Fundamental Determinants of the Accuracy of Equilibrium Constants for Affinity Complexes.

The equilibrium constant of a chemical reaction is arguably the key thermodynamic parameter in chemistry; we naturally expect that equilibrium constants are determined accurately. The majority of equilibrium constants determined today are those of binding reactions that form affinity complexes, such as protein-protein, protein-DNA, and protein-small molecule. There is growing awareness that the determination of equilibrium constants for highly stable affinity complexes may be very inaccurate. However, fundamental (i.e., method-independent) determinants of accuracy are poorly understood. Here, we present a study that explicitly shows what the accuracy of equilibrium constants of affinity complexes depends on. This study reveals the critical importance of the choice of concentration of interacting components and creates a theoretical foundation for improving the accuracy of the equilibrium constants. The predicted influence of concentrations on accuracy was confirmed experimentally. The results of this fundamental study provide instructive guidance for experimentalists independently on the method they use.

Bulk Affinity Assays in Aptamer Selection: Challenges, Theory, and Workflow.

Selection of oligonucleotide aptamers involves consecutive rounds of affinity isolation of target-binding oligonucleotides from a random-sequence oligonucleotide library. Every next round produces an aptamer-enriched library with progressively higher fitness for tight binding to the target. The progress of enrichment can only be accurately assessed with bulk affinity assays in which a library is mixed with the target and one of two quantitative parameters, the fraction of the unbound library (R) or the equilibrium dissociation constant (Kd), is determined. These quantitative parameters are used to help researchers make a key decision of either continuing or stopping the selection. Despite the importance of this decision, the suitability of R and Kd for bulk affinity assays has never been studied theoretically, and researchers rely on intuition when choosing between them. Different approaches used for bulk affinity assays expectedly hinder comparative analyses of selections. Our current work has two goals: to give bulk affinity assays a thorough theoretical consideration and to propose a scientifically justified and practical bulk-affinity-assay approach. We postulate a formal criterion of suitability: a quantitative parameter must satisfy the principle of superposition. R satisfies this principle, while Kd does not, suggesting R as a theoretically preferable parameter. Further, we propose a solution for two limitations of R: its dependence on target concentration and narrow dynamic range. Finally, we demonstrate the use of this algorithm in both computer-simulated and experimental aptamer selection. This study sets a cornerstone in the theory of bulk affinity assays, and it provides researchers with a scientifically sound and instructive approach for conducting bulk affinity assays.

Quantitative Characterization of Partitioning in Selection of DNA Aptamers for Protein Targets by Capillary Electrophoresis.

Partitioning of protein-DNA complexes from protein-unbound DNA is a key step in selection of DNA aptamers. Conceptually, the partitioning step is characterized by two parameters: transmittance for protein-bound DNA (binders) and transmittance for unbound DNA (nonbinders). Here, we present the first study to reveal how these transmittances depend on experimental conditions; such studies are pivotal to the effective planning and control of selection. Our focus was capillary electrophoresis (CE), which is a partitioning approach of high efficiency. By combining a theoretical model and experimental data, we evaluated the dependence of transmittances of binders and nonbinders on the molecular weight of the protein target in two modes of CE-based partitioning: nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) and ideal-filter capillary electrophoresis (IFCE). Our data suggest that as the molecular weight of the protein target decreases: (i) the transmittance for binders remains close to unity in NECEEM but decreases drastically in IFCE and (ii) the transmittance for nonbinders increases orders of magnitude in NECEEM but remains relatively stable at a very low level in IFCE. To determine the optimal CE conditions for a given size of protein target, a balance between transmittances of binders and nonbinders must be reached; such a balance would ensure the collection of binders of sufficient purity and quantity. We conclude that, as a rule of thumb, IFCE is preferable for large-size protein targets while NECEEM should be the method of choice for small-size protein targets.

How to Develop and Prove High-Efficiency Selection of Ligands from Oligonucleotide Libraries: A Universal Framework for Aptamers and DNA-Encoded Small-Molecule Ligands.

