Method for Distinguishing Specific from Nonspecific Protein−Ligand

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Anal. Chem. 2006, 78, 3010-3018

Method for Distinguishing Specific from Nonspecific Protein-Ligand Complexes in Nanoelectrospray Ionization Mass Spectrometry Jiangxiao Sun, Elena N. Kitova, Weijie Wang, and John S. Klassen*

Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2

A new methodology for distinguishing between specific and nonspecific protein-ligand complexes in nanoelectrospray ionization mass spectrometry (nanoES-MS) is described. The method involves the addition of an appropriate reference protein (Pref), which does not bind specifically to any of the solution components, to the nanoES solution containing the protein(s) and ligand(s) of interest. The occurrence of nonspecific protein-ligand binding is monitored by the appearance of nonspecific (Pref + ligand) complexes in the nanoES mass spectrum. Furthermore, the fraction of Pref undergoing nonspecific ligand binding provides a quantitative measure of the contribution of nonspecific binding to the measured intensities of protein and specific protein-ligand complexes. As a result, errors introduced into protein-ligand association constants, Kassoc, as determined with nanoESMS, by nonspecific ligand binding can be corrected. The principal assumptions on which this methodology is based, namely, that the fraction of proteins and protein complexes that engage in nonspecific ligand binding during the nanoES process is determined by the number of free ligand molecules in the offspring droplets leading to gaseous ions and is independent of the size and structure of the protein or protein complex, are shown to be generally valid. The application of the method for the determination of Kassoc for two protein-carbohydrate complexes, under conditions where nonspecific ligand binding is prevalent, is demonstrated. Electrospray ionization mass spectrometry (ES-MS), with its speed, sensitivity, and specificity, is a powerful analytical tool for studying noncovalent biological complexes, such as proteinligand and multiprotein complexes, under physiological or nearphysiological conditions. The ES-MS technique allows for the detection of specific biological complexes in buffered aqueous solutions, the direct determination of their binding stoichiometry,1-3 and the real-time monitoring of reaction dynamics4,5 (e.g., assembly/disassembly of protein complexes). ES-MS also holds * To whom correspondence should be addressed. E-mail: john.klassen@ ualberta.ca. (1) Daniel, J. M.; Friess, S. D.; Rajagopalan, S.; Wendt, S.; Zenobi, R. Int. J. Mass Spectrom. 2002, 216, 1-27. (2) Heck, A. J. R.; van den Heuvel, R. H. H. Mass Spectrom. Rev. 2004, 23, 368-389 (3) Loo, J. A.; Berhane, B.; Kaddis, C. S.; Wooding, K. M.; Xie, Y. M.; Kaufman, S. L.; Chemushevich, I. V. J. Am. Soc. Mass Spectrom. 2005, 16, 998-1008.

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considerable promise as a general method for quantifying association thermochemistry for biological complexes. Association constants, Kassoc, determined directly by ES-MS, for a variety of biological complexes have been reported1 and shown to be in good agreement with values determined using other analytical methods such as isothermal titration calorimetry (ITC). More recently, values of ∆Hassoc and ∆Sassoc for protein-carbohydrate binding were determined from a van’t Hoff analysis of the temperature dependence of Kassoc values measured using variable-temperature ES-MS.6 The successful implementation of the ES-MS technique for detecting specific biological complexes, such as protein-ligand complexes, in solution and quantifying their binding stoichiometry and Kassoc values requires careful control of the experimental conditions to minimize solution and gas-phase processes that may influence the mass spectral data. For example, the relative abundance of a protein in its bound (specifically) and unbound forms may be altered by changes in solution pH brought on by electrochemical processes,7 concentration changes in the ES droplets due to solvent evaporation,8 or gas-phase dissociation reactions in the ion source of the MS.5 ES mass spectra may also be influenced by the formation of nonspecific interactions between analyte molecules during the ES process, as the charged droplets evaporate to dryness, leading to stable gaseous complexes. Nonspecific binding may lead to false positives, i.e. the appearance of complexes that were not present in solution, obscure the true binding stoichiometry in solution, and has been shown to result in values of Kassoc that are artificially high.9 The effects of nonspecific binding are particularly problematic when studying weakly interacting species since the high concentrations of analyte necessary to generate detectable levels of specific complex in solution also tend to promote the appearance of nonspecific complexes in the mass spectra. With the aim of developing strategies to minimize the influence or contribution of nonspecific binding in ES-MS, our laboratory (4) Fandrich, M.; Tito, M. A.; Leroux, M. R.; Rostom, A. A.; Hartl, F. U.; Dobson, C. M.; Robinson, C. V. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 14151-14155. (5) Aquilina, J. A.; Benesch, J. L. P.; Ding, L. L.; Yaron, O.; Horwitz, J.; Robinson, C. V. J. Biol. Chem. 2005, 126, 14485-14491. (6) Daneshfar, R.; Kitova, E. N.; Klassen, J. S. J. Am. Chem. Soc. 2004, 126, 4786-4787. (7) Konermann, L.; Silva, E. A.; Sogbein, O. F. Anal. Chem. 2001, 73, 48364844. (8) Peschke, M.; Verkerk, U. H.; Kebarle, P. J. Am. Soc. Mass Spectrom. 2004, 15, 1424-1434. (9) Wang, W.; Kitova, E. N.; Klassen, J. S. Anal. Chem. 2003, 75, 4945-4955. 10.1021/ac0522005 CCC: $33.50

