pure methanol (HPLC grade). Varying amounts of electrolytes ( N H ~ O A CCsC1, , or (n-C4H&N+Br) were introduced into analyte solutions without changing the analyte concentrations.
RESULTS AND DISCUSSION Proteins are known to exhibit different charge state distribution profiles in electrospray mass spectra acquired on instruments having different configurations, or even when conditions change on the same instrument. For example, the highest number of charges carried by myoglobin was different (+25 and +21) in two different runs several months apart in this laboratory, while a maximum charge state of +29 was reported8 on another instrument in a different laboratory. However, highly reproducible results can be obtained if experiments arecarried out within a relatively short time period (e.g., 1 day) under constant experimental conditions. For this reason, each complete series of solutions containing analyte and various electrolyte concentrations was run in less than 1 h. Addition of strong "spectator" electrolytes (i.e., those which do not participate in the acid-base equilibria of the analyte) to raise the ionic strength of solutions undergoing ESMS resulted in significant suppression of competing ion signals comprising the total electrospray current. These added electrolytes must contribute significantly to the total excess charge in the droplets. Hence, one might expect the concentration of electrolyte to have an important effect not only upon the signal intensity but also upon the analyte charge state distribution because the distribution of charge carriers has changed. To quantify the charge state distribution of the analyte, the term 2,the "average" charge state of analyte A detected by the mass spectrometer was introduced and defined as
where ZAi+,ms is the mass spectrometrically detected signal of analyte ion A in a given charge state and i is the number of charges that the ion carries. Experimental Z values were thus calculated based on the abundances of the individual multiply charged ions arriving at the detector as measured in ES mass spectra. The obtained 2 values are systematically biased slightly toward higher average charge states relative to the average charge states of ions produced in the ion source due to the transmission bias of the quadrupole analyzer. Although conditionswere chosen to minimize collision-induced dissociation (CID), this bias will be compensated to some Analytical Chemism, Vol. 66,No. 21, November 1, 1994
3703
degree by the fact that any CID which does occur will cause a shift in 2 toward lower valuess due to a higher susceptibility of higher charge state ions to CID processes. Positive Ion ESMS Using Myoglobin and Lysozyme as Analytes. Figure 1 gives a comparison of ES mass spectra of 5 X 10-6 M myoglobin obtained at various CsCl concentrations. The intensity of the largest peak in the spectrum decreased from 5406 (arbitrary units) in 10" M CsCl to 282 (arbitrary units) for the same analyte concentration in 1W2 M CsCl. Despite 4 orders of magnitude change in CsCl concentration, the highest abundance analyte ion was that which corresponded to the +15 myoglobin charge state in each case. As shown in Figure 2A, the average detected charge state 2 of myoglobin remainedvirtually constant (14.01-14.70) over an even wider range (from 0 to 0.1 M) of CsCl concentrations than is shown in Figure 1. It should be noted that a minor level of binding of Cs+ to myoglobin was observed at high CsCl concentration where one Cs+ was incorporated into multiply charged analyte ions. For those low-abundance analyte ions containing Cs+ in Figure lD, Z was calculated to be 13.25. A consequence of increasing the electrolyte concentration was that the solution conductivity was raised significantly. The electrospray current I was found by Pfeifer and Hendricks22to be a weak function of the conductivity (a): I Ha" where H a n d n are constants. Because the conductivity is related to electrolyte concentration as u = Amo[Ci], I can be expressed as I H(Am0)"[Cilnwhere Amo is the limiting molar conductivity of electrolyte i and [Ci] is the concentration of electrolyte i.l63l8 In accordance with this relation, the logarithm of the total electrospray current was plotted as a function of log [Ci] in Figure 2B. The slope of the resultant line ( r = 0.957) obtained by use of our experimental data gives a value of about 0.2 for n, which compares favorably with literature values reported in the range of 0.2-0.4.16-22 The effect of electrolyte CsCl concentration on the total ion abundance (sum of myoglobin ions in all charge states) is illustrated in Figure 2C. The increasing CsCl concentration caused the abundance of each individual multiply charged myoglobin ion and therefore the total myoglobin ion abundance to diminish rapidly, whereas the abundance of Cs+ increased concurrently. This observed suppression effect of external electrolyte on multiply charged analyte signal intensity is consistent with previous results obtained for singly charged a n a 1 ~ t e s . lThis ~ behavior can be modeled by an expression analogous to eq 1, which has been adapted to the case of multiply charged ions:
-
-
'A+m
--
kA+
PASkA+[A+]
+ kA2+[AZ+]+ ... + kAI+[Ai+]+ kBt[B+]I (3)
(4) (22) Pfeifer, R. J; Hendricks, C. D. AIAA J . 1968.6, 496-502.
