Electrospray Ionization of Protein Mixtures at Low pH - Analytical

That is, when the data are plotted in terms of the concentration of charge sites, .... Analysis of protein mixtures by electrospray mass spectrometry:...
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Anal. Chem. 2003, 75, 1491-1499

Electrospray Ionization of Protein Mixtures at Low pH Peng Pan and Scott A. McLuckey*

Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-1393

Solutions composed of single proteins and mixtures of proteins are subjected to electrospray ionization to study the influence of protein components on the responses of one another. Protein matrix effects in electrospray ionization are particularly relevant to the development of topdown protein identification methodologies involving protein mixtures, whereby whole protein ions are subjected to tandem mass spectrometry. Emphasis is placed largely on solutions composed of equal parts methanol and water and 1% acetic acid. The results, therefore, are relevant to low-pH solutions with significant organic content, a commonly used set of conditions in electrospray ionization mass spectrometry that tends to denature proteins. Under these conditions, very similar response curves are measured for a variety of proteins after charge normalization. That is, when the data are plotted in terms of the concentration of charge sites, rather than in terms of the concentration of protein molecules, the slopes of the response curves as well as the point at which response becomes less than linear with concentration are similar. Charge normalization is made on the basis of the weighted average charge of a protein, as reflected in the electrospray ionization mass spectrum. When proteins can be regarded as a collection of equivalent charge sites, the signal response from one protein can be used to predict the responses for other proteins. Furthermore, it is also possible to predict the dependence of the signal response for a particular protein in a mixture on the concentration of other proteins in the mixture. Examining signal response on a weighted average charge basis appears to be an effective means for identifying situations in which the protein does not behave as a collection of equivalent charge sites.

The applicability of any ionization method to a mixture of analyte components is determined, in part, by the extent to which the ionization of the individual components is influenced by the presence of the other mixture components (i.e., matrix effects). Electrospray ionization (ESI)1-5 has typically not been applied to * Corresponding author, Phone: (765)494-5270. Fax: (765)494-0239. Email: [email protected]. (1) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71. (2) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Mass Spectrom. Rev. 1990, 9, 37-70. 10.1021/ac020637w CCC: $25.00 Published on Web 02/15/2003

© 2003 American Chemical Society

the ionization of complex protein mixtures due to the multiplecharging phenomenon and the consequent spectral congestion that arises when the charge-state distributions of many proteins overlap in mass-to-charge ratio. For most mass spectrometers, the charge-state overlap problem has been the limiting factor in determining the number of proteins that can be subjected to the ESI simultaneously and subsequently analyzed. This problem, however, can be ameliorated by reducing protein ion charge states via ion/ion reactions6-11 or by use of high resolving power. The ability to subject multiple proteins to ESI simultaneously is of interest from the standpoint of “top-down” approaches to protein identification and characterization.12-16 It is desirable to be able to identify and characterize proteins present in complex mixtures with minimal recourse to separations prior to mass spectrometry. The mixture complexity amenable to mass spectrometry can be limited by the capacity of the mass analyzer to resolve ions derived from the mixture or by the capacity of the ionization method to impart charge to all of the analyte molecules. The former has been addressed by high resolving power mass spectrometry or by use of ion/ion reactions for protein ion charge-state manipulation. The latter has received relatively little attention, although the issue has been raised within the context of the direct top-down analysis of complex protein mixtures using ESI.17 Studies directly focused on matrix effects in ESI have involved study of the ionization of (3) Smith, R. D.; Loo, J. A.; Edmonds, C. G.; Baringa, C. J.; Udseth, H. R. Anal. Chem. 1990, 62, 882-899. (4) Smith, R. D.; Loo, J. A.; Ogorzalek Loo, R. R.; Busman, M.; Udseth, H. R. Mass Spectrom. Rev. 1991, 10, 359-451. (5) Cech, N. B., Enke, C. G. Mass Spectrom. Rev. 2001, 20, 362-387. (6) McLuckey, S. A.; Stephenson, J. L., Jr.; Asano, K. G. Anal. Chem. 1998, 70, 1198-1202. (7) Stephenson, J. L., Jr.; McLuckey, S. A. J. Am. Soc. Mass Spectrom. 1998, 9, 585-596. (8) Stephenson, J. L., Jr.; McLuckey, S. A. J. Am. Chem. Soc. 1996, 118, 73907397. (9) Stephenson, J. L., Jr.; McLuckey, S. A. Anal. Chem. 1998, 70, 3533-3544. (10) Scalf, M.; Westphall, M. S.; Krause, J.; Kaufman, S. L.; Smith, L. M. Science 1999, 283, 194-197. (11) Scalf, M.; Westphall, M. S.; Smith, L. M. Anal. Chem. 2000, 72, 52-60. (12) Kelleher, N. L.; Lin, H. Y.; Valaskovic, G. A.; Aaserud, D. J.; Fridriksson, E. K.; McLafferty, F. W. J. Am. Chem. Soc. 1999, 121, 806-812. (13) Mortz, E.; O’Connor, P. B.; Roepstorff, P.; Kelleher, N. L.; Wood, T. D.; McLafferty, F. W.; Mann, M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 82648267. (14) Meng, F.; Cargile, B. J.; Miller, L. M.; Forbes, A. J.; Johnson, J. R.; Kelleher, N. L. Nat. Biotechnol. 2001, 19, 952-957. (15) Reid, G. E.; Shang, H.; Hogan, J.; Lee, G. U.; McLuckey, S. A. J. Am. Chem. Soc., 2002, 124, 7353-7362. (16) Reid, G. E.; McLuckey, S. A. J. Mass Spectrom. 2002, 37, 663-675. (17) VerBerkmoes, N. C.; Bundy, J. L.; Hauser, L.; Asano, K. G.; Razumovskaya, J.; Larimer, F.; Hettich, R. L.; Stephenson, J. L., Jr. J. Proteome Res. 2002, 1, 239-252.