© 2006 American Chemical Society Published on Web 04/01/2006

Figure 1. Structures of the carbohydrates RTal[RAbe]RMan (1) and globotriaoside Pk (2).

has undertaken a comprehensive study of the phenomenon of nonspecific binding between biopolymers, in particular nonspecific protein-carbohydrate interactions during the ES process.10-12 Some of the factors influencing the formation of nonspecific protein-ligand interactions during nanoflow ES (nanoES) have recently been identified.10 Notably, it was shown that, at a given protein concentration, the fraction of protein engaged in nonspecific ligand binding, as well as the distribution of nonspecifically bound ligands, is determined by the number of free ligand molecules present in the nanoES droplets that ultimately lead to gaseous ions.10 Importantly, the efficiency of nonspecific proteinligand binding, at least for solutions containing a single protein and carbohydrate ligand, was found to be independent of the size and structure of the protein.10 As previously discussed,10,11 these results are consistent with the formation of the nonspecific complexes predominantly via the charge residue model of ES.13 Drawing on the insights gained from these earlier studies, we have developed a simple and straightforward experimental method for distinguishing specific from nonspecific protein-ligand complexes in nanoES-MS. A preliminary description of the method was recently given.14 Here, we provide a more complete description of the methodology, which involves the use of a reference protein added to the nanoES solution to monitor, quantitatively, the extent of nonspecific protein-ligand binding during the nanoES process. The key assumptions upon which the methodology is based have been tested and shown to be generally valid. The effectiveness of the methodology for correcting proteinligand Kassoc values determined under conditions where nonspecific ligand binding is prevalent is demonstrated for mono- and multivalent carbohydrate-binding proteins. MATERIALS AND METHODS Proteins and Carbohydrates. The carbohydrate-binding antibody single-chain fragment, scFv (MW 26 539), was produced using recombinant technology. The scFv was concentrated and dialyzed against deionized water using microconcentrators (Millipore Corp., Bedford, MA) with a molecular weight cutoff of (10) Wang, W.; Kitova, E. N.; Klassen, J. S. Anal. Chem. 2005, 77, 3060-3071. (11) Wang, W.; Kitova, E. N.; Sun, J.; Klassen, J. S. J. Am. Soc. Mass Spectrom. 2005, 16, 1583-1594. (12) Wang, W.; Kitova, E. N.; Klassen, J. S. J. Am. Chem. Soc. 2003, 125, 1363013631. (13) Dole, M.; Mack, L. L.; Hines, R. C.; Ferguson, L. D.; Alice, M. B. J. Chem. Phys. 1968, 49, 2240-2249. (14) Sun, J.; Wang, W.; Kitova, E. N.; Klassen, J. S. Proceedings of the 53rd ASMS Conference on Mass Spectrometry and Allied Topics, 2005.

10 000 and lyophilized prior to MS analysis. The scFv was weighed immediately after removing it from the lyophilizer, dissolved in a known volume of aqueous 50 mM ammonium acetate, and stored at -20 °C if not used immediately. Purified B subunit of Shiga toxin 1 (Stx1), in its lyophilized form, was provided by G. Armstrong (University of Calgary). A stock solution of the Stx1 B subunit at a concentration of 1.15 mg/mL (150 µM) in 50 mM ammonium acetate was prepared. Bovine carbonic anhydrase II (CA, MW 29 089) and bovine ubiquitin (Ubq, MW 8565) were purchased from Sigma-Aldrich and used without any further purification. The synthetic trisaccharides, RTal[RAbe]RMan (1) and 2-trimethylsilylethyl 4-O-[(4-O-R-D-galactopyranosyl)-β-D-galactopyranosyl]-β-D-glycopyranoside (Pk trisaccharide) (2) were provided by D. Bundle (University of Alberta). The structures of these carbohydrates are shown in Figure 1. Any adsorbed water was removed from the ligands prior to the preparation of stock solutions by drying the ligands in a vacuum chamber maintained at ∼5 Torr and 56 °C. Each nanoES solution was prepared from stock solutions of protein and ligand with known concentrations. A 50 mM aqueous solution of ammonium acetate was added to yield a final concentration of 2-3 mM. Mass Spectrometry. All experiments were performed on an Apex II Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer (Bruker, Billerica, MA) equipped with an external nanoES ion source. NanoES was performed using an aluminosilicate capillary (1.0-mm o.d., 0.68-mm i.d.), pulled to ∼4-7-µm o.d. at one end using a P-2000 micropipet puller (Sutter Instruments, Novato, CA). The electric field required to spray the solution was established by applying a voltage of ∼800 V to a platinum wire inserted inside the glass tip. The solution flow rate was typically 20-50 nL/min. The droplets and gaseous ions produced by nanoES were introduced into the mass spectrometer through a stainless steel capillary (i.d. 0.43 mm) maintained at an external temperature of 66 °C. The ion/gas jet sampled by the capillary (48-52 V) was transmitted through a skimmer (0-2 V) and stored electrodynamically in an rf hexapole. A hexapole accumulation time of between 1.5 and 2.0 s was used for all experiments. Ions were ejected from the hexapole and accelerated to ∼-2700 V into a 9.4-T superconducting magnet, decelerated, and introduced into the ion cell. The trapping plates of the cell were maintained at a constant potential of 1.4-1.8 V throughout the experiment. The typical base pressure for the instrument was ∼5 × 10-10 mbar. Data acquisition was controlled by an SGI R5000 computer running the Bruker Daltonics XMASS software, version 5.0. Mass Analytical Chemistry, Vol. 78, No. 9, May 1, 2006