3704 -Analytical Chemlstry, Vol. 68, No. 21, November 1, 1994
Here, IA+,msr IA2+,ms, ...,IA",ms are the mass spectrometrically detected currents of singly charged, doubly charged, and multiply charged analyte ions, respectively. Likewise, pa+, PA2+, ...,PAI+ represent the sampling efficiencies of the mass spectrometer for analyte ions of different charge states, kA+, kA2+, ..., kAi+ represent rate constants for conversion of each ion from the charged droplets into the gas phase, and [A+], [A2+],..., [Ai+] are the concentrations of singly, doubly, and multiply charged ions in solution. All other symbols are the same as those previously defined for eq 1. It should be noted that the sampling efficiency PA(+, is expected to be different for ions of different charge states due to mass spectrometer transmission bias and differing CID susceptibilities. Equation 5 shows that the current for any individual ion at the MS detector is related inversely to the sum of the products of rate constant and concentration for each singly and multiply charged ion, including electrolyte B+. At a constant initial concentration of analyte A, the extent of suppression of IAi+,ms in the presence of B+ is dependent on (i) the concentration of Ai+, (ii) the concentration of electrolyte B+, (iii) the relative ratios of the rate constants for transfer of B+ and analyte Ai+ from charged droplets to the gas phase, and (iv) the total ion current I , which has only a weak dependence on the total electrolyte concentration. Assuming the sampling efficiency PAit, for an ion of charge state i+ to be independent of electrolyte concentration, and considering a possible decrease of the f value (fraction of droplet charge converted to gas-phase ions) at high electrolyte concentrations,18 eq 5 qualitatively accounts for the decrease in the abundance of multiply charged myoglobin ions (Figure 2C) observed as a result of increasing CsCl concentration. To rationalize the nearly constant average detected charge state values reported in Figure 2A, and recognizing f as a coefficient which is common to all desorbed ions, eqs 2 and 5 can be combined to give
Equation 6 would predict that the average detected charge state is independent of the nature and concentration of electrolyte B present in solution. It is known that the increasing ionic strength due to addition of electrolyte would affect, to some degree, the intrinsic pK values of the basic and acidic sites on the analyte. However, the change in pK values over the ionic strength range employed in this experiment is unlikely to be larger than 0.1.23 The resultant shift in pK is thus not likely to exert a large effect on the degree of solution-phase protein protonation. The rate constant kAit is dependent mainly on instrumental conditions and properties of the ionic species and thesolvent. Under the uniform ESMS parameters used in our studies, a constant value of Z is most likely indicative of a rate constant kAf+for each individual multiply (23) Tanford, C. Adu. Protein Chem. 1962, 17, 69-165.