Screening molecular libraries for ligands capable of binding proteins is widely used for hit identification in the early drug discovery process. Oligonucleotide libraries provide a very high diversity of compounds, while the combination of the polymerase chain reaction and DNA sequencing allow the identification of ligands in low copy numbers selected from such libraries. Ligand selection from oligonucleotide libraries requires mixing the library with the target followed by the physical separation of the ligand-target complexes from the unbound library. Cumulatively, the low abundance of ligands in the library and the low efficiency of available separation methods necessitate multiple consecutive rounds of partitioning. Multiple rounds of inefficient partitioning make the selection process ineffective and prone to failures. There are continuing efforts to develop a separation method capable of reliably generating a pure pool of ligands in a single round of partitioning; however, none of the proposed methods for single-round selection have been universally adopted. Our analysis revealed that the developers' efforts are disconnected from each other and hindered by the lack of quantitative criteria of selection quality assessment. Here, we present a formalism that describes single-round selection mathematically and provides parameters for quantitative characterization of selection quality. We use this formalism to define a universal strategy for development and validation of single-round selection methods. Finally, we analyze the existing partitioning methods, the published single-round selection reports, and some pertinent practical considerations through the prism of this formalism. This formalism is not an experimental protocol but a framework for correct development of experimental protocols. While single-round selection is not a goal by itself and may not always suffice selection of good-quality ligands, our work will help developers of highly efficient selection approaches to consolidate their efforts under an umbrella of universal quantitative criteria of method development and assessment.

Necessity and Challenges of Sample Preconcentration in Analysis of Multiple MicroRNAs by Capillary Electrophoresis.

Thousands of putative microRNA (miRNA)-based cancer biomarkers have been reported, but none has been validated for approval by the Food and Drug Administration. One of the reasons for this alarming discrepancy is the lack of a method that is sufficiently robust for carrying out validation studies, which may require analysis of samples from hundreds of patients across multiple institutions and pooling the results together. The capillary electrophoresis (CE)-based hybridization assay proved to be more robust than reversed transcription polymerase chain reaction (the current standard), but its limit of quantification (LOQ) exceeds 10 pM while miRNA concentrations in cell lysates are below 1 pM. Thus, CE-based separation must be preceded by on-column sample preconcentration. Here, we explain the challenges of sample preconcentration for CE-based miRNA analyses and introduce a preconcentration method that can suit CE-based miRNA analysis utilizing peptide nucleic acid (PNA) hybridization probes. The method combines field-amplified sample stacking (FASS) with isotachophoresis (ITP). We proved that FASS-ITP could retain and concentrate both near-neutral PNA with highly negatively charged PNA-miRNA hybrids. We demonstrated that preconcentration by FASS-ITP could be combined with the CE-based separation of the unreacted PNA probes from the PNA-miRNA hybrids and facilitate improvement in LOQ by a factor of 140, down to 0.1 pM. Finally, we applied FASS-ITP-CE for the simultaneous detection of two miRNAs in crude cell lysates and proved that the method was robust when used in complex biological matrices. The 140-fold improvement in LOQ and the robustness to biological matrices will significantly expand the applicability of CE-based miRNA analysis, bringing it closer to becoming a practical tool for validation of miRNA biomarkers.

Spherical-Shape Assumption for Protein-Aptamer Complexes Facilitates Prediction of Their Electrophoretic Mobility.

DNA aptamers are single-strand DNA (ssDNA) capable of selectively and tightly binding a target molecule. Capillary electrophoresis-based selection of aptamers for protein targets requires the knowledge of electrophoretic mobilities of protein-aptamer complexes, while measuring these mobilities requires having the aptamers. Here, we report on breaking this vicious circle. We introduce a mathematical model that allows prediction of protein-aptamer complex mobility, while requiring only three easy-to-determine input parameters: the number N of nucleotides in the aptamer, electrophoretic mobility of N-nucleotide-long ssDNA, and a sum molecular weight of the protein-aptamer complex. The model was derived upon simplifying assumptions of a spherical shape of the protein-aptamer complex. According to this model, the protein-aptamer complex mobility is a linear function of a combination of the three input parameters with empirically determined line's intercept and slope. The intercept and slope were determined using experimental data for seven complexes. The model was then cross-validated with the leave-one-out approach revealing only 2% residual standard deviations for both the slope and the intercept. Such a precise determination of these constants allowed accurate mobility prediction for the excluded complexes with only a 3% maximum deviation from the experimentally determined mobilities. The model was tested by applying it to three protein-aptamer complexes that were not a part of the training/cross-validation set; deviations of the predicted mobilities from the experimentally determined ones were within 5% of the latter. To complete this study, the model was fine-tuned using the 10 complexes. Our results strongly suggest the validity of the spherical-shape assumption for the protein-aptamer complexes when considering complex mobility. The developed model will make it possible to rationally design capillary electrophoresis-based selection of DNA aptamers for protein targets.