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spectra were obtained using standard experimental sequences with chirp broadband excitation. The time domain signal, consisting of the sum of 20-40 transients containing 128K data points per transient, were subjected to one zero-fill prior to Fourier transformation. Determination of Kassoc by Direct ES-MS Measurement. The general equilibrium expression for the association reaction involving a protein (P) and ligand (L) (eq 1) is given by eq 2:

P + L T P-L

(1)

Kassoc ) [PL]equil/[P]equil[L]equil

(2)

If the protein (or protein complex) can bind to multiple molecules of L, there are additional reactions that must be considered. Shown below are the relevant reactions for P binding to between one and N molecules of L:

P + L T P-L

(7a)

P-L + L T P-L2

(7b)

‚

‚

‚

‚

‚

‚

P-LN-1 + L T P-LN The equilibrium concentrations, [PL]equil, [P]equil, and [L]equil, can be calculated from the initial concentration of protein and ligand in solution, [P]o and [L]o, and the relative abundance of the corresponding bound and unbound protein ions measured in the mass spectrum, for example, PLn+ and Pn+ in positive ion mode. In the absence of solution processes (e.g., pH and concentration changes, nonspecific binding) and gas-phase processes (e.g., dissociation of the PLn+ ions in the ion source), which may alter the original distribution of bound and unbound protein, and assuming that the spray and detection efficiencies for the PLn+ and Pn+ ions are similar, the ratio (R) of the ion intensity (I) of the bound and unbound protein ions determined from the mass spectrum is expected to be equivalent to the ratio of the concentrations in solution at equilibrium. Because the Pn+ and PLn+ ions are typically produced by ES or nanoES with a distribution of charge states, the R value is normally calculated as the sum of the intensities of the complex ions, at all charge states, over the sum of the intensities of the protein ions, at all charge states.12 Furthermore, the ion signal in FT-ICR-MS, which was used in the present work, is proportional to the abundance and charge state of the ions. Consequently, the measured ion intensities must be normalized for charge state, n:9

R ) [PL]equil/[P]equil )

∑I(PL

n+

)/n/

n

∑I(P

n+

)/n (3)

n

However, to simplify the proceeding discussion on the influence of nonspecific binding on the measured ion intensities of unbound protein and specific protein-ligand complexes, explicit consideration of the influence of charge state and charge distribution will be omitted from the relevant equations. The equilibrium concentration, [PL]equil, can be determined from the value of R and [P]o using the following expression:

[PL]equil ) R[P]o/(1 + R)

(7N)

The equilibrium concentrations of bound protein can be determined from the relative abundance of the corresponding ions observed in the mass spectrum and eq 8a. The equilibrium concentration of L can then be found from eq 8b.

[P]o ) [P]equil + [PL]equil + [PL2]equil + ... + [PLN]equil (8a) [L]o ) [L]equil + [PL]equil + 2[PL2]equil + ... + N[PLN]equil (8b) If the binding sites are equivalent, Kassoc can be determined for each and any of the ligand binding reactions described by eq 7 using the general expression

Kassoc,p ) Kassoc(N - p + 1)/p

(9)

where p is the number of occupied binding sites.15 Correcting Kassoc for Nonspecific Protein-Ligand Binding. The occurrence of nonspecific protein-ligand binding during the nanoES process will result in changes to the intensities measured for the ions of the unbound protein (P) and specific protein-ligand complexes (PL, PL2, ..., PLN+). The influence of nonspecific ligand binding on the relative abundance of a protein and its 1:1 proteinligand complex is illustrated in Figure 2. Quantitatively, the influence of nonspecific binding on the measured (apparent) intensity (Iapp) for the Pn+ ion can be described by the following expression:

