Kinetic Capillary Electrophoresis Research Articles

Study of molecular interactions by nonequilibrium capillary electrophoresis of equilibrium mixtures: Originations, developments, and applications.

Molecular interactions play a vital role in regulating various physiological and biochemical processes in vivo. Kinetic capillary electrophoresis (KCE) is an analytical platform that offers significant advantages in studying the thermodynamic and kinetic parameters of molecular interactions. It enables the simultaneous analysis of these parameters within an interaction pattern and facilitates the screening of binding ligands with predetermined kinetic parameters. Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) was the first proposed KCE method, and it has found widespread use in studying molecular interactions involving proteins/aptamers, proteins/small molecules, and peptides/small molecules. The successful applications of NECEEM have demonstrated its promising potential for further development and broader application. However, there has been a dearth of recent reviews on NECEEM. To address this gap, our study provides a comprehensive description of NECEEM, encompassing its origins, development, and applications from 2015 to 2022. The primary focus of the applications section is on aptamer selection and screening of small-molecule ligands. Furthermore, we discuss important considerations in NECEEM experimental design, such as buffer suitability, detector selection, and protein adsorption. By offering this thorough review, we aim to contribute to the understanding, advancement, and wider utilization of NECEEM as a valuable tool for studying molecular interactions and facilitating the identification of potential ligands and targets.

Affinity capillary electrophoresis for studying interactions in life sciences.

Affinity capillary electrophoresis (ACE) analyzes noncovalent interactions between ligands and analytes based on changes in their electrophoretic mobility. This technique has been widely used to investigate various biomolecules, mainly proteins, polysaccharides and hormones. ACE is becoming a technique of choice to validate high throughput screening results, since it is very predictively working in realistic and relevant media, e.g. in body fluids. It is highly recommended to incorporate ACE as a powerful analytical tool to properly prepare animal testing and preclinical studies. The interacting molecules can be found free in solution or can be immobilized to a solid support. Thus, ACE is classified in two modes, free solution ACE and immobilized ACE. Every ACE mode has advantages and disadvantages. Each can be used for a variety of applications. This review covers literature of scopus and SciFinder data base in the period from 2016 until beginning 2018, including the keywords "affinity capillary electrophoresis", "immunoaffinity capillary electrophoresis", "immunoassay capillary electrophoresis" and "immunosorbent capillary electrophoresis". More than 200 articles have been found and 112 have been selected and thoroughly discussed. During this period, the data processing and the underlying calculations in mobility shift ACE (ms ACE), frontal analysis ACE (FA ACE) and plug-plug kinetic capillary electrophoresis (ppKCE) as mostly applied free solution techniques have substantially improved. The range of applications in diverse free solution and immobilized ACE techniques has been considerably broadened.

A fast stable discretization of the Constant–Convection–Diffusion–Reaction equations of Kinetic Capillary Electrophoresis (KCE)

A discretization scheme is introduced for a set of convection–diffusion equations with a non-linear reaction term, where the convection velocity is constant for each reactant. This constancy allows a transformation to new spatial variables, which ensures the global stability of discretization. Convection–diffusion equations are notorious for their lack of stability, arising from the algebraic interaction of the convection and diffusion terms. Unexpectedly, our implemented numerical algorithm proves to be faster than computing exact solutions derived for a special case, while remaining reasonably accurate, as demonstrated in our runtime and error analysis.

Plug-plug kinetic capillary electrophoresis for in-capillary exoglycosidase digestion as a profiling tool for the analysis of glycoprotein glycans

An online exoglycosidase digestion was combined with a plug-plug kinetic mode of capillary electrophoresis (CE) for the analysis of glycoprotein-derived oligosaccharides. An exoglycosidase solution and a solution of glycoprotein glycans derivatized with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) were introduced to a neutrally coated capillary previously filled with electrophoresis buffer solution containing 0.5w/v% hydroxypropylcellulose. After immersion of both ends of the capillary in the buffer solutions, a negative voltage was applied for analysis. An APTS group of an oligosaccharide derivative has triply negative charges, which forced saccharide derivatives to anode with fast mobility and pass through the enzyme plug, which are detected at the anodic end. If the terminal monosaccharides of APTS-labeled oligosaccharides are released by the action of an exoglycosidase, the migration times of the oligosaccharides shift to those of digested oligosaccharides. We examined β-galactosidase, α-mannosidase, β-N-acetylhexosaminidase, α-neuraminidase, and α-fucosidase, and found only β-galactosidase and α-neuraminidase showed good reactivity toward APTS-labeled oligosaccharides; the reaction was completed by injecting a 3.6cm long plug of 200 and 50mU/mL concentration of exoglycosidases. In contrast, other exoglycosidases could not react with APTS labeled oligosaccharides at a concentration up to 5U/mL. The β-N-acetylhexosaminidase reaction was successively followed by the electrophoretic mobility of APTS oligosaccharides and stopped for 10min when saccharide derivatives were achieved in the enzyme plug. The reaction of α-fucosidase and α-mannosidase was completed by decreasing the electrophoretic voltage to −2kV when the APTS oligosaccharides were passing through an exoglycosidase plug. We established the CE conditions for all of the glycosidic linkage analysis of glycoprotein glycans.