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Figure 2. For myoglobin dissolved in 50:50 H20/MeOHwith 0.1 % HOAc (v/v) at a concentrationof 5 X M: (A) semiiogartthmic plot of myoglobinaverage detected chargestate (Z) vs CsCi concentration: (B) log-log plot of measuredES current I v s CsCi concentration where the line was generated by a least-square linear regresslon (four data points), with r = 0.957; (C) log-log plot of the total ion abundance of myoglobin(0)and the ion abundance of Cs+(A) vs CsCi concentration. 100
500
1000
1500
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D
Table 1. Effect of NHlOAc Concentratlon on the Average Detected Charge State (2) and Signal Intenrlty of 5 X lod M Myoglobin intensity (arbitrary units) NH40Ac concn (M) average Z highest peak total peak NH4+ peak 0
1W 10-5
10-4 10-3
1 0-2 lo-' 3 X 10-' I
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14.46 14.48 14.18 14.50 14.65 14.25 14.91 14.05
5066 3050 3400 4015 4150 1973 576 182
3.5 x 104 1.76 x 104 2.21 x 104 2.55 x 104 2.73 x 104 1.28 x 104 3.56 x 103 1.02 x 103
0 50 130 100 150 180 450 750
. ion0
11lh
Figure 1. Positive ion electrospray mass spectra of myoglobin (5 X M) dissolved in 5050 H20/MeOHwith 0.1 % HOAc (vlv) wlth (A) lo4, (B) lo4, (C) and (D) M CsCI.
charged ion that is invariant over the entire range of CsCl addition. Also noteworthy is the detection of Cs+(CsCl) cluster ions at low resolution in Figure 1C,D. This observation contrasts with previously reported findings1"'* of a complete absence of alkali halide cluster ions of any size obtained on another instrument. The presence of such cluster ions is taken as supportive evidence for the existence of a final droplet stage where positively charged ions and ion-pairedelectrolyte coexist.
Addition of another electrolyte NH40Ac to myoglobin solutions gave similar results as summarized in Table 1. As the concentration of NH40Ac was raised from to 0.3 M, the average detected charge state ( Z ) of myoglobin exhibited only minor fluctuations (between 14.05 and 14.91). It should also be noted that the pH of the myoglobin solutions increased slightly as the NH40Ac concentration was raised. Again, if eq 6 can be used to rationalize the observation of constant Z values, it is then implied that the ratio C i p ~ t + k ~ t + [ A ~ + ] / & ~ i + k ~ t + [ A ~remained +] virtually constant despite widely varying solution ionic strengths. It thus appears that both the solution equilibrium distribution of protonation and the rate constant for transfer of multiply charged ions into the gas Analytical Chemistry, Vol. 66, No. 21, November 1. 1994
3705
18
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j
,
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o
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Figure 3. For myoglobin dissolved in 50:50 H20/MeOH wlth 0.1 % HOAC(vlv) at a concentration of 5 X loa M: (A) semilogarithmic plot of myoglobin average detected charge state (Z)vs concentration of (rrC,He)4N+CI-; (8)log-log plot of total ton abundance of myoglobin (0)and the ion abundance of (rrC4He)4N+(A)vs concentration of (rrC,Hg)4N+CI-.
o
1 0 ' ~ lod 1c3 io-2 io-1 Concentration of CsCl (M)
ioo
Flgue 4. (A) Sembgarlthmic plot of 1odM lysozymeaveragedetected charge state (Z)in neutral ( 0 )and acidic ( ) solutbns vs concentration of CsCI; (6)log-log plot of the total Ion abundance of lysozyme (0) and the Ion abundance of Cs+ (0)In neutral solutlons and the total Ion abundance of lysozyme (+) and the ion abundanceof Cs+ ( 0 )In acMlc solutions vs concentration of CsCI.