Determination of the Equilibrium Constant and Rate Constant of Protein-Oligonucleotide Complex Dissociation under the Conditions of Ideal-Filter Capillary Electrophoresis.

Ideal-filter capillary electrophoresis (IFCE) allows selection of protein binders from oligonucleotide libraries in a single step of partitioning in which protein-bound and unbound oligonucleotides move in the opposite directions. In IFCE, the unbound oligonucleotide does not reach the detector, imposing a problem for finding the equilibrium constant ( Kd) and rate constant ( koff) of protein-oligonucleotide complex dissociation. We report a double-passage approach that allows finding Kd and koff under the IFCE conditions, i.e. near-physiological pH and ionic strength. First, a plug of the protein-oligonucleotide equilibrium mixture passes to the detector in a pressure-driven flow, allowing for both the complex and free oligonucleotide to be detected as a single first peak. Second, the pressure is turned off and the voltage is applied to reverse the migration of only the complex which is detected as the second peak. The experiment is repeated with a lower voltage consequently resulting in longer travel time of the complex to the detector, greater extent of complex dissociation, and the decreased area of the second peak. Finally, the peak areas are used to calculate the values of Kd and koff. Here we explain theoretical and practical aspects of the double-passage approach, prove its validity quantitatively, and, demonstrate its application to determine Kd and koff for an affinity complex between a protein and its DNA aptamer. The double-passage approach for finding Kd and koff of protein-oligonucleotide complexes under the IFCE conditions is a perfect complement for IFCE-based selection of protein binders from oligonucleotide libraries.

Ideal-filter capillary electrophoresis: A highly efficient partitioning method for selection of protein binders from oligonucleotide libraries.

Selection of affinity ligands for protein targets from oligonucleotide libraries currently involves multiple rounds of alternating steps of partitioning of protein-bound oligonucleotides (binders) from protein-unbound oligonucleotides (nonbinders). We have recently introduced ideal-filter capillary electrophoresis (IFCE) for binder selection in a single step of partitioning. In IFCE, protein-binder complexes and nonbinders move inside the capillary in the opposite directions, and the efficiency of their partitioning reaches 109 , i.e., only one of a billion molecules of nonbinders leaks through IFCE while all binders pass through. The condition of IFCE can be satisfied when the magnitude of the mobility of EOF is smaller than that of the protein-binder complexes and larger than that of nonbinders. The efficiency of partitioning in IFCE is 10 million times higher than those of solid-phase-based methods of partitioning typically used in selection of affinity ligands for protein targets from oligonucleotide libraries. Here, we provide additional details on our justification for IFCE development. We elaborate on electrophoretic aspects of the method and define the theoretical range of EOF mobilities that support IFCE. Based on these theoretical results, we identify an experimental range of background electrolyte's ionic strength that supports IFCE. We also extend our interpretation of the results and discuss in-depth IFCE's prospective in practical applications and fundamental studies.

Ideal-Filter Capillary Electrophoresis (IFCE) Facilitates the One-Step Selection of Aptamers.

Selection of aptamers from oligonucleotide libraries currently requires multiple rounds of alternating steps of partitioning of binders from nonbinders and enzymatic amplification of all collected oligonucleotides. Herein, we report a highly practical solution for reliable one-step selection of aptamers. We introduce partitioning by ideal-filter capillary electrophoresis (IFCE) in which binders and nonbinders move in the opposite directions. The efficiency of IFCE-based partitioning reaches 109 , which is ten million times higher than that of typical solid-phase partitioning methods. One step of IFCE-based partitioning is sufficient for the selection of a high-affinity aptamer pool for a protein target. Partitioning by IFCE promises to become an indispensable tool for fast and robust selection of binders from different types of oligonucleotide libraries.

Direct Quantitative Analysis of Multiple microRNAs (DQAMmiR) with Peptide Nucleic Acid Hybridization Probes.