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relatively small molecules.18-25 However, a consensus has emerged that the main electrospray mechanisms for ionization of small molecules and large molecules (e.g., proteins) are likely to differ.18,22,26-29 Therefore, matrix effects may manifest themselves differently in the application of electrospray to mixtures of large biological molecules versus the application of electrospray to mixtures of relatively small molecules and metals. Two major mechanisms for electrospray have been identified. One is Dole’s charged residue model (CRM).30,31 In this model, evaporation of solvent molecules from a charged droplet steadily decreases its size until it reaches the Rayleigh limit. The parent droplet is broken into many offspring droplets. This sequence continues until the offspring droplets contain only one molecule, if the initial concentration in the condensed phase is sufficiently dilute. As the solvent eventually vaporizes, excess droplet charge tends to condense on the analyte. The other model, proposed by Iribarne and Thomson, is known as the ion evaporation model (IEM).32,33 In the IEM, the charged droplet commences the same solvent evaporation and Coulomb fission steps as in the CRM. However, the IEM holds that, at some intermediate stage in the droplet evolution process, it becomes favorable for an ion present on the surface to evaporate before the Rayleigh limit is reached. This process is most favored for species with relatively small solvation free energies, such as small ions and, particularly, ions derived from species with high surface activities. It is now generally accepted that both mechanisms can give rise to gaseous ions with the IEM considered to be most important for relatively small analyte species and the CRM most important for macromolecules, such as proteins, although models for protein ion evaporation have been proposed.2,18 In 1991, Tang and Kebarle proposed a model for analyte ESI response based on the assumption that the analyte ion evaporation rate from the droplets is proportional to the ion concentration in the droplet.34 The basis for this model was the assumption that the ion evaporation model was the major mechanism for the ion transfer from the droplet to the gas phase. The resulting equation is

I(A+, MS) ) IPfkA[A+]/(kB[B+] + kA[A+])

(1)

where I(A+, MS) represents the mass spectrometrically detected (18) Fenn, J. B. J. Am. Soc. Mass Spectrom. 1993, 4, 524-535. (19) Loscertales, I. G.; de la Mora, J. F. J. Chem. Phys. 1995, 103 (12), 50415060. (20) Constantopoulos, T. L.; Jackson, G. S.; Enke, C. G. J. Am. Soc. Mass Spectrom. 1999, 10, 625-634. (21) Tang, K.; Smith, R. D. J. Am. Soc. Mass Spectrom. 2001, 12, 343-347. (22) Gamero-Castano, M.; de la Mora, J. F. Anal. Chim. Acta 2000, 406, 6791. (23) Gamero-Castano, M.; de la Mora, J. F. J. Mass Spectrom. 2000, 35, 790803. (24) Tang, L.; Kebarle, P. Anal. Chem. 1993, 65, 3654-3668. (25) Enke, C. G. Anal. Chem. 1997, 69, 4885-4893. (26) de la Mora, J. F. Anal. Chim. Acta 2000, 406, 93-104. (27) Kebarle, P.; Peschke M. Anal. Chim. Acta 2000, 406, 11-35. (28) Wang, G.; Cole, R. B. Anal. Chim. Acta 2000, 406, 53-65. (29) Wang, G.; Cole, R. B. Anal. Chem. 1998, 70, 873-881. (30) Dole, M., Mack, L. L.; Hines, R. L.; Mobley, R. C.; Ferguson, L. D.; Alice, M. B. J. Chem. Phys. 1968, 49, 2240-2249. (31) Mack, L. L.; Kralik, P.; Rheude, A., Dole, M. J. Chem. Phys. 1970, 52, 49774986. (32) Iribarne, J. V.; Thomson, B. A. J. Chem. Phys. 1976, 64, 2287-2294. (33) Thomson, B. A.; Iribarne, J. V. J. Chem. Phys. 1979, 71, 4451-4463.