+
phase did not change appreciably with increasing solution has been calculated to be about 1 order of magnitude higher ionic strength. than that of Cs+ due to its low solvation energy and its high Unlike the case of CsCl, the decrease of total ion abundance surface activity.18 As in the previous examples, the calculated upon addition of NH40Ac was not obvious until the con2 stayed more or less constant over the entire range of added centrationexceeded 10-3 M. If the rateconstant kat for NH4+ salt concentrations (Figure 3A). In addition, a sharp decrease is about the same as that for Cs+ as calculated by Tang and of myoglobin intensity did not occur until the concentration Kebarle,16J8then according toeq 5 , one would expect a similar of Bu4N+C1- went above M, where the suppression of degree of analyte suppression from equimolar quantities of analyte signal was more severe than in the case of CsCl at CsCl and NH40Ac. It is possible that the rate constant kNH,+ equivalent concentrations. is somewhatsmaller than kc,+under the employed instrumental To test whether these observations were also applicable to conditions, as evidenced by the observation that INZt+,m is other proteins, lysozyme was chosen as a second test analyte. much lower than for solutions of equal concentration. Figure 4 shows the effect of CsCl addition on the average Alternatively, the increased conductivity due to addition of charge state distribution for neutral and acidic lysozyme B+ (not the same value for Cs+ and NH4+) may contribute solutions. For the neutral lysozyme solution, addition of CsCl to the suppression of analyte signal, which is not reflected in up to a concentration of 10-* M results in a sharp decrease eq 5 . Measurement of solution conductivity, in fact, demof total ion abundance (more than a factor of 130), while for onstrated that addition of CsCl gave rise to a higher acidic solutions CZ,p,msdecreased even more dramatically (a conductivity than addition of an equimolar quantity of NH4factor of 330) from 1.24 X lo4 to 38 (arbitrary units) in OAc. Increasing the solution conductivity has been s h o ~ n * ~ . ~response ~ to CsCl addition (Figure 4B). The average detected to improve the nebulization process that leads to the production charge state Z , on the other hand, showed only minor of smaller size droplets. The efficiency of electrophoretic fluctuations in both cases (Figure 4A). Upon addition of charge separation, however, drops drastically at higher 10-5-10-2 M NH40Ac to either neutral or acidic solutions, conductivitie~,~~ which may be responsible for the breakdown the total ion abundance cIAi+,ms of lysozyme did not decrease of ESMS performance and total loss of analyte signal intensity as steeply (Figure 5B) as in the case of CsCl. In this same from solutions containing greater than 0.3 M NH40Ac. concentration range, the average detected charge state Similar breakdown occurred from solutions containing CsCl exhibited a slight yet steady decrease from 8.61 to 8.15 for at concentrations greater than 0.1 M. Finally, it is also neutral solution, and from 9.36 to 8.36 for acidic solution conceivable that the volatility of NH40Ac may alleviate, to (Figure 5A). some degree, analyte signal suppression as compared to CsCl. The difference in suppression of lysozyme signal intensity The effect of external electrolyte on myoglobin charge state by CsCl and NH40Ac can be rationalized on the basis of kg+ distribution and signal intensity was tested using another salt, differences (lower kB+ for the latter electrolyte) in a manner tetrabutylammonium chloride (Bu4N+Cl-) as the spectator analogous to the previously discussed case of myoglobin. The electrolyte (Figure 3). Bu4N+ has a rate constant, kBt, which observed subtle shift of Z may be rationalized in light of the noticeable changes in solution pH which indicate that NH4(24) Smith, D.P. H.IEEE Trans. I d . Appl. 1986, 22, 527-535. 3700 Analytical Chemistry, Vol. 66, No. 21, November 1, 1994
1.8
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Flgure 5. (A) Semilogarlthmlcplotof 1od M lysozyme average detected charge state (4 Inneutral( 0)and acidic ( ) solutions vs concentratlon of NH40Ac:(6) log-log plot of the total ion abundance of lysozyme in neutral (0)and acidic (A)solutions vs concentration of NH40Ac. 0 HO-I-OH
, /,,,,,,,
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,
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,
, , , , I , , ,
10-3 i o 2 10’1 Concentration of CsCl (M)
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Flgure 7. (A) Semllogarkhmlc plot of the average detected charge state (Z)of phenolphthalein diphosphate (e)and eosin Y (0)vs CsCl concentration: (6) log-log plot of the total ion abundance of PPD (0) and eosln Y (A)vs CsCl concentration. The concentratlonsof PPD and eosin Y were fixed at M in methanol.
