Determination of Ligand− Protein Dissociation Constants by

Anal. Chem. 2004, 76, 7077-7083

Determination of Ligand-Protein Dissociation Constants by Electrospray Mass Spectrometry-Based Diffusion Measurements Sonya M. Clark and Lars Konermann*

Department of Chemistry, The University of Western Ontario, London, Ontario N6A 5B7, Canada

A novel approach for the quantification of ligand-protein interactions is presented. Electrospray ionization mass spectrometry (ESI-MS) is used to monitor the diffusion behavior of noncovalent ligands in the presence of their protein receptors. These data allow the fraction of free ligand in solution to be determined, such that the corresponding dissociation constants can be calculated. A set of conditions is developed that provides an “allowable range” of concentrations for this type of assay. The method is tested by applying it to two different inhibitor-enzyme systems. The dissociation constants measured for benzamidine-trypsin and for N,N′,N′′-triacetylchitotrioselysozyme are (50 ( 10) and (6 ( 1) mM, respectively. Both of these results are in good agreement with previous data from the literature. In contrast to traditional ESI-MSbased methods, the approach used in this work does not rely on the preservation of specific solution-type noncovalent interactions in the gas phase. It is shown that this method allows an accurate determination of dissociation constants, even in cases in which the ion abundance ratio of free to ligand-bound protein in ESI-MS does not reflect the corresponding concentration ratio in solution. The noncovalent binding of low molecular weight ligands to protein receptors plays a central role for many biological processes. Numerous applications, such as the evaluation of novel drug candidates, require the determination of ligand-protein binding affinities. For a ligand-protein complex, LP, that is in equilibrium with free ligand L and free protein P, the binding affinity is reflected in the dissociation constant Kd, defined as Kd ) [L][P]/[LP]. Complexes with large Kd values have a low binding affinity, and vice versa. The dissociation constants observed for various complexes span many orders of magnitude, from subnanomolar values for typical pharmaceutically relevant drugreceptor complexes, to dissociation constants in the millimolar range for many enzyme-substrate systems.1 Electrospray ionization mass spectrometry (ESI-MS) has emerged as a popular method for studying solution-phase noncovalent interactions. Even weakly bound complexes with milli* To whom correspondence should be addressed. Phone: (519) 661-2111, ext. 86313. Fax: (519) 661-3022. E-mail: [email protected]. http:// publish.uwo.ca/∼konerman. (1) Dukhovich, F. S.; Gorbatova, E. N.; Darkhovskii, M. B.; Kurochkin, V. K. Pharm. Chem. J. 2002, 36, 248-254. 10.1021/ac049344o CCC: $27.50 Published on Web 10/23/2004

