DNA Oligonucleotides: A Model System with Tunable Binding

G2-S HDMS hybrid quadrupole time-of-flight mass spectrometer equipped with a LockSpray ESI source and a Z-spray sampling interface (Waters, Manche...
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DNA Oligonucleotides: A Model System with Tunable Binding Strength to Study Monomer−Dimer Equilibria with Electrospray Ionization-Mass Spectrometry Konstantin Barylyuk,† Basri Gülbakan,† Xueshu Xie,‡ and Renato Zenobi*,† †

Department of Chemistry and Applied Biosciences, ETH Zurich, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

‡

S Supporting Information *

ABSTRACT: Electrospray ionization (ESI) is increasingly used to measure binding strengths, but it is not always clear whether the ESI process introduces artifacts. Here we propose a model monomer−dimer equilibrium system based on DNA oligonucleotides to systematically explore biomolecular selfassociation with the ESI-mass spectrometry (MS) titration method. The oligonucleotides are designed to be selfcomplementary and have the same chemical composition and mass, allowing for equal ionization probability, ion transmission, and detection efficiency in ESI-MS. The only difference is the binding strength, which is determined by the nucleotide sequence and can be tuned to cover a range of dissociation constant values. This experimental design allows one to focus on the impact of ESI on the chemical equilibrium and to avoid the other typical sources of variation in ESI-MS signal responses, which yields a direct comparison of samples with different binding strengths. For a set of seven model DNA oligonucleotides, the monomer−dimer binding equilibrium was probed with the ESI-MS titration method in both positive and negative ion modes. A mathematical model describing the dependence of the monomer-to-dimer peak intensity ratio on the DNA concentration was proposed and used to extract apparent Kd values and the fraction of DNA duplex that irreversibly dissociates in the gas phase. The Kd values determined via ESI-MS titration were compared to those determined in solution with isothermal titration calorimetry and equilibrium thermal denaturation methods and were found to be significantly lower. The observed discrepancy was attributed to a greater electrospray response of dimers relative to that of monomers.

N

measurements of binding constants directly from ESI-MS data in a fast, label-free, sensitive, and specific fashion.4,5 Differences in the efficiency of ionization, ion transmission, and detection can, however, skew the peak intensities making the absolute quantification with ESI-MS problematic.4−8 Equal response can be assumed in cases when a small ligand binds to a macromolecular receptor in a confined deep binding pocket as, for instance, in enzyme−substrate, enzyme−inhibitor, and receptor−hormone complexes.4,5 In contrast, different response factors very likely exist in the case of multisubunit protein assemblies or complexes, where the ligand is of comparable size to its receptor.9−13 It is therefore not surprising that many studies report successful determination of equilibrium binding constants in protein−ligand complexes using ESI-MS,14 including works from our group,15,16 while examples of quantifying binding strengths in oligomeric biomolecular complexes are rare.9,12,17,18

oncovalent interactions are the basis of the unique properties and natural activities of biomolecules and play a crucial role in enzymatic catalysis, biospecific recognition, storage of genetic information, and cellular motility.1 In many cases, biomolecular noncovalent complexes are dynamic and exist in chemical equilibrium with binding partners and/or building blocks, providing a basis for regulation and control of cellular processes that they are responsible for. Measuring the binding strength in these complexes is therefore of importance for understanding their function and mechanisms of action as well as for the development of new drugs. Electrospray ionization-mass spectrometry (ESI-MS) is routinely used in modern analytical chemistry not only to detect and identify but also to quantify various chemical substances. Thanks to development of the so-called “native ESI-MS” approach, noncovalent biomolecular assemblies, such as protein−ligand and protein−protein complexes, membrane proteins, molecular machines, and even intact viruses can be preserved upon ionization and ion transfer processes and successfully detected in the ESI mass spectrum.2,3 Peak intensities correlate with the distribution of free binding partners and complexes in solution and give access to © 2013 American Chemical Society

Received: August 22, 2013 Accepted: November 14, 2013 Published: November 14, 2013 11902

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Table 1. Properties of the Oligonucleotides Studied MW sample name

sequence

ssDNA

dsDNA

absorption coefficient (cm−1 mM−1)

melting point (Tm, °C)a

(AT)5 GC(AT)3GC CG(AT)3CG GCA3T3GC (CG)5 GA4T4C G2(AT)3C2

5′p-ATATATATAT-3′OH 5′p-GCATATATGC-3′OH 5′p-CGATATATCG-3′OH 5′p-GCAAATTTGC-3′OH 5′p-CGCGCGCGCG-3′OH 5′p-GAAAATTTTC-3′OH 5′p-GGATATATCC-3′OH

3105.01 3106.99 3106.99 3106.99 3109.95 3106.00 3106.99

6210.03 6213.98 6213.98 6213.98 6219.91 6212.00 6213.98

108.9 99.5 99.5 99.5 85.5 104.2 99.5

9.35 33.31 35.75 37.95 69.78 26.35 28.95

Melting points were predicted using the OligoCalc tool within the framework of the nearest-neighbor model for 25 μM DNA concentration and 100 mM Na+ concentration. a

new models for the prediction of ESI response factors in such analytes is missing. Here we suggest using DNA oligonucleotides as a model system to determine ESI response factors in monomer−dimer equilibria that cover a range of solution-phase binding affinities while keeping the chemical composition and properties of the analytes constant. DNA has the following properties that make it a suitable model system for a systematic assessment of electrospray response factors: (i) DNA duplex formation is a simple monomer−dimer equilibrium between single-stranded and double-stranded DNA (ssDNA and dsDNA, respectively); (ii) the binding affinity is determined by the nucleotide sequence23,24 and (iii) can be reliably predicted or measured, as well as tuned based on the sequence;24 (iv) availability of isobaric oligonucleotides with the same GC/AT content but different sequence allows for elimination of mass discrimination effects during ion transfer and detection.

