Quantifying Protein-Ligand Binding Constants using Electrospray Ionization Mass Spectrometry: A Systematic Binding Affinity Study of a Series of Hydrophobically Modified Trypsin Inhibitors
NanoESI-MS is used for determining binding strengths of trypsin in complex with two different series of five congeneric inhibitors, whose binding affinity in solution depends on the size of the P3 substituent. The ligands of the first series contain a 4-amidinobenzylamide as P1 residue, and form a tight complex with trypsin. The inhibitors of the second series have a 2-aminomethyl-5-chloro-benzylamide as P1 group, and represent a model system for weak binders. The five different inhibitors of each group are based on the same scaffold and differ only in the length of the hydrophobic side chain of their P3 residue, which modulates the interactions in the S3/4 binding pocket of trypsin. The dissociation constants (KD) for high affinity ligands investigated by nanoESI-MS ranges from 15 nM to 450 nM and decreases with larger hydrophobic P3 side chains. Collision-induced dissociation (CID) experiments of five trypsin and benzamidine-based complexes show a correlation between trends in KD and gas-phase stability. For the second inhibitor series we could show that the effect of imidazole, a small stabilizing additive, can avoid the dissociation of the complex ions and as a result increases the relative abundance of weakly bound complexes. Here the KD values ranging from 2.9 to 17.6 μM, some 1–2 orders of magnitude lower than the first series. For both ligand series, the dissociation constants (KD) measured via nanoESI-MS were compared with kinetic inhibition constants (Ki) in solution.
Key wordsNoncovalent interactions Electrospray ionization mass spectrometry Binding affinity Trypsin Protein–ligand complexes Hydrophobic effect
Electrospray ionization mass spectrometry (ESI-MS) is a powerful and increasingly utilized tool for the investigation of noncovalent interactions . This soft ionization technique allows the transfer of noncovalent complexes from solution into the gas phase and their subsequent study by mass spectrometry. To this day, protein–protein, protein–small molecule, protein–DNA, and DNA–small molecule complexes have been successfully detected and studied by this method [2, 3, 4]. Especially in drug discovery ESI-MS is of increasing importance for the investigation of protein-ligand interactions and determination of binding affinities [5, 6]. In recent years, binding affinities (KD) have been successfully determined by ESI-MS for a variety of noncovalent protein-ligand complexes [2, 7, 8]. Other MS-based methods have also been successfully applied for quantifying interactions (e.g., methods dubbed “protein–ligand interactions in solution by MS, titration and H/D exchange” (PLIMSTEX) and “stability of unpurified proteins from rates of H/D exchange” (SUPREX) [9, 10, 11].
Noncovalent interactions are of great importance in nature; for example they play a major role in stabilizing protein conformation. The hydrophobic effect plays an important role in protein folding, in the adhesion of lipid bilayers, nucleic acid structures, and protein-small molecule interactions [12, 13, 14]. Compared with the aqueous environment, the hydration shell is absent in the gas phase, and its not yet fully clear whether the conformation of noncovalent complexes remains unchanged during the transition from solution to vacuum . Some forces such as hydrogen-bonding and electrostatic interactions between two oppositely charged molecules are strengthened in the gas phase, while hydrophobic interactions are weakened and therefore difficult to preserve during ionization and ion transfer [16, 17]. A number of research groups have reported investigations of noncovalent complexes where hydrophobic interactions play a dominant role for the complex stability [18, 19, 20]. In a very recent study, Klassen and co-workers [21, 22] demonstrated the application of ESI-MS to quantify binding strengths of β-lactoglobulin - fatty acid complexes in aqueous solution. For three short fatty acids, association constants smaller than expected were found by the authors (Ka compared with data from a competitive fluorescence assay). They explained this with an in-source dissociation, which reduces the relative abundance of gaseous complex ions measured by ESI-MS. In a previous study of the same research group it was shown that β-lactoglobulin retains the structure of its binding cavity even in the absence of a hydration shell . The authors monitored the dissociation of the fatty acid-protein complexes in a BIRD experiment and extracted the temperature-dependent kinetic parameters. Their results show that the energy required for dissociation correlates with the length of the hydrocarbon fatty acid chain. Surprisingly, quantitative comparison of the dissociation rate constants in the hydrated and dehydrated states showed that the solvated complex is kinetically less stable than the corresponding gaseous ions at all temperature investigated .