Direct quantitative analysis of multiple miRNAs (DQAMmiR) is a hybridization-based assay, in which the excess of the DNA hybridization probes is separated from the miRNA-probe hybrids, and the hybrids are separated from each other in gel-free capillary electrophoresis (CE) using two types of mobility shifters: single-strand DNA binding protein (SSB) added to the CE running buffer and peptide drag tags conjugated with the probes. Here we introduce the second-generation DQAMmiR, which utilizes peptide nucleic acid (PNA) rather than DNA hybridization probes and requires no SSB in the CE running buffer. PNA probes are electrically neutral, while PNA-miRNA hybrids are negatively charged, and this difference in charge can be a basis for separation of the hybrids from the probes. In this proof-of-principle work, we first experimentally confirmed that the PNA-RNA hybrid was separable from the excess of the PNA probe without SSB in the running buffer, resulting in a near 10 min time window, which would allow, theoretically, separation of up to 30 hybrids. Then, we adapted to PNA-RNA hybrids our previously developed theoretical model for predicting hybrid mobilities. The calculation performed with the modified theoretical model indicated that PNA-RNA hybrids of slightly different lengths could be separated from each other without drag tags. Accordingly, we designed a simple experimental model capable of confirming: (i) separation of tag-free hybrids of different lengths and (ii) separation of same-length hybrids due to a drag tag on the PNA probe. The experimental model included three miRNAs: 20-nt miR-147a, 20-nt miR-378g, and 22-nt miR-21. The three complementary PNA probes had lengths matching those of the corresponding target miRNAs. The probe for miR-147a had a short five-amino-acid drag tag; the other two had no drag tags. We were able to achieve baseline separation of the three hybrids from each other. The LOQ of 14 pM along with the high accuracy (recovery >90%) and precision (RSD ≈ 10%) of the assay at picomolar target concentrations suggest that PNA-facilitated DQAMmiR could potentially support practical miRNA analysis of clinical samples.

Aptamer facilitated purification of functional proteins.

DNA aptamers are attractive capture probes for affinity chromatography since, in contrast to antibodies, they can be chemically synthesized and, in contrast to tag-specific capture probes (such as Nickel-NTA or Glutathione), they can be used for purification of proteins free of genetic modifications (such as His or GST tags). Despite these attractive features of aptamers as capture probes, there are only a few reports on aptamer-based protein purification and none of them includes a test of the purified protein's activity, thus, leaving discouraging doubts about method's ability to purify proteins in their active state. The goal of this work was to prove that aptamers could facilitate isolation of active proteins. We refined a complete aptamer-based affinity purification procedure, which takes 4 h to complete. We further applied this procedure to purify two recombinant proteins, MutS and AlkB, from bacterial cell culture: 0.21 mg of 85%-pure AlkB from 4 mL of culture and 0.24 mg of 82%-pure MutS from 0.5 mL of culture. Finally, we proved protein activity by two capillary electrophoresis based assays: an enzymatic assay for AlkB and a DNA-binding assay for MutS. We suggest that in combination with aptamer selection for non-purified protein targets in crude cell lysate, aptamer-based purification provides a means of fast isolation of tag-free recombinant proteins in their native state without the use of antibodies.

Simultaneous Analysis of a Non-Lipidated Protein and Its Lipidated Counterpart: Enabling Quantitative Investigation of Protein Lipidation's Impact on Cellular Regulation.

Here, we introduce protein-lipidation quantitation (PLQ)-the first method for quantitative analysis of both a substrate and a product of protein lipidation in a biologically relevant context. Such analysis is required to study roles of protein lipidation in cellular regulation. In PLQ, the substrate is fused with a fluorescent protein to facilitate quantitative detection of both the nonlipidated substrate and the lipidated product. When expressed in cells with endogenous lipidation activity, the substrate is intracellularly lipidated. Following cell lysis and sampling crude cell lysate for analysis, the substrate and the product are separated by surfactant-mediated capillary electrophoresis (CE) and quantitated by integrating fluorescence intensity over respective CE peaks. In this work, we prove PLQ in principle and demonstrate its robustness to changes in structures of the substrate and lipid donor. Finally, PLQ analysis confirms a hypothesized link between a mutation in p53 and cellular prenylation activity.

High-precision quantitation of a tuberculosis vaccine antigen with capillary-gel electrophoresis using an injection standard.