Several attempts to account for ESI response factors in biomolecular complexes when determining their affinity constant were made.11−13,18 Chitta, Rempel, and Gross12 reported a simultaneous determination of the association constant (Ka) and the electrospray response factor for dimerization of gramicidin. The mathematical model proposed also accounted for in-source collision-induced dissociation (CID) of the dimer. Gabelica and co-workers11,13 suggested two approaches to access equilibrium constants and electrospray response factors simultaneously. In their earlier work,11 complexes between DNA double helix and minor groovebinding drugs as well as between α-cyclodextrin and α,ωdicarboxylic acids were monitored by ESI-MS at various concentrations, while the binding partners were kept at equimolar concentrations. Nonlinear fitting of ESI-MS-derived peak ratios vs concentration yielded Ka and the response factors. However, the fitting procedure is sensitive to deviations from equimolarity and requires very high quality data. In the latter work,13 electrospray response factors were determined independently of binding constants relative to an internal standard, a compound that was kept at constant concentration throughout the experiments and did not bind to either of interacting species. An important prerequisite was that no nonspecific complex formation in electrospray or no in-source CID of the complex took place. Unlike in the other two examples, the absolute response factors of every species could vary with the concentration of analytes as long as the ratio of responses stayed constant. Recently, we also probed the influence of electrospray response factor on the determination of binding constant of pH-dependent dimer−tetramer equilibrium in the lectin protein concanavalin A.18 Because of a much lower ion yield in such large protein complexes, the insufficient data quality prevented us from simultaneous determination of both parameters directly from ESI-MS data. Instead, we fitted the data using several fixed values of the response factor and compared the Ka values to those determined in solution with isothermal titration calorimetry (ITC). The examples discussed above highlight various aspects of the response factors in ESI. Only in a few systematic studies available to date, electrospray response factors were linked to physical parameters of the system, such as analyte polarity,19,20 solvatation energy,10 gas-phase proton affinity,21 electrolyte concentration,22 etc., to yield predictive models that are valid for relatively small molecules giving rise to mostly singly charged ions. Yet, a systematic survey of electrospray response in biomolecular noncovalent complexes that would allow validating or extending the existing models or developing

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EXPERIMENTAL SECTION Double-purified (HPLC + dialysis) DNA oligonucleotides (Table 1) were purchased from Microsynth AG (Balgach, Switzerland). All commercial chemicals and solvents were obtained in the highest available purity and used without further purification. DNA solutions were prepared in 100 mM ammonium acetate buffer (pH 6.91). DNA concentration was measured photometrically (see Table 1 for absorption coefficients) with a Lambda 35 UV−vis spectrophotometer (PerkinElmer Schweiz AG, Schwerzenbach, Switzerland) equipped with a PTP-6 Peltier Temperature Programmer and a temperature-controlled 6 + 1 cell changer. The same apparatus was used to record equilibrium melting curves. Isothermal titration calorimetry experiments were carried out on a VP-ITC microcalorimeter (Microcal/GE Healthcare Europe GmbH, Glattbrugg, Switzerland) at 30 °C. All mass spectrometry experiments were done using a Synapt G2-S HDMS hybrid quadrupole time-of-flight mass spectrometer equipped with a LockSpray ESI source and a Z-spray sampling interface (Waters, Manchester, U.K.). DNA solutions were directly infused at a flow rate of 5 μL min−1 with an external syringe pump (model 22, Harvard Apparatus, Holliston, MA). Electrospray was generated by applying a high voltage of 2.8 kV and −1.8 kV in the positive and negative ion modes, respectively, and the instrumental parameters were carefully optimized to ensure soft ion transmission. A single scan time of 1 s with a 0.015 s interscan delay were used, and mass spectra were typically accumulated for 5 min. Data processing was carried out in MATLAB R2012a (MathWorks, Natick, MA). Further details of the experimental procedures can be found in the Supporting Information. 11903

