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1.
Anal Chem ; 2020 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-32961048

RESUMO

Capillary zone electrophoresis-mass spectrometry (CE-MS) is a mature analytical tool for the efficient profiling of (highly) polar and ionizable compounds. However, the use of CE-MS in comparison to other separation techniques remains underrepresented in metabolomics, as this analytical approach is still perceived as technically challenging and less reproducible, notably for migration time. The latter is key for a reliable comparison of metabolic profiles and for unknown biomarker identification that is complementary to high resolution MS/MS. In this work, we present the results of a Metabo-ring trial involving 16 CE-MS platforms among 13 different laboratories spanning two continents. The goal was to assess the reproducibility and identification capability of CE-MS by employing effective electrophoretic mobility (µeff) as the key parameter in comparison to the relative migration time (RMT) approach. For this purpose, a representative cationic metabolite mixture in water, pretreated human plasma and urine samples spiked with the same metabolite mixture, were used and distributed for analysis by all laboratories. The µeff was determined for all metabolites spiked into each sample. The background electrolyte (BGE) was prepared and employed by each participating lab following the same protocol. All other parameters (capillary, interface, injection volume, voltage ramp, temperature, capillary conditioning and rinsing procedure, etc.) were left to the discretion of the contributing labs. The results revealed that the reproducibility of the µeff for 20 out of the 21 model compounds was below 3.1% vs. 10.9% for RMT, regardless of the huge heterogeneity in experimental conditions and platforms across the thirteen labs. Overall, this Metabo-ring trial demonstrated that CE-MS is a viable and reproducible approach for metabolomics.

2.
Anal Bioanal Chem ; 412(17): 4195-4207, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32377866

RESUMO

To mimic the activity of hyaluronidase in natural environment, the hydrolysis of hyaluronic acid (HA) by hyaluronidase was investigated for the first time in the presence of crowding agents using capillary electrophoresis (CE) as a simple and reliable technique for conducting enzymatic assay. Polyethylene glycol (PEG) 6000 was selected as a model crowder and the hyaluronic acid degradation catalyzed by bovine testes hyaluronidase (BTH) was carried out at different PEG concentrations (0%, 10%, and 17%). After optimization of the CE analytical method and enzymatic assay, the degradation products were monitored at different HA concentrations. At 10% of PEG and 0.3 mg mL-1 of HA, the activity of the enzyme was significantly reduced showing inconvenient interactions of PEG with the hyaluronidase blocking the release of hydrolysis products. A similar reduction of hyaluronidase activity was observed at 1 mg mL-1 of HA due to the presumable formation of the BTH-substrate complex. The experimental curves obtained by CE also evidence that the overall kinetics are governed by the hydrolysis of hexasaccharide intermediates. Finally, the effect of PEG on hyaluronidase activity was evaluated in the presence of natural or synthetic inhibitors. Our results show a significant difference of the inhibitors' affinity toward hyaluronidase in the presence of PEG. Surprisingly, the presence of the crowding agent results in a loss of the inhibition effect of small polycyclic inhibitors, while larger charged inhibitors were less affected. In this work, CE analyses confirm the importance of mimicking the cellular environment for the discovery and development of reliable inhibitors. Graphical abstract.

3.
Anal Chim Acta ; 1085: 117-125, 2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31522725

RESUMO

Capillary electrophoresis (CE) with dual UV and conductivity detection was used for the first time to monitor the functionalization of gold nanoparticles (AuNPs), a process catalyzed by an enzyme, myrosinase (Myr). A thiol glucosinolate (GL-SH) designed by our group was used as substrate. Hydrolysis of free and immobilized GL-SH was characterized using off-line and on-line CE-based enzymatic assays. The developed approaches were validated using sinigrin, a well-referenced substrate of Myr. Michaelis-Menten constant of the synthetized GL-SH was comparable to sinigrin, showing that they both have similar affinity towards Myr. It was demonstrated that transverse diffusion of laminar flow profiles was well adapted for in-capillary Mixing of nanoparticles (AuNPs) with proteins (Myr) provided that the incubation time is inferior to 20 min. Only low reaction volume (nL to few µL) and short analysis time (<5 min) were required. The electrophoretic conditions were optimized in order to evaluate and to confirm the AuNPs stability before and after functionalization by CE/UV based on surface plasmon resonance band red-shifting. The hydrolysis of the functionalized AuNPs was subsequently evaluated using the developed CE-C4D/UV approach. Repeatabilities of enzymatic assays, of electrophoretic analyses and of batch-to-batch functionalized AuNPs were excellent.