Dissociation constant calculation of protein-single-stranded deoxyribonucleic acid interaction by kinetic capillary electrophoresis and its reliability analysis

Dissociation constant calculation of protein-single-stranded deoxyribonucleic acid interaction by kinetic capillary electrophoresis and its reliability analysis

Simultaneous analysis of enzyme structure and activity by kinetic capillary electrophoresis-MS.

To enable the detection of protein conformational isomers, their enzymatic activity and their inhibition in a single experiment, we developed a method based on kinetic capillary electrophoresis coupled on-line with UV detection and ion mobility mass spectrometry (CE-UV-IM-MS). Kinetic CE-UV separated protein conformers and monitored their interconversion dynamics in solution. Ion mobility mass spectrometry analyzed the conformer sizes, exact molecular weights, and structures of an enzyme and of its substrates, inhibitors and corresponding products. This coupled CE-UV-IM-MS system allowed the simultaneous, real-time observation of the effect of small-molecule inhibitors on both the conformational distribution and enzymatic activity of the human tissue transglutaminase TG2. By expanding mass spectrometry profiling of enzymatic reactions beyond proteins and substrates to include protein dynamics, CE-UV-IM-MS opens a new avenue for the modulation and regulation of cellular functions, drug development and protein engineering.

Analysis of DNA in Phosphate Buffered Saline Using Kinetic Capillary Electrophoresis.

Kinetic capillary electrophoresis (KCE) methods are useful in the study of kinetics and equilibrium properties of interactions between DNA and its binding partners (ligands). KCE experiments are typically performed in a narrow set of "conventional" low-conductivity run buffers while DNA-ligand interactions in biological systems occur in physiological fluids, characterized by high ionic strengths. The nature and ionic strength of the buffer, in which DNA-ligand interaction occurs, can significantly influence the binding. Therefore, KCE experiments meant to study such interactions would greatly benefit if they could be performed in physiological buffers, such as phosphate buffered saline (PBS). No previous KCE studies of DNA used PBS as the run buffer. Here, we test the feasibility of using PBS as a KCE run buffer for analysis of DNA and show that its usage under standard KCE conditions renders DNA undetectable. We uncover the causes of this previously unreported detrimental effect and come up with a modification of KCE which allows one to overcome it. We apply the modified KCE method to an experimental model of a platelet-derived growth factor (PDGF) protein and its DNA aptamer, which was selected in PBS, and show that the results obtained in PBS run buffer are much closer to previously reported values than those which were obtained with a conventional low-conductivity capillary electrophoresis (CE) buffer.

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.

Non-Equilibrium Capillary Electrophoresis of Equilibrium Mixtures-Based Affinity Separation and Selective Enrichment of a Long-Length DNA Aptamer

Non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) is a kinetic capillary electrophoresis method used for the affinity analysis of DNA binding to proteins or ligands as well as a rapid selection of DNA aptamers. However, long DNA strands (100-mer or more) are generally difficult to analyse by this method owing to their poor peak separation. Herein, we report optimized conditions (use of a neutral phosphate buffer with an ionic strength of 0.074 as a binding buffer and use of an 80-cm fused silica capillary with a 75-μm internal diameter) for the peak separation of a 100-mer thrombin-binding DNA aptamer-target complex and its consequent enrichment using the NECEEM-based capillary electrophoresis–systematic evolution of ligands by exponential enrichment (CE-SELEX) method.

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.

Virtual issue: structure characterization of biomolecules.