Iapp(Pn+) ) I(Pn+) - f1,PI(Pn+) - f2,PI(Pn+) f3,PI(Pn+) - ‚‚‚ - fi,PI(Pn+) (10a)

(4)

The equilibrium concentration [L]equil can be found from eq 5 and Kassoc can then be determined with eq 6:

where I(Pn+) is the ion intensity of Pn+ expected in the absence of nonspecific binding and fi,P represents the fraction of the protein that is bound nonspecifically to i molecules of L. Given that Σfi,P ) 1 (where i ) 0, 1, 2, ...), eq 10a can be rewritten as

[L]equil ) [L]o - [PL]equil

I(Pn+) ) Iapp(Pn+)/f0,P

Kassoc )

[PL]equil [P]equil([L]o - [PL]equil)

R Kassoc ) R[P]o [L]o 1+R 3012

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(5)

(10b)

(6a) (6b)

where f0,P is the fraction of P that does not undergo nonspecific binding. Similarly, the influence of nonspecific binding on the (15) Wang, W.; Kitova, E. N.; Klassen, J. S. Methods Enzymol. 2003, 362, 376398.

carbohydrate binding revealed that, at low to moderate concentrations (25 µM), the experimentally observed distributions of nonspecific complexes were found to be too broad and irregular to be described by a single λ value. This observation was attributed to the contribution of multiple generations of offspring droplets to the formation of nonspecific protein-ligand complexes. Additionally, the distribution of nonspecifically bound carbohydrates was shown to be sensitive to experimental conditions, such as ion source voltages and the dimensions of the nanoES tip.10,16 Because the experimentally observed distribution of nonspecifically bound ligands is sensitive to the structure of the ligand, the ligand concentrations, and the experimental conditions, the distribution cannot be predicted a priori. Consequently, the only accurate approach for quantifying the contribution of nonspecific protein-ligand binding to a given ES mass spectrum is by directly determining the distribution of nonspecifically bound ligands, i.e., the fi terms. It was also established in an earlier study of nonspecific protein-carbohydrate binding that the fraction of protein that binds nonspecifically to carbohydrates during the nanoES process is insensitive to the nature (size and structure) of the protein.10 Although not shown directly in this previous study, it reasonably follows that the fractions of protein and specific protein-ligand complexes that engage in nonspecific ligand binding are also equivalent; i.e., Σfi,P ) Σfi,PL ) ‚‚‚ ) Σfi,PLN (where i g 1). As shown in the present work, the individual fi terms for all protein species present in the ES solution are also equivalent; e.g., f1,P ) f1,PL ) ‚‚‚ ) f1,PLN. Given that the fi terms are equivalent, vide infra, it follows that the occurrence of nonspecific protein-ligand binding can be assessed qualitatively, and the fi terms assessed quantitatively, by adding a suitable reference protein, Pref, which does not exhibit any specificity for the ligand(s) of interest, to the ES solution. The appearance of Pref bound to i molecules of L, i.e., (PrefLi)n+ ions, in a mass spectrum can serve as an indicator of the formation of nonspecific protein-ligand complexes during the nanoES process. Furthermore, since the fi,Pref terms determined from the mass spectrum are equivalent to the fi terms for the other protein species, they can be used to correct the measured (16) Sun, N.; Kitova, E. N.; Klassen, J. S. Manuscript in preparation.

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Figure 3. NanoES mass spectra obtained from solutions of (a) 11 µM scFv, 11 µM CA, and 93 µM 2 and (b) 11 µM Ubq, 11 µM scFv, and 120 µM 2. The number of molecules of 2 bound (nonspecifically) to the protein ions is indicated by q; the subscript identifies the protein to which 2 is bound, S ) scFv, C ) CA, and U ) Ubq. Table 1. Comparison of the f1 and f2 Terms for Nonspecific Binding of 2 to the Proteins ScFv, CA, and Ubq during the NanoES Process [scFv] (µM)

[CA] (µM)

8 11 8 8

7 11 7 13

12 4 12 17 21 30 4 20 16

[Ubq] (µM)

[2] (µM) 93 93 118 118

11 4 11 17 21 30 8 10 24

f1,CA/f1,scFva,b

f2,CA/f2,scFva,b

1.02 ( 0.06 1.02 ( 0.04 1.07 ( 0.05 0.99 ( 0.02 (1.03 ( 0.03)c

0.94 ( 0.18 1.00 ( 0.13 0.97 ( 0.06 1.01 ( 0.10 (0.98 ( 0.03)c

60 120 120 120 120 120 120 120 120

f1,scFv/f1,Ubqa,b

f2,scFv/f2,Ubqa,b

1.07 ( 0.05 0.96 ( 0.03 1.07 ( 0.05 0.99 ( 0.10 1.03 ( 0.04 0.91 ( 0.18 1.06 ( 0.04 1.02 ( 0.04 0.97 ( 0.08 (1.01 ( 0.06)c

d 1.00 ( 0.18 0.99 ( 0.21 0.97 ( 0.13 d d 0.94 ( 0.15 1.03 ( 0.10 d (0.99 ( 0.03)c

a Values calculated from average f terms taken from 4 or 5 duplicates. b Errors correspond to one standard deviation. c Average values of f i i,CA/ fi,scFv and fi,scFv/fi,Ubq based on all measurements. d The intensities of the corresponding (P + 2(2))n+ ions could not be measured accurately due to low signal-to-noise ratios.