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RESULTS AND DISCUSSION DNA Sequence Design and the Set of Studied Oligonucleotides. There are a number of guidelines we followed in designing the model DNA oligonucleotides used throughout this study. (i) Oligonucleotides must be selfcomplementary to allow for the formation of homodimers. This greatly simplifies experiments because there is no need to prepare precisely equimolar mixtures of binding partners, unlike in ref 11, however, such sequences allow for hairpin structure formation, which may invalidate the two-state assumption for the equilibrium studied. (ii) Oligonucleotide sequences should ensure stable double helix formation at room temperature in solution. To achieve dsDNA stability, the sequence must be sufficiently long (at least four base pairs25) and preferably have terminal GC base pairs to efficiently initiate double helix formation. On the other hand, we did not want the oligonucleotides to be too long, in order to reduce the probability of hairpin formation and ensure that the dsDNA is the most thermodynamically stable structure. Extended poly-G tracts must also be avoided to prevent the formation of Gquadruplex structures. (iii) The Kd values of dsDNA should fall into the high-nanomolar to low-micromolar range, which allows for recording titration curves in the optimal concentration range for ESI-MS. (iv) Oligonucleotides must have the same chemical composition, i.e., the same AT and GC content, so that they produce isobaric ions in ESI-MS and allow for exactly the same ion transmission and detection efficiency in the mass spectrometer. The search for sequences was aided by predictions of DNA thermodynamic properties with the OligoCalc tool.26 Having these considerations in mind, we selected five 10-base pair-long DNA oligonucleotide sequences (Table 1). Two more sequences were added to this set: one with a predicted poor double helix stability, (AT)5, as a negative control, and one positive control sequence with the highest predicted dsDNA stability, (CG)5. ESI-MS Titration Experiments. We measured seven DNA oligonucleotide sequences at DNA concentrations ranging from submicromolar to approximately 25 μM in both positive and negative ion modes with a quadrupole-time-of-flight (Q-TOF) mass spectrometer. Direct infusion of ammonium acetatebuffered solutions of DNA oligonucleotides was carried out under near-native conditions. The sample syringe and the solvent line were both kept at room temperature, while the desolvation and ion source temperatures were set to 30 and 35 °C, respectively. The settings on the ion guides were optimized to provide an efficient yet soft ion transmission regime. In the Synapt G2-S mass spectrometer, a dc offset is applied to almost every individual ion guide. Two more tunable parameters concern the traveling-wave (TW) ion guides: the “wave velocity” and the “wave height”. These parameters may influence the ion transmission efficiency and the extent of fragmentation and therefore need to be optimized. In our experience, the default wave height and wave velocity settings of TW ion guides are optimal in most cases and do not require tuning. In contrast, some dc offsets have a pronounced impact on the survival yield of noncovalent complexes ions because they determine ion kinetic energies. For instance, we found that the sampling cone voltage and collision energy offset values had a major effect on the survival of dsDNA ions in our experiments. Keeping the trap and transfer collision energy offsets low was enough to avoid dissociation of dsDNA ions in these stages. The sampling cone offset of 50 V was found to

provide an optimal balance between, on the one hand, the ion transmission and declustering efficiencies and, on the other hand, DNA duplex survival (Figure S1 in the Supporting Information). All oligonucleotides showed signals from ssDNA and dsDNA in the mass spectrum. While ssDNA gave 2+, 3+, and 4+ ions, with the 3+ charge state dominating the spectrum, dsDNA produced ions with 4+ and 5+ charge states (Figure 1a). The peaks of [ssDNA + 2H]2+ and [dsDNA + 4H]4+ ions overlapped but could still be resolved by analyzing the fine isotopic structure (Figure 1a, inset). The negative ion mode spectra looked very similar, with only a small difference in the

Figure 1. Representative positive-mode ESI mass spectrum of GA4T4C oligonucleotide (CDNA ≈ 1 μM). Both ssDNA and dsDNA are detected as distinct distributions of multiply charged ions Mn+ and Dm+, respectively, with n = 2, 3, 4, and m = 4 and 5 (a). Signals from protonated ions as well as adducts with sodium, potassium, and ammonium are present in the spectrum (a, inset). The signals of protonated ssDNA M2+ and dsDNA D4+ overlap but can be distinguished at the level of the isotopic distributions (a, inset). The MaxEnt3 maximum entropy deconvolution algorithm was utilized to extract total peak intensities of ssDNA and dsDNA (b). The deconvoluted peak positions and intensities were found by MaxEnt3 through iterative fitting of a simulated mass spectrum (a, green trace) to the experimental mass spectrum (a, red trace). 11904

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Figure 2. ESI-MS (a,b) and MaxEnt3-deconvoluted (c,d) spectra of CG(AT)3CG oligonucleotide recorded at two different total DNA concentrations in positive (red traces) and negative (blue traces) ion modes. Intensities of ssDNA and dsDNA peaks changed upon a 10-fold change of DNA concentration in solution (compare top and bottom traces in every panel). Note that not only the protonated peak intensities increased but also those of adducts.

dsDNA peak ratio. We used the MaxEnt3 maximum entropy algorithm for this (Figure 1b). The MaxEnt3 algorithm assumes the simplest mass distribution that is sufficient to describe the observed mass spectrum, the maximum entropy spectrum. The algorithm then simulates the corresponding m/z distribution (Figure 1a, green trace) which is compared to the experimentally measured ESI mass spectrum (Figure 1a, red

relative intensities of peaks of the different charge states. Adducts with sodium, potassium, and ammonium also appeared in the mass spectra along with the all-protonated ions (Figure 1a, inset). The overlap of the ssDNA2+ and dsDNA4+ peaks rendered the data analysis difficult. A signal deconvolution was therefore required to extract the ion intensities and calculate the ssDNA/ 11905

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Figure 3. ESI-MS titration plots obtained in positive (red) and negative (blue) ion modes. Experimentally measured ssDNA/dsDNA peak ratios (circles) were fitted with a nonlinear regression model given by eq 2 (solid lines) accounting for both chemical equilibrium and irreversible loss of a part of dsDNA due to gas-phase dissociation. Prediction boundaries (p = 95%) for new observations are shown by dotted lines. (a) (CG)5; (b) GCA3T3GC; (c) GA4T4C; (d) CG(AT)3CG; (e) G2(AT)3C2; (f) GC(AT)3GC.