© 2004 American Chemical Society

molar dissociation constants can often survive the ESI process.2,3 These assemblies may, therefore, be detected by the direct observation of their intact gas-phase ions.4-8 Due to the inherent selectivity and sensitivity of ESI-MS, this approach is very attractive. Unlike traditional methods for studying noncovalent interactions, ESI-MS does not require optical signal changes upon binding. It also does not involve chemical coupling steps, and it does not suffer from potential artifacts due to binding to column or membrane materials.9 Unfortunately, it is not always clear whether the ion intensity ratio of free to ligand-bound protein observed by ESI-MS directly reflects the Kd value of a complex in solution. For a number of systems, this ratio appears to be in good agreement with solution-phase data.10-13 In other cases, however, deviations of several orders of magnitude have been reported.14,15 These conflicting findings make it clear that a number of factors can influence the appearance of ESI mass spectra, even when “gentle” ionization conditions are used. Dissociation upon transfer into the gas phase can be a significant problem, particularly for complexes that are stabilized by hydrophobic interactions.14,16,17 However, this problem also exists for some electrostatically stabilized assemblies.15 In addition, nonspecific association (2) Kitova, E. N.; Kitov, P. I.; Bundle, D. R.; Klassen, J. S. Glycobiology 2001, 11, 605-611. (3) Griffey, R. H.; Sannes-Lowery, K. A.; Drader, J. J.; Mohan, V.; Swayze, E. E.; Hofstadler, S. A. J. Am. Chem. Soc. 2000, 122, 9933-9938. (4) Ganem, B.; Li, Y.-T.; Henion, J. D. J. Am. Chem. Soc. 1991, 113, 78187819. (5) Katta, V.; Chait, B. T. J. Am. Chem. Soc. 1991, 113, 8534-8535. (6) Loo, J. A. Int. J. Mass Spectrom. 2000, 200, 175-186. (7) Daniel, J. M.; Friess, S. D.; Rajagopalan, S.; Wendt, S.; Zenobi, R. Int. J. Mass Spectrom. 2002, 216, 1-27. (8) Potier, N.; Donald, L. J.; Chernushevich, I.; Ayed, A.; Ens, W.; Arrowsmith, C. H.; Standing, K. G.; Duckworth, H. W. Protein Sci. 1998, 7, 1388-1395. (9) Bohm, H.-J., Schneiger, G., Eds. Protein-Ligand Interactions: From Molecular Recognition to Drug Design; Wiley-VCH: Weinheim, 2003. (10) Wang, W.; Kitova, E. N.; Klassen, J. S. Anal. Chem. 2003, 75, 4945-4955. (11) Wendt, S.; McCombie, G.; Daniel, J.; Kienhofer, A.; Hilvert, D.; Zenobi, R. J. Am. Soc. Mass Spectrom. 2003, 14, 1470-1476. (12) Jorgensen, T. J. D.; Roepstorff, P.; Heck, A. J. R. Anal. Chem. 1998, 70, 4427-4432. (13) Hagan, N.; Fabris, D. Biochemistry 2003, 42, 10736-10745. (14) Robinson, C. V.; Chung, E. W.; Kragelund, B. B.; Knudsen, J.; Aplin, R. T.; Poulsen, F. M.; Dobson, C. M. J. Am. Chem. Soc. 1996, 118, 8646-8653. (15) Mauk, M. R.; Mauk, A. G.; Chen, Y.-L.; Douglas, D. J. J. Am. Soc. Mass Spectrom. 2002, 13, 59-71. (16) Wigger, M.; Eyler, J. R.; Benner, S. A.; Li, W.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 2002, 13, 1162-1169. (17) Li, Y.; Heitz, F.; Le Grimellec, C.; Cole, R. B. Rapid Commun. Mass Spectrom. 2003, 18, 135-137.

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during ESI can result in the formation of ionic aggregates that do not correspond to specific solution-phase complexes.18-24 All of these factors may result in incorrect estimates of solution-phase binding affinities. Consequently, there currently is great interest in MS-based techniques for the quantification of ligand-protein interactions that do not rely on the preservation of specific interactions in the gas phase. Examples of developments in this area include methods involving hydrogen/deuterium exchange25-27 and affinity chromatography.28 Our laboratory has recently proposed an alternative approach for the identification of high-affinity ligands to protein receptors, where ESI-MS is used for monitoring the translational diffusion of analytes.29,30 ESI-MS Taylor dispersion experiments allow the measurement of solution-phase analyte diffusion coefficients.31 In these studies, an initially sharp boundary between two solutions of different analyte concentration is generated within a capillary tube. Laminar flow causes this boundary to undergo dispersion as the solutions flow toward the tube outlet. Radial diffusion continuously changes the flow velocity of individual analyte molecules, thus counteracting the dispersion caused by laminar flow. As a result, intensity-time profiles monitored by ESI-MS at the tube outlet will exhibit relatively steep transitions for analytes with large diffusion coefficients. The profiles for analytes with small diffusion coefficients will be more extended. This concept is illustrated in Figure 1. The application of this technique to study the noncovalent binding of low molecular weight compounds to proteins is straightforward. A potential ligand will have a relatively large apparent diffusion coefficient, DA, as long as it is free (i.e., unbound) in solution; however, DA will be drastically reduced upon binding to a macromolecule. Thus, a relatively extended profile, corresponding to a small diffusion coefficient, will be observed for a ligand in the presence of its protein receptor. In contrast, the profile of the same ligand recorded in a protein-free solution will be much steeper. To ensure that the profile of the potential ligand can be monitored separately from that of the protein, all potential noncovalent interactions are deliberately disrupted immediately prior to ESI. This can be achieved through the addition of a makeup solvent (usually a methanol/acetic acid mixture) at (18) Hu, P.; Ye, Q.-Z.; Loo, J. A. Anal. Chem. 1994, 66, 4190-4194. (19) Cunniff, J. B.; Vouros, P. J. Am. Soc. Mass Spectrom. 1995, 6, 437-447. (20) Juraschek, R.; Dulcks, T.; Karas, M. J. Am. Soc. Mass Spectrom. 1999, 10, 300-308. (21) Zechel, D. L.; Konermann, L.; Withers, S. G.; Douglas, D. J. Biochemistry 1998, 37, 7664-7669. (22) Smith, R. D.; Light-Wahl, K. J.; Winger, B. E.; Loo, J. A. Org. Mass Spectrom. 1992, 27, 811-821. (23) Jorgensen, T. J. D.; Hvelplund, P.; Andersen, J. U.; Roepstorff, P. Int. J. Mass Spectrom. 2002, 219, 659-670. (24) Wang, W.; Kitova, E. N.; Klassen, J. S. J. Am. Chem. Soc. 2003, 125, 1363013631. (25) Powell, K. D.; Ghaemmaghami, S.; Wang, M. Z.; Ma, L.; Oas, T. G.; Fitzgerald, M. C. J. Am. Chem. Soc. 2002, 124, 10256-10257. (26) Zhu, M. M.; Rempel, D. L.; Du, Z.; Gross, M. L. J. Am. Chem. Soc. 2003, 125, 5252-5253. (27) Zhu, M. M.; Rempel, D. L.; Gross, M. L. J. Am. Soc. Mass Spectrom. 2004, 15, 388-397. (28) Schriemer, D. C.; Bundle, D. R.; Li, L.; Hindsgaul, O. Angew. Chem., Int. Ed. 1998, 37, 3383-3387. (29) Clark, S. M.; Konermann, L. J. Am. Soc. Mass Spectrom. 2003, 14, 430441. (30) Clark, S. M.; Konermann, L. Anal. Chem. 2004, 76, 1257-1263. (31) Clark, S. M.; Leaist, D. G.; Konermann, L. Rapid Commun. Mass Spectrom. 2002, 16, 1454-1462.