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ion current of analyte ion A+, f is the fraction of the droplet charge that is converted into gas-phase ions, P is the sampling efficiency of the system, ksubscript represents the ion evaporation rate of the indexed species, and the bracketed ions are the concentrations of the different ions in the electrospray solution. It is assumed that both P and f are essentially independent of the nature of the ions. In this model, the total ion current, I, will be apportioned among the analyte species and solvent and the response of the individual ions depends on the ion evaporation rate constants and ion concentrations. Enke subsequently suggested that the principal phenomenon affecting the relationship between the solution composition and the relative abundance of ions in the mass spectrum is the partitioning of the ionic species between the two phases that are part of the charged droplet system.25 These phases are the interior, solvated, ion-paired phase and the surface excess charge phase. The relative ion abundance observed is proportional to the relative concentration of the ion in the surface excess charge phase. The relative concentration in the surface excess charge phase can be quantitatively predicted by the following equilibrium-based model:

RA ) PfCAKA[Q]/(CAKA + CEKE)

(2)

where the use of P and f follows the convention of Kebarle, KA and KE are the partition coefficients for analyte and electrolyte, respectively, [Q] is the concentration of the excess charge, CA is the analyte concentration, and CE is the electrolyte concentration. On the basis of this equilibrium partition model, Cech and Enke35 suggested that analytes with the highest ESI-MS responses are those with both polar and nonpolar portions. The polar portions are necessary to facilitate ion formation, while the nonpolar portions are responsible for increasing the surface concentration of the analyte molecule on the ESI droplet. This picture, which is also based on the IEM as the major ESI mechanism, predicts that surface activity correlates with signal response in ESI mass spectra. Zhao and Cook36 have recently expanded on the partition model of Enke by considering the influences of ion pairing, surface activity, and electrophoretic migration within the charged droplets on the partition coefficient, KA. Data used to test the models described above were collected using relatively small analyte species that yield singly charged ions. On the basis of current understanding of ESI mechanisms, the ions were expected to be formed largely by ion evaporation. Hence, the models were based on the IEM, as reflected by the use of ion evaporation rate constants in the Tang and Kebarle model and the use of surface ion concentration in the Enke and Zhao and Cook models. Analyte ion evaporation is not expected to play a direct role in analyte ion formation via the charged residue mechanism, and therefore, it is not clear that analyte ion surface concentration should correlate with the responses of species formed via the charged residue mechanism. This study was undertaken, therefore, to explore the effects of analyte species on the signal responses of one another when the analyte ions might be formed by the charged residue mechanism. (34) Tang, L.; Kebarle, P. Anal. Chem. 1991, 63, 2709-2715. (35) Cech, N. B.; Enke, C. G. Anal. Chem. 2000, 72, 2717-2723. (36) Zhao, S.; Cook, K. D. J. Am. Soc. Mass Spectrom. 2001, 12, 206-214.

EXPERIMENTAL SECTION Protein and peptide samples were purchased from Sigma (St. Louis, MO). Perfluoro-1,3-dimethylcyclohexane (PDCH) was purchased from Aldrich (Milwaukee, WI). All samples were used without further purification. Working solutions were prepared daily by dilution of 1-10 mg/mL stock solutions of the proteins or peptides (prepared in a 1% acetic acid solution of 1:1 methanol/ water) to the final molar concentrations. The reported concentrations are calculated from the protein weight and are accurate to within (10%. All experiments were performed using a Hitachi (San Jose, CA) model M-8000 quadrupole ion trap mass spectrometer equipped with electrospray ionization and modified for ion introduction through the ring electrode by atmospheric sampling glow discharge ionization, as discussed in detail elsewhere.37 A Hewlett-Packard series II 1090 HPLC system was used for protein separation. All the sample solutions were injected through a 5-µL Rheodyne model 8125 microscale flow injector to keep the ESI needle position constant. All data were collected using a flow rate of 2 µL/min through an electrospray emitter made of a fusedsilica capillary with o.d. ) 197 µm and i.d. ) 98 µm (Polymicro Technologies, Phoenix, AZ). This electrospray emitter was connected with the flow injector. In the cases of the measurements involving protein mixtures, ion/ion reactions were utilized to minimize charge-state overlap problems, as has been reported previously.38,39 Separate ion sources were used to generate ions of each polarity. Anions from PDCH were formed using glow discharge while cations were formed via electrospray in the positive ion mode. Proton transfer between PDCH anions and protein cations is essentially the exclusive ion/ion reaction path. A typical experiment has a period of cation accumulation time (100-500 ms), followed by anion injection (30-100 ms), mutual storage time to allow for ion/ion reactions (100-300 ms), ejection of anions, and final mass analysis step. Anion accumulation time and ion/ion mutual storage period were adjusted to maximize the signal of the singly charged protein cations. Helium (1 mTorr) was used as bath gas. Each spectrum collected represents the average of 500-1000 individual scans. RESULTS AND DISCUSSION A variety of chemical factors can play a role in the electrospray response for a given species. These include, for example, the extent to which the molecule or atom exists as an ion in solution, the extent to which the atom or molecule is present on the surface of the droplet, the charge affinities of the species present in the gas phase, and the mechanism by which a condensed-phase ion is converted to a gas-phase ion. The mechanism of ionization is dependent, in part, upon solvation energy, which is intimately related to the chemical nature of the ion. Furthermore, the droplet fission process gives rise to small progeny droplets as well as larger parent droplets. It has been shown that uneven partitioning of species present in solution can arise from the repeated droplet (37) Reid, G. E.; Wells, J. M.; Badman, E. R.; McLuckey, S. A. Int. J. Mass Spectrom. 2003, 222, 243-258. (38) Stephenson, J. L., Jr.; McLuckey, S. A. J. Mass Spectrom. 1998, 33, 664672. (39) Stephenson, J. L.. Jr.; McLuckey, S. A. Anal. Chem. 1996, 68, 4026-4032.