PPD (phenolphthalein diphosphate)
0
c
0 c
0
200
-41 o
400
300
500
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Figure 6. Negative ion electrospray mass spectrum of phenolphthalein diphosphate.
loJ M
OAc is not a true “spectator”. In the acidic solutions, as NH40Ac concentration was raised from to M, an increase of pH from 4.1 1 to 5.31 was observed, while addition of CsCl virtually did not affect pH. The pH increase is likely to contribute to the slight but reproducible decreases of the average detected charge state upon addition of NH40Ac to both acidic and neutral lysozyme solutions. Negative Ion ESMS Using Phenolphthalein Diphosphate andEosinY as Analytes. Having shownresultsoflargeprotein molecules in positive ion ESMS, we now turn to smaller molecules forming doubly charged negative ions which do not undergo conformational changes with pH. These analytes contain fixed ionic functional groups that offer improved ion statistics in the negative ion mode, which is prone to problems of in~tability.’2.~~ Figure 6 shows the structure and negative ion ES mass spectrum of phenolphthalein diphosphate (PPD). PPD shows a doubly charged ion (A2-) peak at m/z 238 and a singly charged ion (A-) peak at m / z 477 via deprotonation (25) Wampler, F. M.; Blades, A. T.;Kebarle, P. J. Am. Soc. Muss Spectrom. 1993, 4, 289-295. (26)Varghese, J.; Cole, R. B. J . Chromatogr. 1993, 639, 303-316.
in pure methanol solvent. The additional peaks presumably arise from impurities. The ES mass spectrum of eosin Y has been shown previously,26with doubly charged eosin Y detected at m / z 323, and singly charged eosin Y at m / z 647 (thearyloxy anion is protonated). No triply charged ions were observed for either analyte. In calculating the average charge state distributions based on the mass spectrometrically detected current of negative ions of the analytes PPD and eosin Y, because only singly and doubly charged species were observed, eqs 3, 4, and 6 were simplified and adapted to negative ion detection:
The effects of increasing the electrolyte (CsCl) concentration (in M PPD methanol solutions) on the average detected charge state and total ion abundance are illustrated in Figure 7. The 2 values remained quite constant as the concentration of CsCl in PPD solution increased from l P 5to lCV3 M. Analogous to the previous protein examples, this is in agreement with eq 9, which models 2 as being independent of ionic strength (or total electrolyte concentration). The observed constancy of the average detected charge state implies that, similar to the proteins, the rate constants kA- and kA2for ion desorption from the droplets to the gas phase are independent of changes in electrolyte concentration. MeanAnalytical Chemistry, Vol. $6,No. 21, November 1, 1994
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while, the total intensity of PPD ( Z A - ~+ Z A Z ~ )decreased dramatically from 2150 to 35 (arbitrary units) as a result of increasing the CsCl concentration from to l e 3 M. In fact, above 10-3M CsCl there was no detectable analytesignal. The disappearance of analyte signal can be partially rationalized by the model set forth in eqs 7 and 8. Notably, C1- was observed as a stable signal only when the CsCl concentration was very high, and even then the intensity was rather low compared to Cs+ in positive ion ESMS experiments. Similar results were obtained for eosin Y when the CsCl concentration was increased from 10-5 to M (Figure 7). The average detected charge state showed no significant change (1.1 11.16), while the total ion abundance ( Z A - , ~+~ Z A Z - , ~ )decreased from 1892 to 57 (arbitrary units). These observations in negative ion ESMS confirm that high total electrolyte concentrations severely suppress anionic analyte signal intensity and lead to the breakdown of the electrospray ionization process. As long as signal was detected, however, the average charge state distributions of analytes observed in ESMS remained largely unaffected. In considering the mechanism of ionization, negative ion ESMS is distinguished from positive ion in that the charged droplets carry an excess of negative charges consisting of dissociated analyte and other negatively charged species such as OH-, CH3O-, etc., which are likely to be located near the droplet surface. When CsCl concentration is increased in the bulk solution, there would be a net increaseof C1-concentration in the charged droplets, hence an increased proportion of C1desorption which leads to the observed suppression of analyte signal. The fact that the charge state distributions remained rather constant suggests that the degree of analyte dissociation in the droplet and the relative efficiency of conversion of doubly and singly charged analyte ions into the gas phase were largely maintained. This finding supports the notion previously brought forth in negative ion work in our 1aborat0ry'~J~ that fundamental solvent properties (such as the dielectric constant) are more critical to determining the charge state distribution in negative ion ESMS than solution parameters such as conductivity.