The MaxEnt3-deconvoluted spectra were used to calculate the ssDNA/dsDNA peak ratios. Since not only the protons but also alkali metal and ammonium cations can contribute to the overall charge of the analyte ions (Figures 1 and 2 and Figures S2−S7 in the Supporting Information), all these species were taken into account when calculating the ratio. As expected, the ssDNA/dsDNA peak ratios followed an overall hyperbolic trend when plotted against the total DNA concentration (Figure 3, circles), in agreement with the assumed monomer− dimer equilibrium model. Moreover, the peak ratio decreased more rapidly for the DNA oligonucleotides with higher than expected duplex stability. Similar trends were observed in both positive and negative ion modes (Figure 3, red vs blue) suggesting that the ionization process does not dramatically distort the solution-phase chemical equilibrium. Equilibrium Dissociation Constants from ESI-MS Titration Data. The equilibrium dissociation constants Kd(T) were determined as independent parameters of fitting of the concentration-dependent ssDNA/dsDNA peak ratios R(T,c) using the nonlinear regression model assuming a twostate equilibrium:

trace), and the difference between them is used to guide the algorithm in an iterative search for the maximum entropy spectrum that best reflects the observed data. The relative intensity of dsDNA peaks increased as the total DNA concentration increased, reflecting the shift of the solution-phase monomer−dimer equilibrium toward the formation of dimers (Figure 2). Two notable extremes were the (AT)5 and (CG)5 sequences. The spectra of (AT)5 contained almost exclusively the ssDNA peaks, with negligible peaks of dsDNA showing up at the highest concentration tested (Figure S2 in the Supporting Information). These observations are in line with the expected low solution-phase stability of duplexes for this sequence, because AT-base pairs are stabilized by only two hydrogen bonds and also form less favorable π−π stacking interactions. (AT)5 was therefore not titrated in ESIMS. (CG)5, on the contrary, gave mostly signals of dsDNA ions unless diluted down to low-nanomolar concentrations, reflecting the high solution-phase stability of the duplex formed by DNA oligonucleotide with GC-rich sequence (Figure S3 in the Supporting Information). The other DNA sequences behaved more or less similar to CG(AT)3CG (Figures S4−S7 in the Supporting Information). 11906

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Table 2. Comparison of Dissociation Constants Determined Experimentally for the Studied DNA Oligonucleotides at the Temperature of 30 °C Kd(T), μM ESI-MS sample name (AT)5b GC(AT)3GC CG(AT)3CG GCA3T3GCc (CG)5c GA4T4C G2(AT)3C2

ITC

equilibrium melting curvea

ESI+

ESI−

n.d. 18.2 ± 1.7 7.6 ± 1.0 n.d. n.d. 1.0 ± 0.2 12.0 ± 1.6

n.d. 4.0 ± 0.4 5.5 ± 1.3 0.041 ± 0.100 0.002 ± 0.030 3.9 ± 1.7 11.3 ± 1.7

n.d. 1.2 ± 0.1 0.8 ± 0.4 0.010 ± 0.004 0.012 ± 0.002 0.17 ± 0.05 0.79 ± 0.31

n.d. 1.8 ± 0.1 1.2 ± 0.2 0.007 ± 0.008 0.008 ± 0.006 0.38 ± 0.15 2.0 ± 0.2

Dissociation constant values reported for equilibrium melting curves recorded at DNA concentration of approximately 25 μM. bNo dissociation constant values were determined for (AT)5 because of very low dimer stability under the conditions of the experiments. cThe two oligonucleotides with the most stable duplex showed no signal above the noise in ITC.

a

2M ⇄ D ⎧ [M]2 ⎪ Kd(T ) = [D] ⎪ ⎪ ⎪ c = [M] + 2[D] ⇒ R(T , c) = ⎨ −1 + ⎪ ⎪ IM ⎪ [M] ⎪ R(T , c) = [D] = I ⎩ D

solution: ESI‐MS:

R obs(T , c) =

4 1+

2M V D D → 2M

8c K d(T )

IM R(T , c) + 2δ [M] + 2Δ· [D] = = ID [D] − Δ· [D] 1−δ (2)