Analysis of proteinogenic vaccine antigens in a quality control environment requires an accurate, precise, and reliable method for protein separation and quantitation. While having multiple advantages over the classical SDS-PAGE, capillary gel electrophoresis (CGE) has not yet become a standard tool in vaccine antigen analysis. Here we report on development of a CGE-based method for quantitative analysis of a tuberculosis vaccine fusion antigen protein, H4, currently in clinical trials. We demonstrate that our method can monitor antigen purity and relative quantity with greater precision and accuracy versus SDS-PAGE. In addition, due to use of direct light-absorbance detection, the CGE method is suitable for absolute quantitation, an application for which SDS-PAGE is limited due to the need for staining and limited dynamic range of detection. To further improve the performance of our quantitation method, we introduced Bovine Serum Albumin (BSA) as an injection standard to correct for signal variance associated with the injected sample volume. We found that, for our specific application, BSA was more appropriate as an injection standard versus one provided in a commercial kit, in terms of precision and accuracy for quantitation of H4. In addition to providing better method performance versus SDS-PAGE, CGE is also faster and less resource-intensive. We conclude that CGE should be considered as a replacement for traditional SDS-PAGE methods for vaccine antigen quantitation in a quality-control environment.

Unexpected Electrophoretic Behavior of Complexes between Rod-like Virions and Bivalent Antibodies.

Here we report on the unexpected electrophoretic behavior of complexes between rod-like virus particles (virions) and bivalent antibodies. The multiple complexes formed by the virions and antibodies migrated with electrophoretic mobilities of much greater absolute values than those of the unbound virions or antibodies while typically complexes have mobilities intermediate to those of their components. We hypothesized that the mobilities of unusually high absolute values are caused by the cross-linking of virions by bivalent antibodies into aggregates with prominent side-to-side binding. Theoretically, the mobility of such aggregates should be proportional to the square root of the number of cross-linked virions. The formation of virion aggregates with prominent side-to-side binding was confirmed by atomic force microscopy. The dependence of the aggregate mobility on the number of cross-linked virions can be used to estimate this number.

Predicting Electrophoretic Mobility of Protein-Ligand Complexes for Ligands from DNA-Encoded Libraries of Small Molecules.

Selection of target-binding ligands from DNA-encoded libraries of small molecules (DELSMs) is a rapidly developing approach in drug-lead discovery. Methods of kinetic capillary electrophoresis (KCE) may facilitate highly efficient homogeneous selection of ligands from DELSMs. However, KCE methods require accurate prediction of electrophoretic mobilities of protein-ligand complexes. Such prediction, in turn, requires a theory that would be applicable to DNA tags of different structures used in different DELSMs. Here we present such a theory. It utilizes a model of a globular protein connected, through a single point (small molecule), to a linear DNA tag containing a combination of alternating double-stranded and single-stranded DNA (dsDNA and ssDNA) regions of varying lengths. The theory links the unknown electrophoretic mobility of protein-DNA complex with experimentally determined electrophoretic mobilities of the protein and DNA. Mobility prediction was initially tested by using a protein interacting with 18 ligands of various combinations of dsDNA and ssDNA regions, which mimicked different DELSMs. For all studied ligands, deviation of the predicted mobility from the experimentally determined value was within 11%. Finally, the prediction was tested for two proteins and two ligands with a DNA tag identical to those of DELSM manufactured by GlaxoSmithKline. Deviation between the predicted and experimentally determined mobilities did not exceed 5%. These results confirm the accuracy and robustness of our model, which makes KCE methods one step closer to their practical use in selection of drug leads, and diagnostic probes from DELSMs.

Using nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) for simultaneous determination of concentration and equilibrium constant.

Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) is a versatile tool for studying affinity binding. Here we describe a NECEEM-based approach for simultaneous determination of both the equilibrium constant, K(d), and the unknown concentration of a binder that we call a target, T. In essence, NECEEM is used to measure the unbound equilibrium fraction, R, for the binder with a known concentration that we call a ligand, L. The first set of experiments is performed at varying concentrations of T, prepared by serial dilution of the stock solution, but at a constant concentration of L, which is as low as its reliable quantitation allows. The value of R is plotted as a function of the dilution coefficient, and dilution corresponding to R = 0.5 is determined. This dilution of T is used in the second set of experiments in which the concentration of T is fixed but the concentration of L is varied. The experimental dependence of R on the concentration of L is fitted with a function describing their theoretical dependence. Both K(d) and the concentration of T are used as fitting parameters, and their sought values are determined as the ones that generate the best fit. We have fully validated this approach in silico by using computer-simulated NECEEM electropherograms and then applied it to experimental determination of the unknown concentration of MutS protein and K(d) of its interactions with a DNA aptamer. The general approach described here is applicable not only to NECEEM but also to any other method that can determine a fraction of unbound molecules at equilibrium.