Even when carefully controlling the instrument parameters, complexes that are predominatly stabilized by nonpolar interactions are prone to dissociation in the gas phase . This so-called in-source dissociation can lead to an artificially low binding constants based on the reduced abundance of the complex ions . Stabilization by addition of imidazole to the nanoES solution was presented as a solution for this problem for several weakly bound complexes [26, 27]. The small imidazole molecule acts as a nonspecific, sacrificial ligand and can prevent dissociation of the specifically bound ligand during ES-MS analysis. This can also be thought of as enhanced evaporative cooling of the protein-ligand complex ions in the ion source. The extent of the stabilization depends strongly on the concentration of imidazole. At high imidazole concentrations (>1 mM), the ions of protein-fatty acid, protein-carbohydrate and protein-small molecule complexes can be stabilized , although it is not always possible to prevent dissociation of very labile gas-phase complexes, [26, 28, 29].
To determine binding constants, we used the ES-MS titration method, which has been found suitable for measuring binding strengths, not only in our laboratory but also in other groups [2, 18, 30, 31, 32]. It has also been validated against more established biophysical methods, such as isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), and nuclear magnetic resonance (NMR) spectroscopy [32, 33, 34, 35, 36]. The well-described titration method relies on detection of ions belonging to the complex versus bare proteins. The KD value can then be easily determined from a fit of the intensity ratio of bound and unbound protein as a function of the added ligand. This method assumes that no dissociation takes place during the transmission through the mass spectrometer. The second important assumption is that the intensity ratio observed in the gas phase correlates with the concentration ratio in solution. If a very low-mass ligand is bound to a high-mass protein, the ionization efficiency does not change for the complex vs. the bare protein, and this assumption is fulfilled in almost all cases . As the ESI titration measurements can deliver a “snapshot” of the solution concentrations the KD values determined via nanoESI-MS reflect solution-phase binding affinities.
Chemical structures of the scaffold of the investigated benzamidine- and CMA-type ligands. The residues (R) indicate the substructure of the inhibitor, which binds into the hydrophobic S3/4 pocket of trypsin. This part of the ligand was systematically varied in the size of the hydrophobic chain
The basic question is whether we can observe the expected trend in the binding affinity of the inhibitors, when using ESI-MS as a read-out for the solution phase equilibrium. In the case of the benzamidine-based ligands, the binding affinity based on ESI titration measurements increases with increasing length of the hydrophobic P3 side chain, from 15 to 450 nM. A second question concerns the stability in solution vs. in the gas phase. In some studies it was shown that the gas-phase stability reflects the binding properties in solution [37, 38, 39]. More frequently, however, a correlation between the gas-phase stability and the solution-phase stability is absent [25, 26, 28, 40, 41, 42, 43, 44, 45, 46], for example for leucine-zippers and acyl-CoA binding protein (ACBP) and a series of acyl CoA derivatives . If binding properties in solution correlate with gas-phase stability it has to be assumed that the dominant interactions are very similar in solution and in the gas phase, and that solvent mediation play only a minor role (see Daniel at al. and Sharon and Robinson for comprehensive reviews on this topic [2, 46]). In our study the CID experiments for the benzamidine complexes show a correlation between the binding affinities in solution and the gas phase stability.
The complexes with the CMA inhibitors were prone to in-source dissociation. The main reason may be that complexes stabilized in solution by weak nonpolar interactions exhibit low gas-phase stability . Therefore we have investigated the stabilizing effect of imidazole on this particular model system. Like Klassen and co-workers [21, 22] we found that the addition of imidazole to the nanoESI solution can protect protein-ligand complexes from in-source dissociation during the ESI-MS analysis process. Unlike in the case of the benzamidine series, the trend of higher binding affinity is clearly observed, with the order Gly < d-Ala < d-Leu. No independently measured KDs are available for these systems. Therefore, binding affinities determined by MS were compared with inhibition constants (Ki) determined via an enzyme kinetic inhibition assay.
2.1 Materials and Methods
Bovine pancreas α-trypsin (MW ≈ 23.300 Da), ammonium acetate, and CsI were purchased from Sigma Aldrich (Buchs, Switzerland). Imidazole (99.5 % purity) and DMSO were obtained from Fluka Chemie AG. Water was purified using a Milli-Q Ultrapure water purification system by Millipore (Barnstead, IA / USA). All MS titration experiments were recorded under “native-like” conditions using 50 mM ammonium acetate buffer at pH = 7.8. Stock solutions of ligands were prepared at 40–50 mM concentration in DMSO. Prior to the measurement, the inhibitor solutions were diluted with Milli-Q water to the desired concentration. The protein working solution was made from a 100 μM stock solution in ammonium acetate buffer. The exact trypsin concentration was determined using a UV spectrometer (NanoDrop 1000; Witec AG, Littau, Switzerland).