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Figure 1. Schematic diagram illustrating how solution-phase diffusion coefficients can be measured by ESI-MS (see ref 31). A thin capillary tube is initially filled with solution having a low analyte concentration (yellow). The tube inlet is then connected to a reservoir that contains analyte solution at a somewhat higher concentration (blue), thus generating a sharp boundary at the tube inlet (A). Subsequently, solution is continuously pumped from the reservoir into the tube. Laminar flow leads to a parabolic velocity distribution within the tube; i.e., the flow velocity along the center line is highest, whereas it is zero at the tube walls (as indicated by the arrows in panels B and C). This velocity distribution tends to distort the initially sharp boundary into a parabola. However, radial diffusion of analyte molecules counteracts this process, because the molecules constantly migrate between zones of different flow velocity. This compensating effect is relatively small in the case of slow diffusion (B), but it is large in the case of rapid diffusion (C). Diffusion coefficients can thus be determined by monitoring the analyte signal intensity at the tube outlet by ESI-MS. All of the dispersion profiles obtained in this way have a sigmoidal appearance. An analyte with a small diffusion coefficient shows a more extended profile (B) than one that diffuses more rapidly (C). Note that the effects of axial diffusion are negligible under typical operating conditions.

the tube outlet or by employing harsh declustering voltages in the ion sampling interface. Consequently, this method does not rely on the survival of noncovalent interactions in the gas phase. We previously used this diffusion-based technique for the qualitative detection of ligand-protein interactions.29,30 Here, we demonstrate an extension of this method that allows the quantification of ligand-protein dissociation constants. Two different systems were chosen for the evaluation of the approach presented in this study, namely, benzamidine-trypsin and the complex of N,N′,N′′-triacetylchitotriose (NAG3) with lysozyme. Benzamidine (C6H5C(:NH)NH2, 120.1 Da) is a competitive inhibitor of trypsin (23 293 Da) and many other serine proteases.32,33 Trypsin cleaves peptide bonds on the C-terminal side of arginine and lysine residues. Benzamidine binds specifically to the active site of the enzyme. The amidinium group of benzamidine mimics the guanidinium side chain of arginine, and the noncovalent complex is stabilized by a salt bridge that is reinforced by two hydrogen bonds, linking the amidinium group and Asp189. In addition, there are three other hydrogen bonds connecting the protein to the ligand.34-36 The benzyl ring of (32) Keil, B. In The Enzymes, 3rd ed.; Boyer, P. D., Ed.; Academic Press: New York, 1971; Vol. 3, pp 249-275. (33) Casale, E.; Collyer, C.; Ascenzi, P.; Balliano, G.; Milla, P.; Viola, F.; Fasano, M.; Menegatti, E.; Bolognesi, M. Biophys. Chem. 1995, 54, 75-81. (34) Peters, L.; Frohlich, R.; Boyd, A. S. F.; Kraft, A. J. Org. Chem. 2001, 66, 3291-3298.