where Robs(T,c) is the experimentally observed peak ratio, R(T,c) is the equilibrium concentration ratio (see above), Δ· [D] is the amount of irreversibly dissociated dimer, and δ is the fraction of irreversibly dissociated dimer. This model agreed reasonably well with the experimental data (Figure 3 and Figure S8c,d in the Supporting Information) and allowed us to simultaneously determine both the Kd(T) and the fraction of nonspecifically dissociated DNA duplex δ as independent fitting parameters (Table S1 in the Supporting Information). In the study by Chitta, Rempel, and Gross,12 similar assumptions were made to account for in-source CID of gramicidin dimer. The Kd(T) values extracted from the fit according to eq 2 (Figure 3 and Table 2) suggest that the ESI-MS titration data generally reflected the expected solution-phase stability of dsDNA. Interestingly, the apparent dissociation constants obtained for the negative ion mode were higher by approximately 50% than those of the positive ion mode. This may indicate that due to mechanistic differences in how positive and negative DNA ions are formed in ESI, the electrochemical disturbance of the equilibrium is not equal in positive and negative ion modes. Alternatively, unequal ionization probabilities, i.e., electrospray response factors, of the same species in positive and negative polarity may be responsible for the mismatch in the dissociation constant values found.4 The ESIMS titration method for determining binding affinity is rarely applied in both polarities for the same analyte, due to the sensitivity issues: the positive ion mode is much more common in the analysis of protein complexes, while nucleic acids are usually analyzed in negative polarity only. Remarkably, unlike the Kd(T), values of the parameter δ determined for the DNA oligonucleotides studied were very similar, between 0.2 and 0.3, with slightly smaller numbers for the sequences with higher solution stability (Table S1 in the Supporting Information). The DNA oligonucleotides were of almost the same size, produced ions with very similar charge state distributions, and were analyzed under the same, constant instrumental parameters. Thus, they are expected to experience very similar forces and activation for exactly the same time in the experiment. This suggests that gas-phase CID in the ion

(1)

An attempt to fit the peak ratio versus the total DNA concentration with eq 1 revealed a pronounced deviation of the two-state equilibrium model from experimental data (Figure S8a,b in the Supporting Information). The experimental data points were systematically underestimated by the trend line, especially at higher DNA concentrations (Figure S8a,b in the Supporting Information, residuals). In other words, the peak ratio R = [M]/[D] never dropped to zero. There may be several reasons for the observed discrepancy between the measured data and the two-state equilibrium model. The two-state assumption may not be valid, and the system may actually include multiple equilibrium reactions, such as the formation of ssDNA stem-loops and hairpin-loops or dsDNA hairpin-cross structures and dimers with dangling ends. Noteworthy, we did not detect any peaks corresponding to higher-order DNA oligomers in the ESI mass spectra. Incorporating additional equilibria into the complex equilibrium network will essentially result in a similar overall behavior of the system: new terms in the equation may change the curvature of the titration plot, but the curve will approach zero at infinite DNA concentration (see an example described in the Supporting Information). These considerations suggested that the asymptotic behavior of the measured ssDNA/dsDNA peak ratios might originate from a process affecting the distribution of species detected by the mass spectrometer. We hypothesized that an irreversible dissociation of DNA duplex during either the ionization or ion transmission could systematically distort the actual ssDNA/ dsDNA ratio and explain the observed deviations of experimental data from equilibrium models. 11907

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Figure 4. dsDNA−ssDNA equilibrium in GC(AT)3GC monitored in solution by temperature-resolved UV absorption spectroscopy (a,b) and ITC (c). UV absorption spectra were recorded for 26.8 μM solution of GC(AT)3GC at different temperatures (a). At higher temperatures, the equilibrium shifts toward duplex dissociation, which is manifested in the increase of UV absorbance (ssDNA hyperchromism). Assuming a two-state equilibrium, a fraction of dsDNA at every tested temperature was calculated from UV absorbance data (b, circles) and used to derive an equilibrium melting curve by applying eqs S1 and S2 in the Supporting Information (b, solid line; dotted lines are 95% confidence boundaries for new observations; dashed lines indicate Tm). Kd(T) were then calculated using eq S3 in the Supporting Information (Table 2). Alternatively, Kd(T) was directly determined by ITC (c). The dsDNA dissociation was induced by injecting a 107.2 μM solution of GC(AT)3GC into blank buffer, and the associated heat effects (c, top panel) were used to directly determine ΔH and Kd (c, bottom panel).

source (similar to that observed for gramicidin in ref 12) or at a later stage of ESI-MS experiment is more likely the reason for the observed irreversible DNA duplex dissociation. The isolated ions produced in electrospray are dragged both electrostatically and aerodynamically into the mass spectrometer. As they pass through differential pumping apertures they can undergo collisions with the residual buffer gas and dissociate, a phenomenon that is known as in-source CID.4,8 At the pressures and in densities used in MS, such dissociations are strictly unimolecular. The slight differences in the rate of dsDNA loss due to irreversible gas-phase dissociation can be attributed to variations in the gas-phase stability of DNA duplexes. The gas-phase stability of noncovalent complexes sometimes correlates with that in solution,16 which appears to be true in the present case as well. When conducting native ESI-MS experiments, a common approach to reduce the gas-phase dissociation is to flatten the voltage gradient across the ion guides and to increase the ion source pressure to favor collisional cooling over collisional activation of ions.2,4 However, this is done at the expense of efficient desolvation and ion transmission. The actual settings are therefore a trade-off between softness and efficiency of ion transmission (Figure S1 in the Supporting Information). For some analytes it may be impossible to avoid gas-phase dissociation completely, especially if these are weak noncovalent complexes. In the case of the DNA oligonucleotides studied here, the dsDNA ions were relatively small yet had a relatively high charge density, so that they could reach high velocities and gain enough internal energy through collisions with the residual gas to dissociate. Comparison of the Solution-Phase and ESI-MSDerived Dissociation Constants. To get an insight into