The structural diversity of biomolecules is quite simply enormous. Although many polymeric biomolecules such as proteins, and ribonucleic (RNA) and deoxyribonucleic (DNA) acids have complex but rather well-defined lowest-energy structures, it became increasingly apparent in recent years that many of them have not. Especially for proteins, but also for RNA, it has been shown that they can instead feature a number of significantly different yet similarly stable structures, or even generally lack defined structure at all, which led to the idea that a static view of single, rigid structures may not be sufficient for understanding the properties and function of biomolecules. This fundamental insight into the dynamic nature of biomolecules, and its highly important implications in folding and binding, only became possible with the use of newly developed or refined experimental approaches. This virtual issue in ChemistryOpen on the “Structure Characterization of Biomolecules” highlights the fruitful interplay between chemical synthesis and advanced techniques for biomolecular characterization in solution and in the gas phase, among them, nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), ion mobility spectrometry (IMS), microscale thermophoresis (MST), and kinetic capillary electrophoresis (KCE). Berezovski and co-workers (DOI: 10.1002/open.201400002) used KCE in combination with MS and IMS to study fast conformational dynamics of DNA G-quadruplexes, and demonstrate that rate and equilibrium constants for DNA folding and metal binding can be determined by KCE-MS. They further show that both drift and migration times in IMS and KCE are different for unfolded and folded, metal-bound DNA. Also concerned with DNA binding is the contribution by Diederichsen and co-workers (DOI: 10.1002/open.201400001), who have synthesized two new compounds, each of which comprises four nucleobase recognition units on a rigid template that is based on a cyclopeptide backbone. Binding of these triostin A analogues was studied by MST, temperature-dependent UV spectroscopy, and MS, which showed evidence for self-aggregation of the TTTT but not the ATTA sequence, and specific binding of the ATTA motif to DNA. The work by Konrat and co-workers (DOI: 10.1002/open.201402008) focuses on magnetic resonance based experimental techniques for the structural characterization of weak and transient protein/protein and protein/RNA complexes. They use sophisticated relaxation dispersion experiments to study lowly populated states of the Escherichia coli cold shock protein CspA complexed to RNA, with detection of either protein or RNA signals, and of the homodimeric complex of an oncogenic protein. Moreover, they introduce a new approach that combines NMR with electron paramagnetic resonance (EPR) spectroscopy to obtain structural information on substates of intrinsically disordered proteins that previously escaped detection by conventional techniques. Sharon and co-workers (DOI: 10.1002/open.201402002) have developed an improved rapid mixing device for time-resolved electrospray ionization (ESI) MS measurements to monitor protein folding, unfolding, and enzymatic reactions. Using cytochrome C as a model system, they demonstrate increased signal-to-noise ratio in ESI mass spectra, and a threefold decrease in experimental repeat error compared to previous capillary mixing devices. This virtual issue in ChemistryOpen on the “Structure Characterization of Biomolecules” highlights the fruitful interplay between chemical synthesis and advanced techniques for biomolecular characterization in solution and in the gas phase… Overall, the contributions in this virtual issue beautifully illustrate the value of combining experimental approaches and techniques for the structural characterization of biomolecules to obtain deeper insight into the dynamic nature and function of proteins, nucleic acids, and their complexes. Priv.-Doz. Dr. Kathrin Breuker Guest Editor Editorial Advisory Board Member ChemistryOpen

Cover Picture: Conformational Dynamics of DNA G‐Quadruplex in Solution Studied by Kinetic Capillary Electrophoresis Coupled On‐line with Mass Spectrometry (ChemistryOpen 2/2014)

AbstractNo Abstract

Conformational Dynamics of DNA G-Quadruplex in Solution Studied by Kinetic Capillary Electrophoresis Coupled On-line with Mass Spectrometry.

G-quadruplex-forming DNA/RNA sequences play an important role in the regulation of biological functions and development of new anticancer and anti-aging drugs. In this work, we couple on-line kinetic capillary electrophoresis with mass spectrometry (KCE-MS) to study conformational dynamics of DNA G-quadruplexes in solution. We show that peaks shift and its widening in KCE can be used for measuring rate and equilibrium constants for DNA–metal affinity interactions and G-quadruplex formation; and ion mobility mass spectrometry (IM-MS) provides information about relative sizes, absolute molecular masses and stoichiometry of DNA complexes. KCE-MS separates a thrombin-binding aptamer d[GGTTGGTGTGGTTGG] from mutated sequences based on affinity to potassium, and reveals the apparent equilibrium folding constant (KF≈150 μm), folding rate constant (kon≈1.70×103 s−1 m−1), unfolding rate constant (koff≈0.25 s−1), half-life time of the G-quadruplex (t1/2≈2.8 s), and relaxation time (τ≈3.9 ms at physiological 150 mm [K+]). In addition, KCE-MS screens for a GQ-stabilizing/-destabilizing effect of DNA binding dyes and an anticancer drug, cisplatin.