benzamidine forms favorable van der Waals contacts with the protein, but hydrophobic interactions also contribute to the stability of the complex.36 Lysozyme (14 305 Da) is an enzyme that hydrolyzes polysaccharides containing N-acetylated monomers. The binding specificity of the inhibitor NAG3 (627.6 Da) is generated by an extensive network of hydrogen bonds, both to the protein itself and to bound water molecules.37 It will be shown that diffusion measurements allow the quantification of the binding affinity for both systems, although only one of the complexes is amenable to direct observation by ESI-MS. THEORETICAL BACKGROUND Following a concept borrowed from NMR-based studies,38,39 the apparent diffusion coefficient of a ligand in the presence of its protein receptor, DA, can be approximated as

DA ) fLDL + (1 - fL)DP

(1)

where DL and DP are the diffusion coefficients of the free ligand and the protein, respectively, and where fL is the fraction of ligand that is free in solution. Equation 1 is based on the commonly made assumption that the diffusion coefficient of the protein does not change upon binding to a small ligand.39 Furthermore, it is assumed that the binding equilibrium is fast compared to the time scale of the experiment. This “fast-exchange limit” has been wellcharacterized in NMR studies, which have a typical time scale on the order of 0.01 s. When the association rate constant is diffusionlimited (107-108 M-1 s-1), Kd values down to around 10-5 M are within the fast-exchange limit.40-43 Since the time scale of the diffusion measurements presented here is on the order of 1000 s, dissociation constants as low as 10-10 M should theoretically be accessible in our assays. The measurement of the three diffusion coefficients in eq 1 by ESI-MS allows fL to be determined. Thus, the concentrations of free ligand, [L], free protein, [P], and ligand-protein complex, [LP], can be calculated such that the dissociation constant Kd ) [L][P]/[LP] can be expressed as

Kd )

fL([P]T - (1 - fL)[L]T) (1 - fL)

(2)

concentrations for which dissociation constants can be accurately measured will now be investigated. For any Kd measurement that monitors the fraction of free ligand in solution, it is commonly accepted that fL should be in the range of42

0.1 e fL e 0.9

This constitutes the first condition for the allowable range. Figure 2A shows fL as a function of the ligand and protein concentrations relative to Kd, [L]T/Kd and [P]T/Kd, respectively. It is also necessary to ensure that the macromolecule is not saturated by binding to ligand. Figure 2B shows the fraction of free protein, fP, in a similar diagram, where fP ) ([P]T - (1 - fL)[L]T)/[P]T. The second condition for the allowable range stipulates that

0.1 e fP

(35) Ota, N.; Stroupe, C.; Ferreira-da-Silva, J. M. S.; Shah, S. A.; Mares-Guia, M.; Bunger, A. T. Proteins: Struct. Funct., Genet. 1999, 37, 641-653. (36) Talhout, R.; Engberts, J. B. F. N. Eur. J. Biochem. 2001, 268, 1554-1560. (37) Cheetham, J. C.; Artymiuk, P. J.; Phillips, D. C. J. Mol. Biol. 1992, 224, 613-326. (38) Gounarides, J. S.; Chen, A.; Shapiro, M. J. J. Chromatogr., B 1999, 725, 79-90. (39) Fielding, L. Tetrahedron 2000, 56, 6151-6170. (40) Vogtherr, M.; Fiebig, K. In Modern Methods of Drug Discovery; Hillisch, A., Hilgenfeld, R., Eds.; Birkhauser Verlag: Boston, 2003; pp 183-202. (41) Hajduk, P.; Meadows, R. P.; Fesik, S. W. Q. Rev. Biophys. 1999, 32, 211240. (42) Connors, K. A. Binding Constants; John Wiley & Sons: Toronto, 1987. (43) Feeney, J.; Batchelor, J. G.; Albrand, J. P.; Roberts, G. C. K. J. Magn. Res. 1979, 33, 519-529.