fission process favoring surface-active species on the smaller progeny droplets.21,40 The majority of gas-phase ions are believed to arise from the small relatively highly charged progeny droplets.41 The dimensionality involved makes the derivation of a universal mathematical model to describe electrospray response for all mixtures under all conditions particularly challenging. Therefore, models presented to date apply to a limited subset of conditions. For example, it has been assumed that there were no gas-phase charge-transfer processes involved and that all ions were formed via ion evaporation. If large macroions are largely formed by the charged residue mechanism, it is of interest to determine the influence of macromolecules on the electrospray responses of other macromolecules. This work was motivated primarily by an interest in the influence of proteins on the electrospray responses of one another. Protein data have therefore been collected in the absence of significant concentrations of small molecules other than those of the solvent and added acid. In this study of protein matrix effects, a compromise set of electrospray and instrument operating conditions was established that tends to provide relatively good responses for a range of polypeptides/proteins including Met-Arg-Phe-Ala (MRFA), bradykinin, melittin, bovine insulin, bovine ubiquitin, bovine heart cytochrome c, chicken egg white lysozyme, equine skeletal muscle myoglobin, and serum β-lactoglobulin A. The use of 1% acetic acid in the electrospray solvent was motivated by the need to characterize protein matrix effects when virtually all protein components are expected to be positively charged in solution. This scenario maximizes the likelihood that signals from all proteins present in the mixture will be observed in the ESI mass spectrum, a desirable condition for a comprehensive top-down protein identification strategy. The decision to use electrospray at a flow rate of 2 µL/min followed from the facility with which solutions of different concentrations could be subjected to electrospray under as constant a set of conditions as possible. The use of flow injection permitted the use of constant electrospray needle position and voltage throughout a series of experiments. This avoided ambiguities that could arise from poor reproducibility associated with, for example, use of different nanospray emitters for each solution concentration or different electrospray needle positioning and voltage with each sample loading. Response curves (total signal, as determined by peak areas, versus protein concentration) were generated for each of the proteins examined in this study over a concentration range of 10-8-10-4 M in the absence of any other proteins (data not shown). As is commonly observed,18,24,25 signals were linear with concentration at concentrations less than ∼10-5 M. For all proteins studied, less than linear increases in signal with concentration were first noted at concentrations exceeding 10-5 M. The point at which nonlinearity in signal versus concentration occurred was protein dependent with larger proteins showing rollover in signal versus concentration at lower concentrations (see below). The charge-state distributions of proteins in the nonlinear region of the response curve were also concentration-dependent, as illustrated in Figure 1, which shows electrospray mass spectra of cytochrome c at 1 µM (a), 100 µM (b), and 1 mM (c). The 1 µM spectrum represents the charge-state distribution observed in the (40) Hiraoka, K. Rapid Commun. Mass Spectrom. 1992, 6, 463-468. (41) Kebarle, P.; Ho, Y. In Electrospray Ionization Mass Spectrometry; Cole, R. B., Ed.; John Wiley and Sons: New York, 1997; Chapter 1.