CONCLUSION Addition of spectator electrolytes to analyte solutions was found to have an insignificant effect upon analyte charge state distributions observed in positive and negative ion ESMS. These observations indicate that an increased concentration of ionic species which do not participate in the association/ dissociation equilibria of the analyte does not significantly influence the process of charge attachment or departure leading up to production of multiply charged ions. In both the positive and negative ion modes, the total abundance of singly and multiply charged analyte ions was suppressed dramatically in the presence of increasing concentrations of electrolyte, which is consistent with previous ESMS studies16.18of monocationic (27) Iribarne, J. V.; Thomson, 9. A. J . Chem. Phys. 1976,64, 2287-2294. (28) Thomson, 9. A.; Iribarne, J . V. J . Chem. Phys. 1979, 71, 4451-4463. (29) Dole, M.; Mack, L. L.; Hines, R. L.; Mobley, R. C.; Fcrguson, L. D.; Alice, M.9. J . Chem. Phys. 1968, 49, 2240-2249.
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AnalyticalChemlstry, Vol. 66, No. 21, November 1, 1994
analytes. The detected abundance of each multiply charged ion appears to follow a dependence on ESMS parameters similar to that of singly charged ions. The fact that the degree of multiple charging in the gas phase was found to be independent of electrolyte concentration and accompanying changes in desorbed ion abundance is a new finding to be placed in context with previous results showing substantial shifts in charge state distributions with varying solvent composition4J9 and P H . ~ J ' The foregoing observations in positive and negative ion ESMS and the model given in eqs 5 and 8 for multiply charged cationic and anionic analyte systems, respectively,may provide further insight into the mechanism of formation of multiply charged ions. Some models of the positive ion electrospray process might depict the analyte charge state distribution as being largely dependent on the level of excess protons located near the droplet surface. At higher spectator electrolyte concentrations, protons constitute an increasingly minor portion of the excess charge in generated droplets. In fact, Cs+ has a lower solvation energy than H+, which may further contribute to an increasing proportion of Cs+ near the droplet surface. As a result, protein molecules might be expected to attach fewer protons and exhibit an overall shift of analyte charge states toward lower values, yet in our experiments, this was not observed. This finding implies that a change in the cationic species comprising the charge excess in droplets does not significantly influence the charge state distribution of protonated analyte species (although introduced cations may themselves participate in charge attachment to analyte molecules). A further conclusion which sheds additional light on an ambiguity in a previous paper' is that a larger absolute number of solution-phase anions (created by introducing higher concentrations of ion-paired spectator species) does not cause the desorption profile of analyte species to shift toward lower charge states (fewer protons attached to analyte molecules). Kinetic and equilibrium considerations governing proton attachment and departure from analyte molecules during the electrospray process thus are evidently not perturbed by changes in the solution conductivity. If these results are considered while bearing in mind the tenets of ion evaporation t h e ~ r y , ~the ~ Jrate ~ ~constants ~* for desorption of each multiply charged analyte ion from droplets into the gas phase, kAt+ and k ~ t - appear , to remain constant despite increasing solution ionic strength. On the other hand, if single ion in a droplet theory17J9 is maintained, all multiply charged analyte ions would ultimately originate from droplets where external electrolyte was entirely ion-paired. Hence, the lack of shift in analyte charge state distribution with increasing solution ionic strength would be attributable to similar droplet environments which differed only in the number of ion-paired salt molecules present.
ACKNOWLEDGMENT Financial support for this research was provided by the Louisiana Education Quality Support Fund through Grant LEQSF(1991-94)-RD-A-36. Received for review April 4, 1994. Accepted July 12, 1994.' *Abstract published in Advance ACS Abstracts, September 15, 1994.