(4)

Although these first two conditions are general and apply to the measurement of Kd values by many different techniques, there are additional considerations that have to be taken into account for the approach used here. For the experiments carried out in the present work, the ligand concentration changes by a factor of 2 over the course of the measurement. Thus, if the average total ligand concentration is [L]0T, the value of [L]T will change from 2/ [L]0 to 11/ [L]0 , while [P] remains constant. It will now be 3 3 T T T determined what ranges of ligand and protein concentrations are acceptable for a reliable determination of Kd. Changes in [L]T will affect the value of fL. Under certain conditions, this could represent a problem, because large changes in fL preclude an accurate in measurement of DA (see eq 1). If fL varies between f low L a solution containing 2/3[L]0T, and f high in a solution containing L - f high 11/3[L]0T, then ∆fL represents the difference |f low L L |. The relative change in the fraction of ligand that is free in solution is then given by ∆fL/f 0L, where f 0L is the fraction of ligand free in solution when [L]T ) [L]0T. ∆fL/f 0L is plotted as a function of [L]T/Kd and [P]T/Kd in Figure 2C. Model calculations show that a reasonable third boundary condition for the allowable range is given by

∆fL where [L]T and [P]T are the total ligand and protein concentrations, respectively. The described approach imposes certain restrictions on both the ligand and the protein concentrations that can be used in the binding assays. The “allowable range” of

(3)

fL0

e 0.15

(5)

Finally, [L]T is used to calculate Kd according to eq 2. Therefore, the experiment should be done under conditions for which the measured Kd does not show a pronounced variation when the calculation is based on either [L]T ) 2/3[L]0T or [L]T ) 11/3[L]0T. For [L]T ) 11/3[L]0T, the relative deviation in the 0 high is the difference calculated Kd is ∆Khigh d /Kd, where ∆Kd between the Kd calculated using [L]T ) [L]0T and [L]T ) 11/3[L]0T, and K0d is the dissociation constant calculated for [L]T 0 ) [L]0T. A graphic representation of ∆Khigh d /Kd is given in Figure 2D. A virtually identical plot is observed when a value of [L]T ) 2/ [L]0 is used to calculate the relative deviation in K , i.e., for 3 d T 0 low is the difference between determining ∆Klow d /Kd, where ∆Kd the Kd calculated from [L]T ) [L]0T and that calculated from [L]T ) 2/3[L]0T (data not shown). It is reasonable to express this Analytical Chemistry, Vol. 76, No. 23, December 1, 2004

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Figure 2. Visualization of four conditions that have to be satisfied for the determination of Kd values by ESI-MS-based diffusion measurements. The two axes represent the total ligand and protein concentrations relative to Kd, that is, [L]T/Kd and [P]T/Kd, respectively. (A) fraction of free ligand, fL; (B) fraction of free protein, fP; (C) relative change in fL when the ligand concentration changes by a factor of 2, ∆fL/f 0L; (D) relative 0 deviation ∆Khigh d /Kd. (E) “Allowable range” (brown) for Kd determinations, based on conditions 3-6 (see text for more details). (F) Same as (E), plotted on a linear scale. Crosses correspond to experiments on benzamidine-trypsin; circles represent NAG3-lysozyme measurements.

fourth boundary condition as

∆Klow d K0d ∆Khigh d K0d

e 0.1

e 0.1

(6a)

(6b)

The allowable region based on a combination of conditions 3-6 is shown on a logarithmic scale in Figure 2E and on a linear scale in Figure 2F (brown areas). If the ligand and protein concentrations are chosen to be within this region, Kd values can be calculated on the basis of eqs 1 and 2 by using the approximation that [L]T ) [L]0T. The experiments described below demonstrate that the definition of the allowed region is fairly rigorous, meaning that even concentration combinations that are close to the edge of the brown areas in Figure 2E,F still allow Kd values to be determined accurately. To illustrate the implications of these allowed regions, consider the following example. For a dissociation constant of 10 µM, a protein concentration of 60 µM represents the center of the x axis in Figure 2F. The corresponding allowed range for [L]T/Kd includes all values below ∼1.5; i.e., the ligand concentrations used have to be less than ∼15 µM. Similarly, for a Kd value of 10 nM and a protein concentration of 60 nM, the ligand concentrations would have to be less than ∼15 nM. These considerations make it clear that the range of dissociation constants that is accessible by diffusion measurements will ultimately be limited by the sensitivity of the instrumentation used. Roughly speaking, the mass spectrometer has to be able to provide dispersion profiles with acceptable signal-to-noise ratio for ligand and protein concentrations that are on the same order of magnitude as the dissociation constant of the system. In other words, at the current 7080