2.2 Mass Spectrometry
ESI spectra were acquired with a hybrid quadrupole time-of-flight mass spectrometer (Q-TOF ULTIMA; Waters/Micromass, Manchester, UK) in the positive ion mode. The instrument was controlled via the MassLynx ver. 4.0 software. In order to obtain a good signal-to-noise ratio, 100 scans were accumulated for one spectrum. The mass spectrometer is equipped with an automated chip-based nanoESI system (Nanomate 100; Advion Biosciences, Ithaca, NY, USA). It has a 96-well sample plate, a rack of 96 disposable, conductive pipet tips, and a nanospray chip containing 20 × 20 nozzles of 5 μm diameter. For investigation of noncovalent complexes, appropriate instrumental conditions have to be found. In this case the desolvation must be sufficiently complete in order to get narrow peaks for the detected species but not to dissociate the noncovalent complex. This can take an influence on peak broadening because of the adduct formation with salt and buffer molecules from the spray solution. The settings described below were found to be a good compromise between the intact complex detection and sufficient desolvation of analytes. For all nanoESI-MS measurements the voltage was set to 1.8–1.9 kV and a gentle backing pressure of 5 bar on the spray tip was used to assist the liquid sample flow. The source temperature was kept at 21 °C. To prevent dissociation of the noncovalent complexes, the mass spectrometer was run with gentle desolvation parameters. The cone and first ion tunnel RF1 voltages, the parameters that control the kinetic energy of the ions in the source region of the mass spectrometer, were optimized to 40 V and 35 V. After this stage, the ion beam passed a hexapole collision cell filled with argon (purity 5.0; PanGas). Collision-induced dissociation (CID) used in MS/MS experiments were preformed by adjusting the acceleration collision energy (CE) voltage until full dissociation of the parent complex ions was achieved. Calibration of the mass spectrometry instrument was performed using CsI clusters. The concentration of CsI was 2 μg/uL dissolved in water/2-propanol (1/1, vol/vol).
2.3 Data Processing
It was assumed here that the ionization efficiency for the bare protein and the complex is equal, which allowed us to use the intensity ratios of free protein over complex instead of their concentrations in solution. The KD calculations and the fitting of the titration curves were performed using MATLAB software (2010a, The MathWorks, Natick, MA, USA).
2.4 Kinetic Inhibition Assay
Kinetic inhibition of bovine trypsin was determined photometrically at 405 nm using the chromogenic substrate Pefachrom tPa (LoxoGmbH, Dossenheim, Germany) according to the protocols described by Stürzebecher et al.  under the following conditions: 50 mM Tris/HCl (pH 8.0), 154 mM NaCl, 5 % DMSO, and 0.1 % polyethylene glycol (PEG) 8000 at 25 °C using different concentrations of substrate and inhibitor. Ki values were determined at least in triplicate.
3 Results and Discussion
3.1 KD-Determination of the Benzamidine-Based Inhibitors by the nanoESI-MS Titration Method
The stabilizing effect of imidazole, explained in detail below, was also tested on this particular model system. To test the influence of imidazole, a small stabilizing solution additive, we chose the d-Val-inhibitor. However, upon addition of 10 mM imidazole to the nanoES solution, no change of the complex ions to the free protein was observed (data not shown). The only observable effect of imidazole was a shift of the charge state distribution to lower charge states. Since the ESI titration measurements were performed under “soft” sampling conditions and the benzamidine inhibitors bind specifically into all trypsin pockets, we assume that complexes are stable and not prone to dissociation in the gas phase.
List of calculated dissociation constants (KD) for five benzamidine based ligands and trypsin determined by the nanoESI titration method, and Ki values from a kinetic inhibition assay determined in solution for trypsin and thrombin
Benzamidine series (R)
Kinetic inhibition assay trypsinb
Kinetic inhibition assay thrombinb
15.0 ± 2.5
0.95 ± 0.18
0.12 ± 0.01
79.3 ± 5.9
1.43 ± 0.18
0.89 ± 0.12
87.1 ± 15.2
0.52 ± 0.11
1.29 ± 0.35
193.5 ± 17.8
1.95 ± 0.77; 5 ± 3c
5.2 ± 0.8
449.7 ± 75.9
15.9 ± 3.06; 37 ± 3c
3.7 ± 0.6
The discrepancy between KD and Ki values can be easily explained by different assay conditions: for the MS-based KD determination, ammonium acetate was used as a buffer, while for the kinetic inhibition assay, a buffer solution of 50 mM Tris/HCl (pH 8.0), 154 mM NaCl, 5 % DMSO, and 0.1 % polyethylene glycol (PEG) was used. Different buffer solutions may have a substantial influence on the binding affinities for benzamidine-trypsin complexes. Since benzamidine inhibitors are very potent, the Ki determination using chromogenic substrate are close to the instrument limit of detection. The consequence is that even marginal measurement inaccuracies would have significant influence on the Ki values. This might be a possible explanation for observing higher binding affinity for d-Val instead of d-Cha. However, with the exception of d-Val, the KDs and the Ki values that were determined show that relative binding affinities of the different inhibitors can be successfully determined, and that the KD and Ki values decrease with larger hydrophobic side chain.