Conformational Dynamics of DNA G-Quadruplex in Solution Studied by Kinetic Capillary Electrophoresis Coupled On-line with Mass Spectrometry.

Invited for this months cover is the group of Prof. Maxim V. Berezovski. The cover picture shows the two-dimensional separation of unfolded (green) and folded (red) forms of G-quadruplex (GQ) DNA. The first dimension is kinetic capillary electrophoresis (KCE) separation of unfolded and folded DNA with different K+ concentrations in solution; the second dimension is ion mobility mass spectrometry separation of DNA conformers in the gas phase. DNA folding into a compact GQ structure is mediated by K+ ions. For more details, see the Full Paper on p. 58 ff.

Minimizing adsorption of histidine-tagged proteins for the study of protein–deoxyribonucleic acid interactions by kinetic capillary electrophoresis

Affinity interactions between DNA and proteins play a crucial role in many cellular processes. Kinetic Capillary Electrophoresis is a highly efficient tool for kinetic and equilibrium studies of protein–DNA interactions. Recombinant proteins, which are typically used for in vitro studies of protein–DNA interactions, are often expressed with a His tag to aid in their purification. In this work, we study how His tags affect Kinetic Capillary Electrophoresis analysis of protein–DNA interactions. We found that the addition of a His tag can increase or decrease protein adsorption to a bare-silica capillary wall, dependent on the protein. For Kinetic Capillary Electrophoresis measurements, it is essential to have as little protein adsorption as possible. We screened a number of capillary coatings to reduce adsorption of the His-tagged DNA mismatch repair protein MutS to the capillary wall and found that UltraTrol LN was the most effective coating. The effectiveness of the coating was confirmed with the prevention of adsorption of His-tagged fat mass and obesity-associated protein. Under typical conditions, the coating reduced protein adsorption to a level at which accurate Kinetic Capillary Electrophoresis analysis of protein–DNA interactions was possible. We further used Kinetic Capillary Electrophoresis to study how the His tag affected Kd of protein–DNA interactions for the MutS protein. Using UltraTrol LN, we found that the effect of the His tag was insignificant.

Volatile Kinetic Capillary Electrophoresis for Studies of Protein–Small Molecule Interactions

Kinetic capillary electrophoresis (KCE) is a toolset of homogeneous affinity methods for studying kinetics of noncovalent binding. Sensitive KCE measurements are typically done with fluorescence detection and require a fluorescent label on a smaller-sized binding partner. KCE with fluorescence detection is difficult to use for study of protein-small molecule interactions since labeling small molecules is cumbersome and can affect binding. A combination of KCE with mass-spectrometry (KCE-MS) has been recently suggested for label-free studies of protein-small molecule interactions. The major obstacle for studies by KCE-MS is a buffer mismatch between KCE and MS; MS requires volatile buffers while KCE of protein-ligand interactions is always run in near-physiological buffers. Here we asked a simple question: can protein-ligand interactions be studied with KCE in a volatile buffer? We compared three volatile buffers (ammonium acetate, ammonium bicarbonate, and ammonium formate) with a near-physiological buffer (Tris-acetate) for three protein-ligand pairs. The volatile buffers were found not to significantly affect protein-ligand complex stability; moreover, when used as CE run buffers, they facilitated good-quality separation of free ligands from the protein-ligand complexes. The use of volatile buffers instead of Tris-acetate in detection of small molecules by MS improved the detection limit by approximately 2 orders of magnitude. These findings prove the principle of "volatile" KCE, which can be easily coupled with MS to facilitate label-free kinetic studies of protein-small molecule interactions.

A semipermanent coating for preventing protein adsorption at physiological pH in kinetic capillary electrophoresis

Protein adsorption to the inner capillary wall hinders the use of kinetic capillary electrophoresis (KCE) when studying noncovalent protein-ligand interactions. Permanent and dynamic capillary coatings have been previously reported to alleviate much of the problems associated with protein adsorption. The characteristic limitations associated with permanent and dynamic coatings motivated us to look at a third type of coating - semipermanent. Here, we demonstrate that a semipermanent capillary coating, designed by Lucy and co-workers, comprised of dioctadecyldimethylammonium bromide (DODAB) and polyoxyethylene (POE) stearate, greatly reduces protein adsorption at physiological pH - a necessary requirement for KCE. The coating (i) does not inhibit protein-DNA complex formation, (ii) prevents the adsorption of the analytes, and (iii) supports an electoosmotic flow required for many applications of KCE. The coating was tested in three physiological buffers using a well-known DNA aptamer and four proteins that severely bind to bare silica capillaries as standards. For every protein, a condition was found under which the semipermanent coating effectively suppresses protein adhesion. While no coating can completely prevent the adsorption of all proteins, our findings suggest that the DODAB/POE stearate coating can have a broad impact on CE at large, as it prevents the absorption of several well studied, highly adhesive proteins at physiological pH.