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stage of development, the approach presented here will be most useful for measuring Kd values in the high nanomolar to millimolar range. EXPERIMENTAL SECTION Chemicals. Hen egg white lysozyme, bovine pancreas R-trypsin, ammonium acetate, and benzamidine hydrochloride were purchased from Sigma (St. Louis, MO). Glacial acetic acid and HPLCgrade methanol were products of Fisher Scientific (Nepean, ON, Canada). NAG3 was obtained from Seikagaku America (East Falmouth, MA), and lithium chloride was from Merck (Darmstadt, Germany). All chemicals were used without further purification. Solutions were prepared in freshly distilled water prepurified by reverse osmosis. The lysozyme concentration was determined by the absorption at 280 nm using an extinction coefficient of 37 932 M-1 cm-1.44 Since the trypsin used had a purity of >99%, solutions were made up to the desired concentration by weight. No significant autolysis of trypsin solutions was observed by ESI-MS over a period of 8 h. Nevertheless, fresh solutions were made every 5 h in order to avoid small changes in [P]T due to autolysis. Solution pH values were measured using an Accumet pH meter (Fisher Scientific, Nepean, ON, Canada). ESI-MS. Mass spectra and dispersion profiles were acquired on an API 365 triple quadrupole mass spectrometer (Sciex, Concord, ON, Canada) in positive ion mode using pneumatically assisted ESI (ion spray). Dispersion profiles were recorded by selected ion monitoring with a dwell time of 200 ms. Prior to data analysis, groups of 5 data points were averaged to give an effective dwell time of 1 s. The deconvolution of mass spectra was carried out using the BioMultiView software package supplied by the instrument manufacturer. Diffusion Measurements. Dispersion profiles were recorded as described previously.31 Slightly different Teflon flow tubes (44) Gill, S. C.; von Hippel, P. H. Anal. Biochem. 1989, 182, 319-326.

(Upchurch, Oak Harbor, WA) with lengths of ∼3 m and inner diameters around 265 µm were used for individual experiments. All solutions containing benzamidine or trypsin also contained 10 mM ammonium acetate, giving a pH of 6.5, whereas those containing NAG3 or lysozyme contained 1 mM ammonium acetate for a pH of 6.7. Each experiment involved the use of two solutions, termed 1 and 2, between which the initially sharp boundary was generated. For benzamidine/trypsin experiments, the flow tube solutions 1 and 2 and the makeup solvent were introduced at 7 µL/min, giving a total flow rate of 14 µL/min at the ion source. The makeup solvent used consisted of methanol/acetic acid (90:10 v/v). Benzamidine was detected as a singly protonated ion. A combination of orifice, ring, and skimmer voltages of 5, 95, and 0 V, respectively, resulted in the best signal stability for benzamidine. Apparent diffusion coefficients of benzamidine in the presence of trypsin were determined by using benzamidine concentrations of 5 and 10 µM in solutions 1 and 2, respectively, corresponding to [L]0T ) 7.5 µM. The trypsin concentration was identical in both solutions (details are given in the Results and Discussion section). Dispersion profiles of trypsin were recorded by monitoring the intensity of [M + 10H]10+ ions, using protein concentrations of 5 and 10 µM in solutions 1 and 2, respectively. The corresponding concentrations used for the determination of the benzamidine diffusion coefficient in the absence of protein were 1 and 2 µM. For NAG3/lysozyme experiments, a flow rate of 10 µL/min was used for both the flow tube solution and for the makeup solvent. The latter consisted of water/methanol/acetic acid (5:85:10 v/v/v) and contained 5 mM LiCl; NAG3 was detected as singly a lithiated ion. The orifice, ring, and skimmer voltages used were 100, 300, and 0 V, respectively. The experiments were carried out with [L]0T ) 7.5 µM. Lysozyme dispersion profiles were recorded by monitoring [lysozyme + 9H]9+ as a function of time, using protein concentrations of 5 and 10 µM in the two solutions, respectively. In addition, both solutions contained 1 mM ammonium acetate. The reported diffusion coefficients, fL, and Kd values represent the average of at least three independent measurements. Errors correspond to one standard deviation. All experiments were carried out at room temperature (22 ( 2 °C). RESULTS AND DISCUSSION Benzamidine-Trypsin. In favorable cases, the dissociation constant of a noncovalent ligand-protein complex can be determined directly from the ESI mass spectrum of the corresponding ligand-protein mixture. This approach requires the ion abundance ratio of ligand-bound to free protein to reflect the concentration ratio of the two species in solution. Kd can then be calculated on the basis of the observed peak intensities.10-13 Figure 3 provides an example of a ligand-protein system in which this simple method fails. It shows the ESI mass spectrum of 10 µM trypsin in the presence of 50 µM benzamidine, recorded under “soft” ionization conditions. The solution also contained 10 mM ammonium acetate. On the basis of reported Kd values between 18 and 39 µM,32,45 the ion abundance ratio of ligand-bound to free protein would be expected to be at least 1:1. Surprisingly, no ions corresponding to the benzamidine-trypsin complex are observed. (45) Rauh, D.; Klebe, G.; Sturzebecher, J.; Stubbs, M. T. J. Mol. Biol. 2003, 330, 761-770.

