Drug binding to plasma proteins influences processes such as liberation, adsorption, disposition, metabolism, and elimination of drugs, which are thus one of the key steps of a new drug development. As a result, the characterization of drug-protein interactions is an essential part of these time- and money-consuming processes. It is important to determine not only the binding strength and the stoichiometry of interaction, but also the binding site of a drug on a protein molecule, because two drugs with the same binding site can mutually affect free drug concentration. Capillary electrophoresis-frontal analysis with mobility shift affinity capillary electrophoresis is one of the most used affinity capillary electrophoresis methods for the characterization of these interactions. In this study, a well-known sensitivity problem of most capillary electrophoresis-frontal analyses using ultraviolet detection is solved by its combination with contactless conductivity detection, which provided sixfold lower limits of quantitation and detection. Binding parameters of the human serum albumin-salicylic acid model affinity pair were evaluated by this newly developed approach and by the classical approach with ultraviolet detection primarily used for their mutual comparison. The results of both approaches agreed well and are also in agreement with literature data obtained using different techniques.
- MeSH
- elektrická vodivost MeSH
- elektroforéza kapilární metody MeSH
- krevní proteiny * MeSH
- lidé MeSH
- lidský sérový albumin * MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The process of choosing the most proper technique for studying the molecular interactions is based on critical factors such as instrumentation complexity, automation, experimental procedures, analysis time, consumables, and cost-value. This review has tracked the use of affinity capillary electrophoresis (ACE) and microscale thermophoresis (MST) techniques in the evaluation of molecular binding among different molecules during the 5 years 2016-2021. ACE has proved to be an attractive technique for biomolecular characterization with high resolution efficiency where small variations in several controlling factors can much improve such efficiency compared to other analytical techniques. Meanwhile, MST has proved its higher sensitivity for smaller amounts of complex non-purified biosamples without affecting its robustness while providing high through output. However, the main motivation to review both techniques in the proposed review was their capability to carry out all experiments without the need for immobilizing one interacting partner, besides a great flexibility in the use of buffering systems. The proposed review demonstrates the importance of both techniques in different areas of life sciences. Moreover, the recent advances in exploiting ACE and MST in other research interests have been discussed.
- MeSH
- elektroforéza kapilární * metody MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Monitoring metabolite uptake and excretion in the culture medium is a noninvasive technique that is used for the metabolic study of cleaving embryos after in vitro fertilization. Low sample consumption, the versatility of the detection, and optimal sensitivity and selectivity are essential elements for extracellular metabolome analyses, and can be conveniently achieved by combining CE with mass spectrometric detection. This paper reports a method for amino acid determination in a limited volume sample (8 μL) of spent culture media collected after the cultivation of in vitro fertilized embryos. Special attention was focused on the sample preparation procedure. The sample was processed with acetonitrile, which facilitates online sample preconcentration via field-amplified sample stacking, and undesired sample evaporation was significantly reduced by the simultaneous addition of dimethyl sulfoxide. Key parameters that affected electrophoretic separation and mass spectrometric detection were investigated, including the type of buffers and organic solvent, optimization of their concentrations, and finally the settings for their ionization. The separation and quantification of 19 amino acids were achieved using 15% acetic acid as the background electrolyte with a sheath liquid consisting of an equimolar mixture of methanol and water. The applicability of the optimized system was demonstrated by determining the amino acid profile in 40 samples of spent cultivation medium in this pilot study. This developed method also has great potential for amino acid analyses in minute sample volumes of other biological matrices.
In this study, two capillary electrophoresis-based ligand binding assays, namely, mobility shift affinity capillary electrophoresis (ms-ACE) and capillary electrophoresis-frontal analysis (CE-FA), were applied to determine binding parameters of human serum albumin toward small drugs under similar experimental conditions. The substances S-amlodipine (S-AML), lidocaine (LDC), l-tryptophan (l-TRP), carbamazepine (CBZ), ibuprofen (IBU), and R-verapamil (R-VPM) were used as the main binding partners. The scope of this comparative study was to estimate and compare both the assays in terms of their primary measure's precision and the reproducibility of the derived binding parameters. The effective mobility could be measured with pooled CV values between 0.55% and 7.6%. The precision of the r values was found in the range between 1.5% and 10%. Both assays were not universally applicable. The CE-FA assay could successfully be applied to measure the drugs IBU, CBZ, and LDC, and the interaction toward CBZ, S-AML, l-TRP, and R-VPM could be determined using ms-ACE. The average variabilities of the estimated binding constants were 64% and 67% for CE-FA and ms-ACE, respectively.
- MeSH
- akutní myeloidní leukemie * MeSH
- elektroforéza kapilární metody MeSH
- ibuprofen MeSH
- izotachoforéza * MeSH
- lidé MeSH
- lidský sérový albumin metabolismus MeSH
- reprodukovatelnost výsledků MeSH
- tryptofan MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
CE/frontal analysis (CE/FA) is probably one of the most frequently used modes of CE for studying affinity interactions. It is typically performed with classic UV-Vis detection that suffers from low concentration sensitivity. To overcome this limitation, the applicability of CE/FA in combination with ESI-MS detection for the investigation of drug-HSA interactions was demonstrated. The developed new method combines the advantages of CE/FA, such as low sample consumption and no labeling or immobilization of interacting partners, with the benefits of MS detection, such as higher selectivity and sensitivity; moreover, it can be used for molecules lacking a fluorophore or chromophore. The binding parameters of tolbutamide (TL) and glimepiride (GLP), first- and second-generation antidiabetics that differ strongly in their solubility in aqueous solutions, were investigated by this CE/FA-MS method. This method, in contrast to the CE/FA method with the most commonly used UV-Vis detection, is more sensitive; an almost three times lower LOD was reached. The binding parameters of TL and GLP were investigated by this CE/FA-MS method and compared with the literature data. The binding constant value of TL obtained by UV-Vis detection was lower than the value obtained by the method hyphenated with MS detection, which is probably given by the influence of the ESI parameters on the stability of drug-HSA complex. In addition, the ratio of TL and HSA concentrations was divergent in both of the experimental approaches. Finally, it can be concluded that both detection methods have their strengths and weaknesses.