3.2 Gas Phase Stability versus Binding Affinity
Several groups have tried to find a correlation between the gas-phase stability and the type of interactions involved in a complex. As already mentioned in the Introduction, it is important to note that the gas-phase stabilities of noncovalent complexes generally do not correlate with solution binding affinities.
3.3 KD-Determination of the CMA series by nanoESI-MS Titration Method
The stabilization effect of imidazole in the presence of 25 μM Gly-inhibitor is illustrated in Figure S1 (Supporting Information). The nanoESI measurements were acquired under identical conditions as those in Figure 6 but now in the presence of 10 mM imidazole in the ES-solution. Upon its addition to the nanoES solution, nonspecific adducts between the imidazole molecules and trypsin-ligand complexes can be formed. The nonspecific interactions should be kinetically less stable compared to specific trypsin-inhibitor complexes. After loss of the nonspecific interactions in the hexapole region, the internal energy of trypsin-ligand ions in the source should be lowered, thereby stabilizing the complex as shown in the spectra, the addition of imidazole to Gly-inhibitor/trypsin nanoES solution results in a double increase in the relative abundance of the complex ions. The important effect of this small molecule is the constant relative complex abundance measured over the longer accumulation time (data not shown). Also, the addition of imidazole reduces the average charge state distribution of the Gly-inhibitor/trypsin complex from n = 9–7 to n = 8–6 (Figure S1, Supporting Information). This stabilizing effect can be explained through enhanced cooling from imidazole evaporation that delays the dissociation. Coulomb repulsion can be minimized due to the reduced net charge state of the protein that stabilizes the complex . Due to the high gas-phase basicity of imidazole (217 kcal mol–1), it is able to strip protons from the protonated protein ions in the gas phase . As mentioned above, this observed charge stripping might play an important role in stabilizing of the complex, because the lower charge states are less susceptible to collisional dissociation. However, the appearance of the lower charge states is probably not the main mechanism for the complex stabilization, since SF6 provides similar complex stabilization without any shift of charge state distribution .
Another explanation for observing a drop in relative abundance of complex ions might be due to electrochemical reactions that occur where the electrode contacts the solution of the ES ion source. Products of such electrochemical reactions can alter the solution composition and affect the relative abundance of CMA complexes in solution and protonated complex ions during acquisition of spectra. As already shown  the solution composition and the resulting nanoESI spectra can be time dependent, with changes in the spectra being ascribed to on-going (electro)chemical reactions upstream in the capillary. It is conceivable that addition of imidazole can buffer a shift of the solution phase equilibrium, although the detailed mechanism of how this occurs is unknown.
This observation indicates that imidazole is a suitable additive for protecting the CMA-complexes during the nanoES process. Because the stabilization effect of imidazole on Gly-trypsin system appeared rather effective, we decided to perform further titration experiments with other CMA-inhibitors in the presence of imidazole. All titration experiments for CMA-complexes were carried out in positive ion mode. The only difference to the previous experiments is a higher concentration of the ligand (1–30 μM), required to observe the weaker interactions and to obtain useful free protein-to-complex ratios.
List of calculated dissociation constants (KD) for five CMA based ligands determined by the nanoESI titration method and kinetic inhibition assay (Ki) determined in solution. For both methods the binding affinity increasing in order Gly < d-Ala < d-Leu
CMA series (R)
Kinetic inhibition assay trypsinb
Kinetic inhibition assay thrombinb
17.63 ± 1.2
6.6 ± 0.8
0.052 ± 0.005
2.9 ± 0.2
0.4 ± 0.09
0.259 ± 0.024
12.4 ± 0.2
6 ± 0.2
0.788 ± 0.070
8.4 ± 1.4
3.1 ± 1
2.2 ± 0.4
24.4 ± 0.49
9.9 ± 0.1
1.5 ± 0.1
The relative difference in the KD values determined by nanoESI-MS and the characterized Ki values are equivalent, considering the error margins of the methods. These results confirm the same trend observed in the kinetic inhibition assay as under the experimental MS conditions, demonstrating the ability of quantitative ESI-MS measurement to clearly distinguish between ligand affinities.