Figure 3. (A) ESI mass spectrum of 10 µM trypsin measured in the presence of 50 µM benzamidine. The data were recorded using “soft” ionization conditions (orifice, ring, and skimmer potentials of 0, 80, and 0 V, respectively). The protonation state of the most intense peak, corresponding to [trypsin + 9H]9+ ions, is indicated. (B) Deconvoluted mass distribution of the data depicted in panel A. Peaks labeled with “#” in panel B are separated by 18 Da and have been tentatively assigned to water adducts. The relative contribution of these peaks could be drastically reduced by using higher orifice voltages (data not shown). “X” indicates the position where the benzamidine-trypsin complex would be expected.

The same result was obtained at increased benzamidine concentrations, at which more than 96% of the protein is expected to be bound to the ligand. The complex was also undetectable when the experiments were repeated in negative ion mode (data not shown). The benzamidine-trypsin complex is stabilized by a salt bridge, intermolecular hydrogen bonds, and by van der Waals forces.35 In addition, hydrophobic interactions play a role,36 thus lending some support to the notion that complexes exhibiting this type of interaction are difficult to observe by ESI-MS.14,16 Nonetheless, the total lack of benzamidine-trypsin complex ions in our experiments is unexpected. We cannot rule out the possibility that instruments equipped with different types of ion sources, sampling interfaces, and mass analyzers may provide results that differ from those shown in Figure 3. It is noted, however, that other noncovalent complexes are readily observable under the conditions employed here (see below). In any case, data like those depicted in Figure 3 demonstrate the need for methods that allow studies on ligand-protein complexes without relying on the preservation of noncovalent interactions in the gas phase. Figure 4 demonstrates the application of the proposed diffusion-based approach to the benzamidine-trypsin system, for a protein concentration of 80 µM. As predicted by eq 1, the dispersion profile of benzamidine in the presence of trypsin (Figure 4A) is intermediate in steepness between that of benzamidine alone (Figure 4B), and that of trypsin alone (Figure 4C). This behavior clearly confirms that benzamidine does, indeed, Analytical Chemistry, Vol. 76, No. 23, December 1, 2004

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Figure 4. ESI-MS dispersion profiles of (A) benzamidine in the presence of 80 µM trypsin, (B) benzamidine, and (C) trypsin. The benzamidine concentration change in (A) was from 5 to 10 µM, for an [L]0T value of 7.5 µM in eq 2. Solid lines are fits to the experimental data based on eq 17 of ref 31.

Figure 6. (A) ESI mass spectrum of 10 µM lysozyme measured in the presence of 10 µM NAG3. The data were recorded using the same “soft” instrument settings as for the spectrum shown in Figure 3. Ions corresponding to free lysozyme and to the noncovalent NAG3lysozyme complex are labeled as “lyso” and “NAG3 lyso”, respectively. (B) Deconvoluted mass distribution of the data depicted in panel A. Figure 5. Apparent diffusion coefficient, DA, of benzamidine measured for different trypsin concentrations. Also shown are the Kd values measured for 20, 50, and 80 µM trypsin. The dotted line indicates the diffusion coefficient of trypsin.