Two-peak approximation in kinetic capillary electrophoresis

Kinetic capillary electrophoresis (KCE) constitutes a toolset of homogeneous kinetic affinity methods for measuring rate constants of formation (k(+)) and dissociation (k(-)) of non-covalent biomolecular complexes, C, formed from two binding partners, A and B. A parameter-based approach of extracting k(+) and k(-) from KCE electropherograms relies on a small number of experimental parameters found from the electropherograms and used in explicit expressions for k(+) and k(-) derived from approximate solutions to mass transfer equations. Deriving the explicit expressions for k(+) and k(-) is challenging but it is justified as the parameter-based approach is the simplest way of finding k(+) and k(-) from KCE electropherograms. Here, we introduce a unique approximate analytical solution of mass transfer equations in KCE termed a "two-peak approximation" and a corresponding parameter-based method for finding k(+) and k(-). The two-peak approximation is applicable to any KCE method in which: (i) A* binds B to form C* (the asterisk denotes a detectable label on A), (ii) two peaks can be identified in a KCE electropherogram and (iii) the concentration of B remains constant. The last condition holds if B is present in access to A* and C* throughout the capillary. In the two-peak approximation, the labeling of A serves only for detection of A and C and, therefore, is not required if A (and thus C) can be observed with a label-free detection technique. We studied the proposed two-peak approximation, in particular, its accuracy, by using the simulated propagation patterns built with the earlier-developed exact solution of the mass-transfer equations for A* and C*. Our results prove that the obtained approximate solution of mass transfer equations is correct. They also show that the two-peak approximation facilitates finding k(+) and k(-) with a relative error of less than 10% if two peaks can be identified on a KCE electropherogram. Importantly, the condition of constant concentration of B is always satisfied in macroscopic approach to studying kinetics at equilibrium (MASKE) whether or not B is in excess to A* and C*, and, thus, the two-peak approximation is applicable to MASKE. It completes a toolset of fitting-free methods for processing MASKE data and makes MASKE a simple practical method for finding k(+) and k(-) of "fast", "slow", and "intermediate-rate" non-covalent interactions.

Pressure-Based Approach for the Analysis of Protein Adsorption in Capillary Electrophoresis

Protein adsorption to inner capillary walls creates a major obstacle in all applications of capillary electrophoresis involving protein samples. The problem is especially severe in kinetic capillary electrophoresis (KCE) techniques, which are used to study protein-ligand interactions at physiological conditions and, thus, cannot utilize extreme pH. A variety of coatings exist to reduce protein adsorption in CE, each expressing a unique surface chemistry that interacts with individual proteins differently. Here we introduce a simple pressure-based method for the qualitative assessment of protein adsorption that can facilitate the direct antiadhesive ranking of several coatings toward a protein of interest. In this approach, a short plug of the protein is injected into a capillary and propagated through with a pressure low enough to ensure adequate Taylor dispersion. The experiment is performed with a nonmodified commercial instrument in a pseudo-two-detector approach. The two detectors are mimicked by using two different distances from the capillary inlet to a single detector. If the peak area and shape do not change with changing distance, the protein does not adsorb appreciably, while a decreasing peak area with increasing distance infers inner surface adsorption. The magnitude change of the peak area between the two distances along with the overall peak shape is used to gauge the extent of protein adsorption. By using this method, we ranked antiadhesive properties of different wall chemistries for a series of proteins. The described method will be useful for optimizing protein analysis by CE and, in particular, for KCE experiments that investigate how proteins interact with their respective ligands.

Kinetic Capillary Electrophoresis with Mass‐Spectrometry Detection (KCE‐MS) Facilitates Label‐Free Solution‐Based Kinetic Analysis of Protein–Small Molecule Binding

Tandem tracker: Here we introduce a method for studying the kinetics of protein-small-molecule interactions based on kinetic capillary electrophoresis (KCE) separation and MS detection. Due to the variety of KCE methods and MS modes available, the KCE-MS tandem is a highly versatile platform for label-free, solution-based kinetic studies of affinity interactions.