how well ESI-MS titration reflects the solution-phase equilibrium between ssDNA and dsDNA, we measured the solution-phase Kds with two widely used methods: equilibrium thermal denaturation27 and isothermal titration calorimetry (ITC) (Figure 4).28 The hyperchromism of ssDNA allows for monitoring thermal denaturation of DNA oligonucleotide duplexes by UV absorption spectroscopy (Figure 4a,b, Figures S9 and S10 in the Supporting Information). Melting points of dsDNA depend on the DNA sequence and can be predicted based on the so-called nearest neighbor model (NNM).23−27 Ammonium acetate is, however, not equivalent to sodium chloride with respect to DNA duplex stabilization, and the thermodynamic parameters proposed by the NNM may not be directly applicable. Equilibrium melting curves were recorded for different concentrations of DNA (Figure S11 in the Supporting Information). The concentration range between low-micromolar and approximately 100 μM was tested. The melting point shifted to higher values at greater DNA concentrations indicating that the observed melting reaction was bimolecular.27 The linear fit of the reciprocal melting point versus the logarithm of the total DNA concentration (Figure S12 in the Supporting Information) allows one to determine thermodynamic parameters of the reaction, ΔH° and ΔS°, from the slope and intercept, which can subsequently be used to calculate the dissociation constant at a given temperature, Kd(T).27 Herein, the dependence is given only qualitatively, to illustrate the effect, but no number was extracted because of the low number of data points acquired. Instead, we determined the fraction of dsDNA at 30 °C from the equilibrium melting curves recorded for approximately 25 μM DNA solutions and used it to 11908

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Table 3. Relative Electrospray Response Factors and Fractions of dsDNA Lost Due to Irreversible Gas-Phase Dissociation Obtained by Fitting ESI-MS Titration Data with Equation 5 with Explicitly Given Kd(T) Values Determined in ITC Titration and Equilibrium Thermal Denaturation Experiments (see Table 2 for Reference) ESI+

ESI− δ

response factor oligonucleotide sequence

a

value

error

GA4T4C G2(AT)3C2 GC(AT)3GC CG(AT)3CG

0.21 0.09 0.11 0.12

0.01 0.01 0.01 0.01

GA4T4C G2(AT)3C2 GC(AT)3GC CG(AT)3CG GCA3T3GCa (CG)5a

0.06 0.09 0.38 0.16 0.42 2.89

0.00 0.01 0.01 0.01 0.11 0.29

value

δ

response factor error

ITC Titration 0.23 0.01 0.38 0.06 0.32 0.01 0.37 0.01 Equilibrium Melting Curve 0.25 0.02 0.38 0.06 0.29 0.01 0.36 0.01 0.14 0.03 0.00 0.03

value

error

value

error

0.46 0.18 0.10 0.21

0.12 0.03 0.01 0.01

0.27 0.36 0.43 0.32

0.06 0.06 0.03 0.03

0.14 0.19 0.47 0.28 0.37 6.69

0.03 0.03 0.02 0.02 0.21 2.54

0.31 0.35 0.37 0.31 0.19 0.00

0.05 0.06 0.02 0.03 0.03 0.08

The fitting was less reliable due to high binding strength in these two oligonucleotides.

calculate Kd(T) according to eq S3 in the Supporting Information.27 The calculated values are given in Table 2 with 95% confidence intervals. ITC is considered as the gold-standard for determining binding constants of receptor−ligand interactions.28 It is also applied to characterize monomer−dimer equilibria, although fairly rarely. While a macromolecular receptor at a set concentration is titrated with low-molecular weight ligands in the former case, the dimer dissociation is induced by diluting a concentrated solution of macromolecules in the latter case (Figure 4c).28 We measured ITC titration curves for six of the seven oligonucleotides; (AT)5 was not titrated. It is worth noting here that ITC curves had in general quite poor signal-tonoise ratios due to the inherently low sensitivity of the method.28 While four DNA sequences produced measurable signals (Figure S13 in the Supporting Information), (CG)5 and GCA3T3GC repeatedly gave signals only close to the noise level making it very difficult to extract any reliable Kd(T). High stability of dsDNA form for these two samples was also the reason for the low accuracy of Kd(T) determination based on equilibrium melting curves.27 The fractions of dsDNA used for calculating Kd(T) were measured quite far from the melting temperature. The error of such calculations propagates in a nonlinear fashion and increases dramatically when moving away from the melting point, thereby increasing the uncertainty of the results.27 Table 2 summarizes the values of Kd(T) obtained with the different methods used in this study. The values of dissociation constants obtained in solution phase with ITC and from equilibrium melting curves were in a reasonable agreement. ESI-MS-derived dissociation constants all were significantly lower, by a factor of approximately 10, than the values obtained in solution phase. This is a surprising result considering that we found quite a pronounced loss of dimers due to irreversible gasphase dissociation. Also, no reliable correlation could be established between the values obtained in ESI-MS experiments and those from ITC or the equilibrium melting curve method (Figure S14 in the Supporting Information). There may be several explanations for the unexpectedly low dissociation constants measured with ESI-MS: a higher ionization efficiency of the dimers compared to the monomers,