form a noncovalent complex with trypsin. The diffusion coefficients DA, DL, and DP can be determined from fits to the experimental profiles depicted in Figure 4, resulting in values of (3.9 ( 0.5), (7.9 ( 0.5), and (0.96 ( 0.05) × 10-10 m2 s-1, respectively. According to eq 1, these diffusion coefficients correspond to fL ) 0.43 ( 0.08, thus yielding a Kd value for benzamidine-trypsin of (60 ( 20) µM. A decrease of the protein concentration shifts the binding equilibrium more toward the dissociated species, thus resulting in an increased DA value for benzamidine. This trend is depicted in Figure 5. In addition, Figure 5 shows that the measured dissociation constant is independent of the protein concentration used in the assay, within error limits. Note that all three combinations of ligand and protein concentrations used in Figure 5 fall within the allowed range, as indicated by the crosses in Figure 2E,F. Averaging the data obtained from these three sets of experiments, ESI-MS-based diffusion measurements result in a Kd value for benzamidine-trypsin of 50 ( 10 µM. This is in reasonable agreement with dissociation constants from the literature, which range from 18 to 39 µM.32, 45 NAG3-Lysozyme. In contrast to the benzamidine-trypsin system, NAG3-lysozyme is “well-behaved”, in the sense that gasphase ions of this complex can be directly observed by ESI-MS.4 A mass spectrum recorded for NAG3 and lysozyme concentrations of 10 µM, in the presence of 1 mM ammonium acetate, is depicted in Figure 6. It is noted that these data were acquired by using the same “gentle” interface voltages that had been employed for the benzamidine-trypsin system in Figure 3. Close inspection of 7082 Analytical Chemistry, Vol. 76, No. 23, December 1, 2004

Figure 7. ESI-MS dispersion profiles of (A) NAG3 in the presence of 42 µM lysozyme, (B) NAG3, and (C) lysozyme. Further explanations are given in the text and in the caption of Figure 4.

Figure 6A reveals that the peak intensity ratio of NAG3 bound to free lysozyme decreases with increasing charge state. This trend is not surprising, given the fact that higher charge states will be more affected by collision-induced dissociation in the ion-sampling interface. The lowest charge state that allows a reliable determination of peak intensities is 8+, in which case the measured ratio (0.91) corresponds to a Kd value of 6 µM.10 The average ratio that takes into account all charge states (0.62, obtained from the deconvoluted mass distribution in Figure 6B) results in a somewhat higher dissociation constant of 10 µM. Both of these results are in line with literature values for the NAG3-lysozyme system, ranging from 5 to 19 µM.46 Figure 7 depicts dispersion profiles recorded in order to determine the Kd value of NAG3-lysozyme by diffusion measurements. Figure 7A represents a NAG3 profile recorded in the presence of 42 µM lysozyme, which corresponds to a diffusion (46) Imoto, T.; Johnson, L. N.; North, A. C. T.; Phillips, D. C.; Rupley, J. A. In The Enzymes; Boyer, P. D., Ed.; Academic Press: New York, 1972; Vol. 7, pp 666-836.

coefficient of DA ) (1.7 ( 0.1) × 10-10 m2 s-1. The diffusion coefficients of free NAG3 and lysozyme were found to be DL ) (4.5 ( 0.1) × 10-10 m2 s-1 (Figure 7B) and DP ) (1.2 ( 0.2) × 10-10 m2 s-1 (Figure 7C), respectively. These data result in an fL value of 0.15 ( 0.01 and a dissociation constant of (6 ( 1) µM. Within error limits, the same Kd value was determined at protein concentrations of 34 and 53 µM, indicated by the filled circles in Figure 2E,F. This measured dissociation constant is consistent with the corresponding literature values, and it agrees reasonably well with the Kd value determined directly from the ESI mass spectrum, as discussed in the previous paragraph. CONCLUSIONS Diffusion measurements by ESI-MS allow the determination of ligand-protein dissociation constants, even in cases in which the ratio of free to ligand-bound protein in the mass spectrum does not reflect the solution-phase binding equilibrium. The diffusion-based approach employed in this study is inherently simple; it does not involve any isotopic labeling or chemical immobilization steps while taking advantage of the sensitivity and selectivity of ESI-MS. It appears that this novel technique could provide a valuable addition to currently existing methods for the

quantification of ligand-protein interactions and also for monitoring the interactions of ligands with other macromolecular receptors, such as nucleic acids. The direct observation of noncovalent complexes by ESI-MS remains an extremely valuable approach; however, future work is required to develop a better understanding of the factors determining the ability of noncovalent assemblies to survive the ESI process. ACKNOWLEDGMENT We thank Yuhong Liu and Derek J. Wilson for help with some of the control experiments for this study. We also thank John S. Klassen for helpful discussions regarding the detectability of the trypsin-benzamidine complex in the gas phase. This work was financially supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canada Foundation for Innovation (CFI), the Provincial Government of Ontario, and the Canada Research Chairs Program.

Received for review May 4, 2004. Accepted September 9, 2004. AC049344O

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