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Figure 1. Electrospray ionization mass spectra of cytochrome c at concentrations of (a) 1 µM, (b) 100 µM, and (c) 1 mM.

linear response region whereas the 100 µM and 1 mM spectra are examples of charge-state distributions observed in the nonlinear response region where the observed charge-state distribution is sensitive to concentration. Interestingly, in this case, two distinct charge-state distributions become apparent for highconcentration cytochrome c solutions even under the denaturing conditions used here. The lower charge-state distribution composed of (M + 8H)8+, (M + 7H)7+, and (M + 6H)6+ ions is the distribution typically observed under nondenaturing conditions (aqueous solution near pH ) 7) and is associated with the native form of the protein.42 The tendency for observation of lower charge states at high protein concentrations in electrospray mass spectrometry has been noted previously.3,43,44 Several explanations for this behavior have been offered.3,18,43,44 If the charged residue model holds for proteins, the most likely cause arises from the fact that the limited excess charge arising from protons must be distributed among an increasing quantity of protein, thereby resulting in lower average charge per protein. The fact that proteins are typically observed as multiply protonated species in positive electrospray and that the charge-state distribution can be dependent upon protein concentration gives rise to a level of complexity that is absent with small singly charged ions. To allow for a direct comparison of the behavior of species of different charge, we make a simplifying first-order approximation that each protein molecule is simply a collection of equivalent charge sites. With respect to the consumption of the excess charge associated with a charged (42) Chowdhury, S. K.; Katta, V.; Chait, B. T. J. Am. Chem. Soc. 1990, 112, 9012-9013. (43) Chowdhury, S. K.; Katta, V.; Chait, B. T. Rapid Commun. Mass Spectrom. 1990, 4, 81-87. (44) Wang, G., Cole, R. B. Anal. Chem. 1994, 66, 3702-3708.

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droplet, a protein ion with 10 charges is equivalent to 10 singly charged ions. To compare the concentration dependences of the signals from different proteins, we charge-normalize the data by multiplying the concentration of protein molecules by the weighted average charge derived from the electrospray mass spectrum, defined as qav ) ∑qiWi/∑Wi, where qi is the net charge of the ith charge state and Wi is its relative signal abundance.44-46 It is recognized that the weighted average charge determined from the electrospray mass spectrum may not reflect the weighted average charge of the ions formed prior to sampling into the mass spectrometer. The latter value is the most appropriate normalization factor to use. However, it is assumed that, in the absence of strongly basic molecules in the electrospray that may strip charge from protein ions, the use of the weighted average charge based on the mass spectrum is a reasonable approximation to the weighted average charge of the gaseous ions formed early in the electrospray process. The charge-normalized comparison shows the data in terms of the concentration of analyte charge sites rather than the concentration of analyte molecules. Using charge normalization, we find that signal saturation with concentration occurs at similar levels for all proteins studied. Figure 2 illustrates this observation for three species. Figure 2a shows plots of relative abundance versus concentration for MRFA, a singly protonated peptide and for cytochrome c and myoglobin. The proteins show rollover in signal versus concentration between concentrations of 10 and 20 µM whereas the same plot for MRFA shows rollover in signal versus concentration between the 200 and 300 µM data points. After multiplying the protein concentrations by the weighted average charges of the ions observed in the respective mass spectra, similar points of rollover are noted in the plot of relative abundance versus chargenormalized concentration, as shown in Figure 2b. Figure 2b shows the point of intersection for each analyte of the line determined from the linear signal versus concentration region and the horizontal line representing the signal saturation level. These points are observed in the range from 150 to 270 µM, and presumably represent the condition where the concentration of available charge sites is roughly equivalent to the concentration of excess charge. It is possible to estimate the point at which the concentration of available protein charge sites equals the concentration of excess charge based on approximate relationships for droplet charge and radius described by Loscertales and de la Mora.19 It is the charge concentration of the initially formed electrospray droplet that determines the upper limit to ionization. That is, the first droplet to form establishes the number of charges and number of molecules involved in the subsequent sequence of droplet fission events. Droplet radius, R, is given as18 3

R ≈ xVf/K

(3)

where Vf is the flow rate,  is the permittivity of the solvent, and (45) Iavarone, A. T.; Jurchen, J. C.; Williams, E. R. Anal. Chem. 2001, 73, 14551460. (46) Iavarone, A. T.; Williams, E. R. Int. J. Mass Spectrom. 2002, 219, 63-72. (47) Wypych, G., Ed. Handbook of Solvents; William Andrew Inc.: New York, 2001.

Figure 2. Analyte ion abundance versus concentration curves for MRFA (unfilled diamonds), cytochrome c (unfilled circles), and myoglobin (filled circles) before (a) and after (b) normalization on the basis of weighted average charge. The concentration listed in (a) is in terms of molecules per unit volume whereas the concentration listed in (b) is in terms of charges per unit volume.

K is the conductivity of the solution. Droplet charge, Q, is given as19

Q ≈ 0.7 × 8πx0γR3

(4)

where 0 is the permittivity of vacuum and γ is the surface tension of the solvent. The molar concentration of excess charge of the initially formed droplet, Cq, (that is, the moles of elementary charges per liter) is approximately given by

Cq ≈ (4.2/eN)x0γK/Vf

(5)

where e is the elementary charge and N is Avogadro’s number. For a flow rate of 2 µL/min, which applies to the data of Figure 2, and values of /0 ) 51, γ ) 0.047 N/m, and K ) 0.4 S/m, as calculated from available literature data,47,48 the molar excess charge concentration is estimated to be 150 µM. This estimate is in accord with the experimental results, which show that when the concentration of charge sites reaches the vicinity of 100-300 µM, signal levels become nonlinear with charge site concentration (48) Atkins, P. W. Physical Chemistry, 6th ed.; W. H. Freeman and Co.: New York, 1999.