In conclusion, we have applied nanoESI-MS for the investigation of a series of hydrophobically modified ligands interacting with trypsin. The size of the hydrophobic side chain (R) that binds in the S3/4 pocket of trypsin was systematically increased. The different substituents (R = Gly, d-Ala, d-Val, d-Leu, d-Cha) have a significant influence on the binding constants. The quantification of binding affinities was possible using the titration method. In the case of the benzamidine series the trend to higher binding affinity with increasing hydrophobic P3 side chain is strong, ranging from 450 nM to 15 nM. The binding affinities measured by ESI-MS titration and kinetic inhibition constants for the benzamidine-trypsin complexes show, with the exception of d-Val, the same relative ordering. Collision-induced dissociation experiments across the benzamidene type series clearly show the correlation between the binding affinity and the gas phase stability. More collision energy is necessary to dissociate the complex with higher binding affinity and vice versa.
The CMA-inhibitors served as a model system for a series of less potent complexes, which are prone to in-source dissociation. This effect causes a reduced relative abundance of the gaseous complex ions and leads therefore to artificially lower binding affinities in the measurements. Upon addition of imidazole, a stabilizing solution additive, the relative abundance of the non-dissociated could be increased. Compared to benzamidine-inhibitors the CMA ligands did not show the clear trend towards higher binding strengths with longer side-chains. The increased binding affinity is observed for Gly < d-Ala < d-Leu.
For the CMA series the relative difference for KD and the characterized Ki values are equivalent, which demonstrates the ability of quantitative ESI-MS to distinguish between ligand affinities.
The authors acknowledge support for this work by the CHEBANA-Project (grant no. 264772).
- 13.Pace, C., Shirley, B., McNutt, M., Gajiwala, K.: Forces contributing to the conformational stability of proteins. FASEB J. 10(1), 75–83 (1996)Google Scholar
- 16.Jørgensen, T.J.D., Delforge, D., Remacle, J., Bojesen, G., Roepstorff, P.: Collision-induced dissociation of noncovalent complexes between vancomycin antibiotics and peptide ligand stereoisomers: Evidence for molecular recognition in the gas phase. Int. J. Mass Spectrom. 188, 63–85 (1999)CrossRefGoogle Scholar
- 20.Barylyuk, K., Balabin, R., Grönstein, D., Kikkeri, R., Frankevich, V., Seeberger, P., Zenobi, R.: What happens to hydrophobic interactions during transfer from the solution to the gas phase? The case of electrospray-based soft ionization methods. J. Am. Soc. Mass Spectrom. 22(7), 1167–1177 (2011)CrossRefGoogle Scholar
- 22.El-Hawiet, A., Kitova, E., Liu, L., Klassen, J.: Quantifying labile protein-ligand interactions using electrospray ionization mass spectrometry. J. Am. Soc. Mass Spectrom. 21(11), 1893–1899 (2010)Google Scholar
- 30.Bovet, C., Wortmann, A., Eiler, S., Granger, F., Ruff, M., Gerrits, B., Moras, D., Zenobi, R.: Estrogen receptor–ligand complexes measured by chip-based nanoelectrospray mass spectrometry: an approach for the screening of endocrine disruptors. Protein Sci. 16(5), 938–946 (2007)CrossRefGoogle Scholar
- 31.Jecklin, M., Touboul, D., Bovet, C., Wortmann, A., Zenobi, R.: Which electrospray-based ionization method best reflects protein-ligand interactions found in solution? A comparison of ESI, nanoESI, and ESSI for the determination of dissociation constants with mass spectrometry. J. Am. Soc. Mass Spectrom. 19(3), 332–343 (2008)CrossRefGoogle Scholar
- 50.Brandt, T., Holzmann, N., Muley, L., Khayat, M., Wegscheid-Gerlach, C., Baum, B., Heine, A., Hangauer, D., Klebe, G.: Congeneric but still distinct: How closely related trypsin ligands exhibit different thermodynamic and structural properties. J. Mol. Biol. 405(5), 1170–1187 (2011)CrossRefGoogle Scholar
- 52.Lorenzen, K., Duijn, E.V.: Native mass spectrometry as a tool in structural biology. Curr. Protoc. Protein Sci. 62, 17.12.1–17.12.17 (2010)Google Scholar