intensities for the unbound protein and specific protein-ligand complexes for nonspecific binding (via eq 10) and, thereby, provide more reliable values of Kassoc. RESULTS AND DISCUSSION Influence of Protein Structure on Magnitude of fi Terms. The starting point of the present study was to more firmly establish that the distribution of the intensities (i.e., the magnitude of the fi terms) of complexes formed between a nonspecific ligand and a protein during nanoES is independent of the size and structure of the protein. To demonstrate this directly, the distributions of carbohydrate molecules bound nonspecifically to three proteins (CA, scFv, Ubq) during the nanoES process were compared over a range of concentrations. NanoES mass spectra were acquired for solutions of 2 (93, 118 µM) with scFv and CA (7-13 µM) and for solutions of 2 (60, 120 µM) with scFv and Ubq (4-30 µM). The carbohydrate 2 is not known to bind specifically to any of these proteins in aqueous solution. Shown in Figure 3a is an illustrative mass spectrum acquired in positive ion mode for a solution containing 2 (93 µM), scFv (11 µM), and CA (11 µM). The scFv and CA proteins, which have similar MWs, are observed predominantly in their protonated form, i.e., (P + nH)n+ ≡ Pn+, at charge states n ) 9-11. Also 3014 Analytical Chemistry, Vol. 78, No. 9, May 1, 2006

present in the mass spectrum are protonated protein ions bound to one and two molecules of 2, i.e., (P + 2)n+ and (P + 2(2))n+. Visual inspection of the mass spectrum reveals that the distributions of nonspecifically bound molecules of 2 are similar for the scFv and CA ions. The similarity in the distributions of nonspecific ligands is more clearly seen by comparing the corresponding fi values determined for CA and scFv. Listed in Table 1 are the ratios, f1,CA/f1,scFv and f2,CA/f2,scFv, determined for each of the solutions investigated. The average values of f1,CA/f1,scFv and f2,CA/ f2,scFv, based on the four experiments, are 1.03 ( 0.03 and 0.98 ( 0.03, respectively. These results indicate that, within the precision of the measurements, the distributions of 2 bound nonspecifically to CA and scFv are identical. Shown in Figure 3b is an illustrative mass spectrum acquired for a solution of 2 (120 µM) with scFv (11 µM) and Ubq (11 µM). Ions corresponding to the unbound Ubq and scFv (i.e., Ubqn+ and scFvn+) are observed, along with ions corresponding Ubq and scFv bound to one and two molecules of 2. Because the MW of Ubq is roughly one-third that of scFv, the gaseous bound and unbound Ubq ions are produced at lower charge states, n ) 5 and 6, compared to the scFv ions. Again, similar distributions of nonspecifically bound 2 are observed; the average f1,scFv/f1,Ubq and