mass discrimination during ion transfer, unequal efficiency of detection of the species, or an association of the monomers into the dimers in ESI droplets.4 We can safely rule out mass discrimination effects during ion transfer and detection because all the relevant signals appeared within the same narrow m/z range (Figures 1 and 2 and Figures S2−S7 in the Supporting Information). The ion guide transmission profile was tuned to ensure optimal ion transfer for the target mass range. The total ion current and base peak intensity in the spectra were also well below the detector saturation limit, and the Synapt G2-S mass spectrometer features approximately 5 orders of magnitude of linear dynamic range, according to the specifications given by the manufacturer. Nonspecific aggregation of analytes during the electrospray process is often observed in ESI-MS, especially at high concentrations, because analytes are essentially concentrated in the shrinking electrospray droplets and electrostatic interactions are simultaneously facilitated due to the polarity change during desolvation.4,8 In the present case, these phenomena should have taken place for every ssDNA and dsDNA molecule regardless of sequence or binding affinity. Consequently, if nonspecific aggregates would be significant it would manifest itself not only as nonspecific dimers but also higher-order associates. However, no DNA trimers or higherorder oligomers were found in ESI mass spectra. Moreover, almost no dimer was present in ESI mass spectra of (AT)5, an oligonucleotide with a very low duplex stability (Figure S2 in the Supporting Information). All these findings speak against nonspecific aggregation of ssDNA during ESI. The observed discrepancy between the solution-phase and ESI-MS-derived dissociation constants must therefore be attributed to a difference in electrospray response of ssDNA and dsDNA. Relative Electrospray Response Factors. Electrospray response factors are usually introduced in the form of proportionality coefficients ri to convert analyte concentration ci into signal intensity Ii:4,8,11−13,18 Ii = rci i

(3)

For the case of ssDNA−dsDNA equilibria, the observed intensity ratio is therefore a product of equilibrium 11909

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Figure 5. Relative electrospray response factors (a,b) and fractions of dsDNA lost in electrospray due to irreversible gas-phase dissociation (c,d) estimated for the four studied DNA oligonucleotides (GA4T4C, G2(AT)3C2, GC(AT)3GC, and CG(AT)3CG) by fitting the ESI-MS-derived data with eq 5 using Kd(T) values obtained from ITC titration (a,c) and equilibrium melting curves (b,d). The data corresponding to the positive and negative ion modes are shown in red and blue, respectively. The error bars indicate 95% confidence intervals.

equilibrium melting curves could still be done but also revealed poor correlation (Figure S15 in the Supporting Information). To cope with the large uncertainty of this three-parametric fitting of the data, we inserted explicit Kd(T) values measured in solution into the model. The overall quality of the fit was then comparable with that obtained when eq 2 was applied (see the Discussion, Figure 3, and Figure S8 in the Supporting Information). This allowed us to extract values for the relative electrospray response factor for the four oligonucleotides as well as the values for the parameter δ (Table 3, Figure 5). We found that the extracted values of the response factors were all below 1 (∼0.1−0.4) suggesting a higher ionization efficiency for the DNA duplex, which is consistent with the low apparent Kd(T) values determined by fitting the ESI-MS titration data with eq 2. The values of response factors corresponding to the negative ion mode data were lower compared to those of the positive ion mode, in line with our finding that dissociation constants were greater in the case of negative-polarity data. The parameter δ assumed values in the range between 0.2 and 0.4. Remarkably, similar values of the response factor and δ were found by Chitta, Rempel, and Gross for gramicidin.12 They used a definition of response factor that is reciprocal to the one used here and found it to be 1.3 ± 0.2 in ethanol and 5 ± 1 in n-propanol; df, which is equivalent to δ used here, amounted to 0.19 ± 0.01 and 0.15 ± 0.01, respectively. In our own study of concanavalin A dimer-tetramer equilibrium, F = 0.25 was

concentration ratio and the relative electrospray response factor: IM r [M] [M] = M· =F = F · R (T , c ) ID rD [D] [D]

(4)

We derived a model that accounted for chemical equilibrium, relative electrospray response factor, and irreversible gas-phase dissociation by combining eqs 2 and 4, similar to ref 12, and used it to fit the ESI-MS titration data. It is important to note that the relative electrospray response factor appears in this model only to scale the equilibrium concentration ratio and not the whole eq 2, because the term δ describes the process that occurs after ion formation: R obs(T , c) =

IM F ·R(T , c) + 2δ = ID 1−δ

(5)

We applied eq 5 to fit the data on the four oligonucleotides for which solution-phase Kd(T) were determined reliably: GA4T4C, G2(AT)3C2, GC(AT)3GC, and CG(AT)3CG. The data quality was however not high enough to allow for accurate simultaneous determination of all three parameters, which resulted in a large uncertainty of the fitted Kd(T) and F parameters (Table S2 in the Supporting Information). Nonetheless, a qualitative comparison of the dissociation constants with those determined with ITC titration and 11910