Figure 3. Responses for each protein present in a four-component mixture as a function of concentration over a range for which linearity is observed before (a) and after (b) ion abundances were normalized on the basis of weighted average charge. (filled circles, myoglobin; unfilled circles, cytochrome c; filled squares, ubiquitin; unfilled squares, insulin)

regardless of whether the charge sites are distributed as one charge per molecule or multiple charges per molecule. The slopes of the response curves in the linear region are also affected by the multiple-charging phenomenon. However, on a charge site normalized basis, similar responses are observed. This is suggested in the slopes of the linear regions of the curves in Figure 2b. However, the data of Figure 2b were collected using different single-component solutions on different days and under somewhat different instrument tuning conditions. A better experiment for comparing the slopes of the response curves is to examine species under identical experimental conditions, as is the case when an equimolar mixture of analytes is examined. Figure 3a shows the responses for each protein present in a fourcomponent mixture as a function of concentration over a range for which linearity is observed. Mass spectra were collected after an ion/ion reaction period was used to simplify interpretation of the data (see Experimental Section). There is a trend showing increasing slope with increasing protein size (i.e., myoglobin > cytochrome c > ubiquitin > insulin). Figure 3b shows the response curves after charge normalization. In this case, the charge normalization was applied to the protein signals by dividing them by the weighted average charge so that the same concentration scale would apply to all proteins. The slopes for the four proteins are indistinguishable after charge normalization. This result, as well as that of Figure 2, suggests that a very simple model can approximate response versus concentration under the Analytical Chemistry, Vol. 75, No. 6, March 15, 2003

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specialized conditions used here (i.e., low pH, protein mixture components of mass of >5 kDa, high protein solubility, no other strongly basic or surface-active components at high concentration, and excess charge). In the absence of basic mixture components other than the proteins, the excess charge condition applies when the total excess surface charge, Q, greatly exceeds the sum of the charges associated with the protein mixture components, ∑Cnqav,n, where Cn symbolizes the solution concentration of a protein component and qav,n represents its weighted average charge. The response for any given protein is approximated as,

Rn ) kproteinCnqav,nE

(6)

where kprotein is a phenomenological ionization rate constant that, in the simplest case, is equal for all proteins and E is an overall efficiency of ion sampling, transmission, and detection. The kprotein term is introduced here to put response in units of charge per unit time (i.e., ion current) and to account explicitly for any chemical factors that may affect protein ionization. A number of factors are likely to play roles in determining the rate constant and they may differ, at least in part, from those that determine small ion desorption rates if the different ionization mechanisms are at work for small ions and macroions. The E term can be broken down into component parts such as fPTD, where, following the convention of Kebarle,34 f is the efficiency of converting the excess surface charge into gas-phase ions amenable to mass spectrometry and P is the efficiency of the gas-phase ion transfer into the mass spectrometer. The terms T and D account for ion transmission and detection efficiencies within the mass spectrometer. (Note that eq 6 can be rearranged as Rn/qav ) kproteinCnE, which is the relationship reflected in Figure 3b.) At high protein concentration, i.e., Cnqav,n > Q, Rn becomes independent of Cn:

Rn ) kproteinQE

(7)

That the response is often noted to decrease at high concentrations (e.g., >500 µM) may indicate a breakdown in the assumptions that kprotein or one or more of the component factors of E are constant as a function of concentration. The fact that the very simple picture represented by eq 6 applies reasonably well to the proteins studied here may be due to the relatively restricted, but widely used, set of conditions employed to collect these data (i.e., low pH, high protein solubility, etc.). Nevertheless, it is noteworthy that no significant surface activity effect is noted in the data presented for the protein mixture. Several research groups have reported the effect of analyte polarity and hydrophobicity on ESI response for relatively small molecules. In 1983, Iribarne and Dziedzic’s studies of atmospheric pressure ionization mass spectrometry indicated that the analytes with significant nonpolar portions have higher mass spectral response than highly polar analytes. They suggested that this enhanced response was a result of nonpolar analytes preferring the air-liquid interface at droplet surfaces.49 In 1993, Tang and Kebarle proposed that surface affinity could be correlated with (49) Iribarne, J. V.; Dziedzic, P. J. Int. J. Mass Spectrom. Ion Phys. 1983, 50, 331-347.