f2,scFv/f2,Ubq values are 1.01 ( 0.06 and 0.99 ( 0.03, respectively, Table 1. It should be noted that these measurements were performed using relatively short accumulation times (e2 s) in the rf hexapole of the ion source. The use of longer accumulations (>2 s) leads to fi,scFv/fi,Ubq values of *1.0 (data not shown). It has been previously shown that at accumulation times of >2 s, ions undergo significant collisional heating while stored within the hexapole.10,17 This heating is sufficient to cause the dissociation of noncovalent complexes with relatively low gas-phase stabilities. A recent study revealed that the gas-phase stability (kinetic and energetic) of nonspecific protein-carbohydrate complexes is sensitive to size and charge state of the complexes, with small and low charge-state complexes being less stable.11 The nonequivalent fi values determined for Ubq and scFv ions at longer accumulation times can, therefore, be reasonably explained by greater dissociation of the Ubq complexes, relative to the scFv complexes, in the ion source. These results provide compelling evidence that, in a given nanoES-MS experiment, the efficiency of nonspecific ligand binding is insensitive to the size (at least for MWs between 8000 and 30 000) and structure of proteins and their specific complexes. Therefore, in principle, any protein that does not interact with the ligand(s) and proteins(s) of interest in solution can serve as Pref. However, when using a Pref with a low MW, relative to the protein(s) and protein complex(es) of interest, source conditions must be carefully controlled to avoid in-source dissociation of nonspecific (and possibly specific) protein-ligand complexes, which may result in nonequivalent fi values. Influence of [Pref] on Nonspecific Protein-Ligand Binding. It was also important to establish whether there are restrictions on the concentration of Pref that can be used in these measurements. To assess the influence of protein concentration on nonspecific binding, titration experiments, in which the nonspecific ligand concentration was kept constant and the protein(s) concentration was varied, were performed. One of the experiments involved titrating a solution of 1 (68 µM) with CA at concentrations ranging from 5 to 27 µM. Shown in Figure 4a is a plot of the fraction of CA participating in nonspecific binding with 1, i.e., Σfi,CA (where i g1) versus protein concentration. It can be seen that, at low protein concentrations (e10 µM), the fraction of nonspecifically bound CA is relatively constant (Σfi,CA ≈ 0.5), independent of concentration. However, at higher protein concentrations, there is a noticeable decrease in the fraction of nonspecifically bound CA, with the Σfi,CA value decreasing to ∼0.3 at 27 µM CA. Analysis of the influence of protein concentration on the fi values determined for solutions of 2 with Ubq and scFv (at the concentrations indicated in Table 1) yields similar results, Figure 4b. There are at least two possible explanations for the apparent decrease in the extent of nonspecific ligand binding at higher protein concentrations. First, the transfer efficiency of carbohydrate molecules from the larger nanoES droplets to the offspring droplets, which ultimately yield the gaseous ions, may be sensitive to protein concentration, at least over certain concentration ranges. At sufficiently high concentrations, the protein molecules can compete with the carbohydrates for positions on the surface of (17) Hakansson, K.; Awxlsson, J.; Palmblad, M.; Hakansson, P. J. Am. Soc. Mass Spectrom. 2000, 11, 210-217.

Figure 4. Influence of protein concentration on the fraction of protein undergoing nonspecific ligand binding. (a) Plot of Σfi (where i g 1) versus CA concentration with 1 at fixed concentration of 68 µM. (b) Plot of Σfi (where i g 1) versus total protein concentration ([scFv] + [Ubq]) at fixed concentration of 2 (120 µM); (b) equimolar concentration of scFv and Ubq; (0) 4 µM scFv, 8 µM Ubq; (O) 20 µM scFv, 10 µM Ubq; (4)16 µM scFv, 24 µM Ubq.

the nanoES droplets and, thereby, suppress the transfer of carbohydrates to the offspring droplets during fission, resulting in a decrease in the number of ligand molecules that ultimately end up in the nanodroplets.18 Second, the probability that the offspring droplets contain two or more protein molecules will increase with protein concentration. The ligand molecules within the offspring droplets may then be partitioned between the available protein molecules in the droplets, leading to a decrease in the extent of nonspecific ligand binding. The results of this and previous studies10,14 indicate that the extent of nonspecific protein-ligand binding is sensitive to the concentrations of both protein and (nonspecific or free) ligand in solution. Higher ligand concentrations serve to increase the fraction of protein (complex) undergoing nonspecific binding, while higher protein concentrations serve to decrease the efficiency of nonspecific binding. Consequently, the adverse effects of nonspecific protein-ligand binding may be reduced simply by adding a noninteracting protein at high concentration to the nanoES solution. However, there are practical limitations to the protein concentration that can be used since high concentrations will also have the deleterious effect of reducing the signal-to-noise ratio (S/N) of the protein and specific complex ions of interest. (18) Benkestock, K.; Sundqvist, G.; Edlund, P. O.; Roeraade, J. J. Mass Spectrom. 2004, 39, 1059-1067.

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Figure 5. NanoES mass spectra obtained from the solution of (a) 9 µM scFv, 10 µM 1, and 10 µM CA, (b) 7 µM scFv, 15 µM 1, and 2 µM CA, and (c) 17 µM scFv, 42 µM 1, and 8 µM CA. The number of molecules of 1 bound to the protein ions is indicated by q; the subscript identifies the protein to which 1 is bound, S ) scFv and C) CA.

Correcting Kassoc Values for Protein-Ligand Binding, As Determined by nanoES-MS, for Nonspecific Ligand Interactions. To demonstrate the effectiveness of the proposed methodology for identifying the formation of nonspecific protein-ligand complexes, and correcting protein-ligand Kassoc values, as determined by nanoES-MS, for nonspecific binding, binding affinity measurements were performed for two protein-carbohydrate complexes. One of the complexes investigated involved the binding of scFv with the specific trisccharide ligand, 1. A Kassoc value of 1.17 ((0.03) × 105 M-1 has been determined for this complex at 298 K by ITC.19 To monitor the formation of nonspecific complexes between scFv and 1 during the nanoES, CA, which has no known affinity for 1 in aqueous solution, was added to the nanoES solution and served as Pref. NanoES mass spectra were measured for three solutions containing different initial concentrations of scFv, CA, and 1. Shown in Figure 5a is a mass spectrum measured for a near-equimolar (∼10 µM) solution of scFv, CA, and 1. The protonated scFvn+ and (scFv + 1)n+ ions, (19) Bundle, D. R. Unpublished data.