Prediction of protein-DNA complex mobility in gel-free capillary electrophoresis.

Selection of protein binders from highly diverse combinatorial libraries of DNA-encoded small molecules is a highly promising approach for discovery of small-molecule drug leads. Methods of kinetic capillary electrophoresis provide the high efficiency of partitioning required for such selection but require the knowledge of electrophoretic mobility of the protein-ligand complex. Here we present a theoretical approach for an accurate estimate of the electrophoretic mobility of such complexes. The model is based on a theory of the thin double layer and corresponding expressions used for the mobilities of a rod-like short oligonucleotide and a sphere-like globular protein. The model uses empirical values of mobilities of free protein, free ligand, and electroosmotic flow. The model was tested with a streptavidin-dsDNA complex linked through biotin (small molecule). The deviation of the prediction from the experimental mobility did not exceed 4%, thus confirming that not only is the model adequate but it is also accurate. This model will facilitate reliable use of KCE methods for selection of drug leads from libraries of DNA-encoded small molecules.

Pre-equilibration kinetic size-exclusion chromatography with mass spectrometry detection (peKSEC-MS) for label-free solution-based kinetic analysis of protein-small molecule interactions.

Here we introduce pre-equilibration kinetic size-exclusion chromatography with mass-spectrometry detection (peKSEC-MS), which is a label-free solution-based kinetic approach for characterizing non-covalent protein-small molecule interactions. In this method, a protein and a small molecule are mixed outside the column and incubated to approach equilibrium. The equilibrium mixture is then introduced into the SEC column to initiate the dissociation process by separating small molecules from the complex inside the column. A numerical model of a 1-dimensional separation was constructed to simulate mass chromatograms of the small molecule for varying rate constants of binding.

Emulsion PCR significantly improves nonequilibrium capillary electrophoresis of equilibrium mixtures-based aptamer selection: allowing for efficient and rapid selection of aptamer to unmodified ABH2 protein.

Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM), a homogeneous approach to select DNA aptamers, is among the most efficient partitioning techniques. In contrast with surface-based systematic evolution of ligands by exponential enrichment (SELEX) approaches, the ability of NECEEM to select aptamers to unmodified proteins in solution is preferable for identifying aptamers for eventual in vivo use. The high stringency and low sample volumes of NECEEM, although generally beneficial, can result in binding of very few aptamers, requiring highly efficient amplification to propagate them. When amplified with standard PCR, detectable library enrichment can fail due to the fast conversion of the aptamers into byproducts and preferential amplification of nonbinders. As an alternative, we proposed the use of emulsion PCR (ePCR), which is known to reduce byproduct formation, as a PCR mode for coupling with NECEEM partitioning. For the first time, we tested the advantages of ePCR in NECEEM-based aptamer selection to a medically relevant DNA repair enzyme, ABH2. We report that the combination of ePCR with NECEEM allowed for the selection of aptamers in the first three rounds of SELEX, while SELEX with conventional PCR failed in a number of attempts. Selected aptamers to an unmodified ABH2 protein have potential use in diagnostics and as leads for anticancer cotherapies, used as enhancements of alkylating agents in chemotherapy.

Kinetic size-exclusion chromatography with mass spectrometry detection: an approach for solution-based label-free kinetic analysis of protein-small molecule interactions.

Studying the kinetics of reversible protein-small molecule binding is a major challenge. The available approaches require that either the small molecule or the protein be modified by labeling or immobilization on a surface. Not only can such modifications be difficult to do but also they can drastically affect the kinetic parameters of the interaction. To solve this problem, we present kinetic size-exclusion chromatography with mass spectrometry detection (KSEC-MS), a solution-based label-free approach. KSEC-MS utilizes the ability of size-exclusion chromatography (SEC) to separate any small molecule from any protein-small molecule complex without immobilization and the ability of mass spectrometry (MS) to detect a small molecule without a label. The rate constants of complex formation and dissociation are deconvoluted from the temporal pattern of small molecule elution measured with MS at the exit from the SEC column. This work describes the concept of KSEC-MS and proves it in principle by measuring the rate constants of interaction between carbonic anhydrase and acetazolamide.