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ESI response.24,50 In a recent paper35 and a more recent review,5 Cech and Enke related ESI response to nonpolar character of small peptides. In that work, six equimolar tripeptides with different side chains were electrosprayed. The results showed that more hydrophobic side chains resulted in higher ESI responses. In a more recent paper,51 Cech et al. also predicted ESI response from chromatographic retention time. For the series of small peptides studied, higher ESI response was observed for analytes with longer reversed-phase HPLC retention times (higher hydrophobicities). If proteins have the same ESI mechanism, the same trend observed for small peptides might also be expected for proteins, i.e., proteins with higher hydrophobicities (which correlate with longer reversed-phase HPLC retention times) should have greater ESI responses. Figure 4 shows a plot of charge-normalized abundances for equimolar concentrations (5 µM) of bradykinin, insulin, ubiquitin, cytochrome c, melittin, and myoglobin versus reversed-phase HPLC retention times. The retention times of these six polypeptide/proteins increase in the order of myoglobin > melittin . cytochrome c = ubiquitin = insulin . bradykinin. Based on Enke’s studies of small peptides, the ESI responses of these six equimolar polypeptides/proteins would be expected to increase in the same order. The charge-normalized plot shows no significant dependence of signal on retention time. Under the condition of excess charge, little is expected in the way of matrix effects for mixtures composed only of proteins. Each protein is expected to behave according to the approximation of eq 6. The electrospray mass spectrum of any protein at a known concentration can be used to determine the product kproteinE in eq 6, which, in turn, can be used to predict the response for any other protein. (An accurate absolute measurement of kproteinE requires that the relationship between detector input and output be known. In the absence of this information, relative values of kproteinE can be used for a fixed set of instrument operating conditions.) As an example, Figure 5 shows both the observed response of cytochrome c over the range of 1-10 µM and the predicted response derived from the electrospray mass spectrum of a 1 µM solution of myoglobin and eq 6. The predicted and observed slopes are the same within the error of the measurement, which reflects the near equivalence of the kproteinE term for each protein. Extension to the condition of mixtures of proteins at concentrations sufficiently high that all of the excess charge is consumed by proteins is straightforward. The response for protein A is approximated as

∑C q

RA ) kproteinQECAqav,A/

n av,n

(8)

In the simple case of a two-component mixture of protein A and protein B,

RA ) kproteinQECAqav,A/(CAqav,A + CBqav,B)

(9)

If CA is kept constant while CB is increased, when CB . CA, (50) Tang, L. Kebarle, P. Anal. Chem. 1993, 65, 972A-985A. (51) Cech, N. B.; Krone, J. R.; Enke, C. G. Anal. Chem. 2001, 73, 208-213.

Figure 4. Charge-normalized abundances for equimolar concentrations (5 µM) of bradykinin, insulin, ubiquitin, cytochrome c, melittin, and myoglobin versus RP-HPLC retention time.

Figure 5. Abundance of cytochrome c measured as a function of concentration (data points) and the predicted response based upon a value of kproteinE derived from the response of a 1 µM solution of myoglobin and eq 6.

eq 9 can be simplified to

RA ≈ kproteinQECAqav,A/CBqav,B

(10)

Under these conditions, the response for protein A is inversely related to the concentration of protein B. Rearrangement of eq 10 leads to the relationship between the charge-normalized abundance of the minor component and the concentration of the major component, i.e.

RA(qav,B/qav,A) ≈ kproteinQECA/CB

(11)

Figure 6 gives results for experiments that reflect the conditions just described. It shows plots of reciprocal response versus

concentration for two different binary mixture experiments. In one case, 1 µM cytochrome c was subjected to electrospray in mixtures with myoglobin over a range of myoglobin concentrations of 1050 µM. In the other case, 1 µM myoglobin was subjected to electrospray in mixtures with cytochrome c over a range of cytochrome c concentrations of 10-50 µM. In both cases, mass spectra were collected after an ion/ion reaction period to simplify data interpretation. Figure 6a shows plots of the reciprocal total cytochrome c ion abundance derived from 1 µM cytochrome c as a function of myoglobin concentration (10-50 µM) and reciprocal total myoglobin ion abundance derived from 1 µM myoglobin as a function of cytochrome c concentration (10-50 µM). Both show the expected inverse relationship between the signal of the minor component and the concentration of the major component. When Analytical Chemistry, Vol. 75, No. 6, March 15, 2003

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Figure 6. (a) Abundances of the ions of the minor components of binary protein mixtures as a function of the concentration of the major component. (filled circles, myoglobin; unfilled circles, cytochrome c). (b) Charge-normalized abundances of the ions of the minor components of binary protein mixtures as a function of the concentration of the major component.