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as well as the protonated CAn+ ions are clearly evident in the mass spectrum. However, there are no detectable (CA + 1)n+ ions, indicating that nonspecific binding of 1 with CA (and, consequently, with scFv and (scFv + 1)) during the nanoES process is insignificant. The Kassoc value derived from the measured (apparent) R value (Rapp) is 0.94 ((0.04) × 105 M-1, which is in reasonable agreement with the ITC-derived value. The spectra shown in Figure 5b,c were acquired for solutions with higher concentration of 1 (15 and 42 µM). It can be seen that, in addition to the CAn+, scFvn+, and (scFv + 1)n+ ions, there are also (scFv + 2(1))n+ and (CA + 1)n+ ions clearly evident in the mass spectra, indicating the occurrence of nonspecific binding of the protein/specific complex with 1. Ignoring the contribution of nonspecific binding (i.e., calculating R from measured ion intensities) leads to Kassoc values that are artificially large, by as much as 100%, Table 2. It is worth pointing out that the addition of CA to the solution increases the total protein concentration and, for reasons discussed above, leads to a decrease in the degree of nonspecific binding. In the absence of CA or some other Pref in solution, even larger errors in the Kassoc values for the (scFv + 1) complex would be expected. Correcting the Rapp values for nonspecific binding, using eq 10, leads to Kassoc values of 1.16 ((0.12) × 105 and 1.13 ((0.10) × 105 M-1 (Table 2). The average value of Kassoc, based on all three nanoES-MS measurements, after correction, is 1.08 ((0.12) × 105 M-1, a result that is in excellent agreement with the ITC value. A second protein-carbohydrate complex investigated involved the interaction of the B subunit of Stx1 with 2. It is known that, in aqueous solution at neutral pH, the B subunits assemble into a stable homopentamer, B5.20 X-ray analysis of the cocrystal obtained for B5 with 2 has shown that each of the B subunits possesses three binding sites for 2.21,22 One of the binding sites (referred to as site 2) is believed to represent the dominant interaction site.20,23 Based on ITC measurements, the value of Kassoc per subunit (site 2) is estimated to be in the (0.5-1) × 103 M-1 range.23 The ITC measurements also revealed that binding of 2 to B5 is noncooperative. Due to the low affinity exhibited by the B5 complex for 2, relatively high concentrations of 2 are required to produce detectable levels of complex. As a result, the binding stoichiometry and Kassoc values, as revealed by nanoES-MS, are expected to be obscured by nonspecific binding. To determine the Kassoc values for the binding of 2 with B5, nanoES mass spectra were measured for equimolar solutions of B5 and scFv (5 µM) and 2 at concentrations ranging from 20 to 200 µM. Shown in Figure 6a is a mass spectrum acquired for a solution containing 20 µM 2. The B5 homopentamer is observed predominantly in its unbound form, B5n+, where n ) 11-13, with only a small fraction bound to a single molecule of 2, (B5 + 2)n+. The scFvn+ ions are observed exclusively in their unbound form, indicating that nonspecific ligand binding under these conditions is insignificant. Based on the intensity ratios for the B5n+ and (B5 + 2)n+ ions, a value of 0.26 ((0.03) × 103 M-1 is obtained for (20) Shimizu, H.; Field, R. A.; Homans, S. W.; Donohue-Rolfe, A. Biochemistry 1998, 37, 11078-11082. (21) Bast, D. J.; Banerjee, L.; Clark, C.; Read, R. J.; Brunton, J. L. Mol. Microbiol. 1999, 32, 953-960. (22) Ling, H.; Boodhoo, A.; Hazes, B.; Cummings, M. D.; Armstrong, G. D.; Brunton, J. L.; Read, R. J. Biochemistry 1998, 37, 1777-1788. (23) St. Hilaire, P. M.; Boyd, M. K.; Toone, E. J. Biochemistry 1994, 33, 1445214463.

Table 2. Apparent and Corrected (for Nonspecific Ligand Binding) KAssoc Values, Determined by NanoES-MS, for Binding of scFv with 1 at 298 Ka [scFv]o (µM)

[1]o (µM)

[CA] (µM)

Rappb

Rb

Kassoc,appc (×10-5 M-1)

Kassocc (×10-5 M-1)

9 7 17

10 15 42

10 2 8

0.63 ( 0.02 1.90 ( 0.16 3.67 ( 0.38

0.63 ( 0.02 1.38 ( 0.14 3.25 ( 0.28

0.94 ( 0.04 1.84 ( 0.20 1.29 ( 0.14

0.94 ( 0.04 1.16 ( 0.12 1.13 ( 0.10 1.08 ( 0.12 d

a A K 5 -1 was determined by ITC, ref 19. b R and R assoc value of 1.17 ((0.03) × 10 M app calculated with eq 3 with and without correction for nonspecific ligand binding, respectively. c Kassoc and Kassoc,app calculated from R and Rapp values, respectively, using eq 6b. d Average of the Kassoc values.