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ESI process on the quantitation of biomolecular interactions. The DNA duplex stability can be tuned conveniently by varying the nucleotide sequence. A set of seven isobaric oligonucleotides with dissociation constants spanning the Kd range from low-nanomolar to millimolar was generated and tested both in positive and negative polarity with the ESI-MS titration method. A clear advantage of this system was that the nature of analytes remained unchanged, allowing for direct comparison of the results obtained for different samples and opening new possibilities for systematic experiments. The overall equilibrium behavior of the system was successfully captured by the ESI-MS titration method, highlighting its potential for the analysis of multisubunit biomolecular noncovalent complexes. We tested several possible mathematical models to describe the reactions and processes responsible for peak intensities observed in ESI mass spectra, including competing equilibrium reactions in solution prior to electrospray analysis and irreversible reactions in the gas phase as well as unequal ionization efficiency. The model incorporating a two-state solution-phase equilibrium followed by irreversible partial DNA duplex dissociation in the gas phase was found to most adequately describe the experimental data and allowed us to isolate the processes of interest. The error of Kd values determined from the direct ESI-MS titration data was as low as in gold-standard solution-phase methods. Direct comparison of ESI-MS-derived dissociation constants to those measured in solution using two orthogonal methods, isothermal titration ITC and equilibrium thermal denaturation, revealed that the apparent binding affinities determined by ESI-MS titration were higher (Kd lower) than those measured in solution. We attributed this to different electrospray response factors and found them all to be similar, in the range of 0.1−0.4, with a greater electrospray response found for dsDNA. The observed similarities in relative electrospray response factors and rates of irreversible gas-phase dissociation of dsDNA, as well as between positive and negative ion modes, further supported the applicability of ESI-MS titration method for quantitation of binding affinities in multisubunit biomolecular complexes: the ESI-related factors were uniform across the whole range of studied analytes and allowed to differentiate them based on their intrinsic solution-phase stability. Further investigations are required to propose a mechanistic explanation for the finding that dsDNA exhibits a higher electrospray response relative to ssDNA. Nevertheless, as this study clearly reveals and some previous work also suggested,12,18 a greater electrospray response should be expected for biomolecular oligomeric complexes relative to their subunits, which should be taken into account when determining the binding strength in such complexes by the direct ESI-MS titration method.

shown to give better agreement between dissociation constants determined by ESI-MS and reference ITC titration measurements.18 The bias in relative electrospray response factors toward more efficient dimer ionization is somewhat surprising. According to the equilibrium partitioning model proposed by Enke,19 analytes with greater polarity tend to preferentially reside in the bulk of ESI droplets while less polar compounds migrate to the surface layer and therefore have higher probability to acquire charge and become ionized. Looking at DNA oligonucleotides, we expected dsDNA to be more polar than ssDNA because of the large, relatively nonpolar surface, which is sequestered from solution in a double helix due to base stacking. The phosphate backbone that is located on the periphery of the double helix in a regular fashion also masks the nucleobases and contributes to overall polarity of the molecule. We therefore initially anticipated seeing a higher probability of ionization for the relatively less polar ssDNA. The experiment however showed the opposite. A possible explanation might be that both ssDNA and dsDNA are located in the bulk of electrospray droplets and get ionized according to the so-called charge residue model.6,8,29 Perhaps, more rigid and extended dsDNA exposes a larger surface area and provides more sites where charges may bind, which results in a greater ionization probability. An alternative explanation is the effect of analyte concentration in shrinking droplets during the electrospray process, which shifts the equilibrium toward association. This would, however, manifest itself in a correlation between the response factors and binding constants, which was not found here. Also, mechanistic studies of the time scale of droplet evolution suggest that Coulomb explosions in electrospray may happen too fast to allow for a shift in equilibrium.5,8,30 The typical lifetime of an electrospray droplet between two consecutive fissions is on the order of 10−100 μs.5,8,29 Short DNA oligonucleotides, such as those studied in the present work, have an association rate constant on the order of 106 to 105 M−1 s−1 at 30 °C and in the presence of 1 M NaCl,31 and it should be even lower under the solution conditions used here. Peschke, Verkerk, and Kebarle showed that the error in Kd introduced by an artificially high association due to a putative concentration increase in shrinking electrospray droplets is negligibly small for molecules with such association rate constants.5 Moreover, simple estimates show that at lowmicromolar analyte concentrations typically used in ESI, the small-volume droplets that produce the ions contain on average no more than one analyte molecule, which is definitively not enough to produce a shift in chemical equilibrium toward association.5 Thus, it is still a question of future research to discover the actual reason for a greater electrospray response of biomolecular multisubunit complexes relative to their building blocks.

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S Supporting Information *

CONCLUSIONS In the present study, a convenient and flexible model system to probe the influence of the electrospray ionization process on solution-phase chemical equilibria of biomolecular oligomeric complexes was proposed and characterized. The system is based on self-complementary DNA oligonucleotides with the same size, mass, and chemical composition but different sequence. This system design allowed us to rule out mass discrimination effects originating from unequal ion transfer and detection efficiencies and focus exclusively on the impact of the

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. 11911

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ACKNOWLEDGMENTS This work was funded by SNF Grant 200020-124663. B.G. acknowledges an ETH Postdoctoral Fellowship. The authors thank the group of Prof. Dr. Donald Hilvert for sharing their UV−vis spectrophotometer and the Microanalysis Lab at ETH Zurich for providing access to the ITC microcalorimeter. Very fruitful discussions with Dr. Pablo Martinez-Lozano Sinues, Dr. Volker Neu, and Dr. Alexander Sobol are gratefully appreciated.

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