plotted in terms of the reciprocal of the non-charge-normalized abundance, it appears as though myoglobin has a greater influence on cytochrome c than vice versa. However, when abundance is adjusted for weighted average charge ratio, qav,B/qav,A, the difference disappears (see Figure 6b). An experiment similar to that of the binary mixture just described was conducted with a series of four protein component mixtures composed of cytochrome c, ubiquitin, and insulin each at 1 µM and myoglobin at concentrations of 10-50 µM. Mass spectra were recorded after an ion/ion reaction period to simplify spectral interpretation. Figure 7a shows plots for the reciprocal abundances of the ions derived from cytochrome c, ubiquitin, and insulin as a function of myoglobin concentration. The insulin signal is the most sensitive of the three minor protein components to changes in myoglobin concentration. However, when adjusted for weighted average charge ratio, it is apparent that all three minor components show a similar sensitivity to myoglobin concentration. The slopes for cytochrome c and insulin are the same within experimental error whereas the slope for ubiquitin is statistically different. However, in a similar experiment with a three-component mixture involving ubiquitin and insulin as the minor components and cytochrome c as the major component, the charge-normalized plots for ubiquitin and insulin showed the same slopes (data not shown). Different instrument conditions were used to conduct the experiment that gave rise to Figure 7 and the experiment 1498 Analytical Chemistry, Vol. 75, No. 6, March 15, 2003

Figure 7. (a) Abundances of the ions derived from the electrospray ionization of the minor components of quaternary protein mixtures as a function of the concentration of the major component myoglobin (unfilled squares, insulin; filled squares, ubiquitin; unfilled circles, cytochrome c) (b) Charge-normalized abundances of the ions derived from the electrospray ionization of the minor components of quaternary protein mixtures as a function of the concentration of the major component.

mentioned above, which highlights the fact that different E factors can apply to different proteins, depending upon tuning conditions. The studies related here have indicated that plotting protein electrospray data on a charge-normalized basis is a useful way to compare directly responses from different proteins. Significant deviations from “statistical” behavior (i.e., when a protein response differs from that expected on the basis of a collection of equivalent charge sites, as determined by the weighted charge average) indicate that a protein differs from others in some way. For example, we have noted in positive ion studies that will be detailed elsewhere that proteins with pI values less than the solution pH show lower responses after charge normalization than those with pI values equal to or greater than the solution pH. Presumably this observation is related to the extent to which proteins exist as cations in solution. Another example is illustrated by the data in Figure 8, which summarize post-ion/ion reaction mass spectral abundances of the components of a protein mixture in two different solvent systems. It was noted that lysozyme consistently showed a response lower than expected from 1% acetic acid 1:1 methanol/water solutions. Figure 8a summarizes the chargenormalized abundances of a six-component equimolar mixture composed of insulin, ubiquitin, cytochrome c, lysozyme, myoglobin, and β-lactoglobulin A versus protein mass at a total protein concentration of 5 µM in 1:1 methanol/water, 1% acetic acid. Based

normalized abundance versus protein mass for a nominally 99% water/1% acetic acid solution. In this case, it is apparent that lysozyme yields signals much closer to the expected result, thereby suggesting that a solvent effect, possibly associated with lysozyme solubility, is responsible for the lower than expected signal in Figure 8a.

Figure 8. (a) Charge-normalized abundances of six proteins in an equimolar mixture in 1:1 methanol/water, 1% acetic acid. (b) Chargenormalized abundances of six proteins in an equimolar mixture in 99% water/1% acetic acid.

on the average weighted charge states of the protein components, the charge site concentration is roughly 60 µM, which is less than that required for a significant protein-protein matrix effect. The relatively low lysozyme signal therefore is not likely to be due to a lack of available charge. Having ruled out an instrument discrimination effect by examining the mixture spectrum over a wide range of instrumental conditions, the mixture was analyzed under different solvent conditions. Figure 8b shows the charge-

CONCLUSIONS Evaluating protein response versus concentration on the basis of weighted average charge is a useful way to compare the responses of different proteins and to evaluate the effect of protein mixture components on the responses of one another. This very simple picture regards the protein as a collection of equivalent charge sites with the number being determined by the weighted average of the charge observed in electrospray mass spectra collected under relatively dilute conditions. At relatively low pH and under conditions in which protein solubility is high, very similar responses are observed for different proteins on a chargenormalized basis. Under these conditions, it is possible to predict the concentration-dependent response for a protein based on the response observed for a different protein. Furthermore, it is also possible to predict the effect of high concentrations of one protein on the responses of all other proteins. Interestingly, the responses of proteins observed under the conditions used in this study appear not to be related to surface concentration. However, not surprisingly, preliminary results suggest that solution pH and protein solubility can lead to significant deviations from the expected protein responses based on responses from highly soluble proteins known to be cations in solution. Plotting data on a charge-normalized basis appears to be an effective way to study the roles of these and other factors that affect the electrospray responses of proteins. ACKNOWLEDGMENT This research was sponsored by the National Institutes of Health, Grant GM 45372. Received for review October 14, 2002. Accepted January 14, 2003. AC020637W

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