Figure 6. NanoES mass spectra obtained from solutions of (a) 5 µM scFv, 5 µM Stx1 B5, and 20 µM 2, (b) 5 µM scFv, 5 µM Stx1 B5, and 100 µM 2, and (c) 5 µM scFv, 5 µM Stx1 B5, and 143 µM 2. The number of molecules of 2 bound to the protein ions is indicated by q; the subscript identifies the protein to which 2 is bound, S ) scFv, B ) B5. Table 3. Apparent and Corrected (for Nonspecific Ligand Binding) KAssoc Values for Binding of B5 Homopentamer of Stx1 with 2 in Aqueous Ammonium Acetate Solutions at 298 Ka [B5]o (µM)

[2]o (µM)

[scFv] (µM)

Kassoc(1),appb (×10-3 M-1)

5 5 5 5

20 100 143 200

5 5 5 5

0.26 ( 0.03 0.54 ( 0.20 0.41 ( 0.12 0.44 ( 0.11

Kassoc(2),appb (×10-3 M-1)

0.70 ( 0.11 0.48 ( 0.11

Kassoc(1)b (×10-3 M-1)

Kassoc(2)b (×10-3 M-1)

0.26 ( 0.03 0.29 ( 0.08 0.37 ( 0.07 0.17 ( 0.05 0.27 ( 0.08 b

0.42 ( 0.11 0.37 ( 0.10 0.39 ( 0.04 b

a K 3 -1 b assoc value of (0.5-1) × 10 M , determined by ITC, is reported in ref 23. Kassoc and Kassoc,app calculated from R and Rapp values, respectively, using eq 6b. c Average of the corrected Kassoc values.

Kassoc(1), which is the affinity of 2 for a single B subunit. At higher concentrations 2, g100 µM, B5n+, (B5 + 2)n+, and (B5 + 2(2))n+ ions were observed, along with ions corresponding to scFvn+, (scFv + 2)n+, and (scFv + 2(2))n+. From the measured ion intensities, Kassoc(1)app and Kassoc(2)app values in the range of (0.410.70) × 103 M-1 are obtained, Table 3. Correction for nonspecific binding yields values ranging from 0.17 to 0.42 (×103) M-1. The average values of Kassoc(1) and Kassoc(2) are 0.27 ((0.08) × 103 and

0.39 ((0.04) × 103 M-1, respectively. These are among the smallest Kassoc values determined by the direct ES-MS technique.24 Within experimental error, the values of Kassoc(1) and Kassoc(2) are identical, which is consistent with noncooperative binding.23 The nanoES-MS-derived Kassoc values are somewhat lower than the ITCderived value. However, it should be noted that, due to the weak (24) Griffey, R. H.; Sannes-Lowery, K. A.; Drader, J. D.; Mohan, V.; Swayze, E. E.; Hofstadler, S. A. J. Am. Chem. Soc. 2000, 122, 9933-9938.

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binding of 2 and B5, it is difficult to obtain a reliable Kassoc value by ITC.25,26 CONCLUSIONS A simple, straightforward method for distinguishing between specific and nonspecific protein-ligand complexes in nanoES-MS is described. The method involves the addition of an appropriate reference protein (Pref), which does not bind specifically to any of the solution components, to the nanoES solution to monitor nonspecific ligand binding. The principal assumption on which this methodology is based, namely, that the distribution of ligand molecules bound nonspecifically to proteins and specific protein complexes during the nanoES process is determined by the number of free ligand molecules in the nanodroplets that yield gaseous ions and is independent of the size and structure of the protein or protein complex, is shown to be generally valid. It follows that the fraction of Pref undergoing nonspecific ligand binding provides a quantitative measure of the contribution of nonspecific binding to the measured intensities of proteins and (25) Bundle, D. R. Methods Enzymol. 1994, 247, 288-305. (26) Christensen, T.; Toone, E. J. Methods Enzymol. 2003, 362, 486-504.

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specific protein-ligand complexes of interest. As a result, errors introduced by nonspecific ligand binding into Kassoc values, as determined with nanoES-MS, for specific protein-ligand complexes, can be corrected. Ideally, a Pref with a MW similar to that of the proteins and protein complexes of interest should be used to minimize errors that can result from gas-phase dissociation reactions in the ion source. High concentrations of Pref, which help to minimize the adverse effects of nonspecific protein-ligand binding, are recommended. However, the chosen Pref concentration must provide a reasonable S/N for the Pref ions as well as for the protein and specific complex ions of interest. The application of the method for the determination of Kassoc for two proteincarbohydrate complexes, under conditions where nonspecific ligand binding is prevalent, was demonstrated and shown to yield binding affinities which are in agreement with values obtained from ITC measurements.

Received for review December 13, 2005. Accepted March 8, 2006. AC0522005