Assuntos
Glucosinolatos/metabolismo , Glicosídeo Hidrolases/análise , Ouro/metabolismo , Nanopartículas Metálicas/química , Compostos de Sulfidrila/metabolismo , Biocatálise , Condutividade Elétrica , Eletroforese Capilar , Glucosinolatos/química , Glicosídeo Hidrolases/metabolismo , Ouro/química , Hidrólise , Estrutura Molecular , Espectrofotometria Ultravioleta , Especificidade por Substrato , Compostos de Sulfidrila/química
4.
Bioconjug Chem ; 30(5): 1385-1394, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30933500

RESUMO

Fluorescein isothiocyanate (FITC) is one of the most extensively used fluorescent probes for the labeling of biomolecules. The isothiocyanate function reacts with lysine residues of proteins to provide a chemically stable thiourea linkage without releasing any byproduct. However, diversification of isothiocyanate-based reagents is still hampered by the lack of mild conditions to generate isothiocyanate chemical functions, as well as by their poor stability and limited solutions available to increase water solubility, restricting the use of isothiocyanate labeling to highly water-soluble fluorophores. Inspired by plant biological processes, we report a safe and biocompatible myrosinase-assisted in situ formation of isothiocyanate conjugates from a highly water-soluble and stable glucosinolate precursor. This method was applied for the fluorescence labeling of a plasmatic protein and fluorescence imaging of living cells.


Assuntos
Fluoresceína-5-Isotiocianato/síntese química , Corantes Fluorescentes/síntese química , Glicosídeo Hidrolases/química , Células HEK293 , Humanos , Solubilidade
5.
Carbohydr Res ; 475: 56-64, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30836261

RESUMO

The activity of eukaryote hydrolase-type of hyaluronidases was studied using a miniaturized capillary electrophoresis (CE) assay developed in our laboratory. Few nanoliters of reagents are sufficient and no labeling is required for this assay. The effect of natural and original synthetic effectors of hyaluronidase was evaluated. These di- and trisaccharides from linkage region of proteoglycans were synthesized in 30-40 steps from monomeric units using classical protection, deprotection, glycosylation and deoxygenation reactions. The influence of the chain length (di/trisaccharide), the modification type (methoxy/deoxy) and its position (2/4/6) was studied. The inhibition and/or activation percentages were determined at two concentrations of effectors; 0.2 mM and 2 mM. The half maximal effective concentration (EC50) values were evaluated (n = 2) for the most effective inhibitors (∼1 mM) and activators (∼0.2 mM). Results showed that hyaluronidase was mostly inhibited in a concentration-dependent fashion by a deoxy modification and activated by a methoxy modification. Trisaccharides were found to be more effective on hyaluronidase activity than disaccharides. Position 4 was found to be more favorable for hyaluronidase activity than position 6 and the activity in position 2 was negligible. For a better understanding of the enzyme function mode, the inhibition constant (Ki) was also evaluated by CE (Ki ∼ 2 mM). These results are of great interest especially as few activators of hyaluronidase are presented in the literature.


Assuntos
Eletroforese Capilar , Ensaios Enzimáticos , Inibidores Enzimáticos/farmacologia , Hialuronoglucosaminidase/antagonistas & inibidores , Oligossacarídeos/farmacologia , Animais , Configuração de Carboidratos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Humanos , Hialuronoglucosaminidase/metabolismo , Oligossacarídeos/química , Relação Estrutura-Atividade
6.
Anal Chim Acta ; 1049: 115-122, 2019 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-30612642

RESUMO

Nucleosides and their analogues play a crucial role in the treatment of several diseases including cancers and viral infections. Their therapeutic efficiency depends on their capacity to be converted to the active nucleoside triphosphates form through successive phosphorylation steps catalyzed by nucleoside/nucleotide kinases. It is thus mandatory to develop an easy, rapid, reliable and sensitive enzyme activity tests. In this study, we monitored the three-step phosphorylation of thymidine to thymidine triphosphate respectively by (1) human thymidine kinase 1 (hTK1), (2) human thymidylate kinase (hTMPK) and (3) human nucleoside diphosphate kinase (hNDPK). Free and immobilized kinase activities were characterized by using the Michaelis-Menten kinetic model. Flow Injection Analysis (FIA) with High-Resolution Mass Spectrometry (HRMS) was used as well as capillary electrophoresis (CE) with UV detection. The three-step cascade phosphorylation of thymidine was also monitored. FIA-HRMS allows a sensitive and rapid evaluation of the phosphorylation process. This study proposes simple, rapid, efficient and sensitive methods for enzyme kinetic studies and successive phosphorylation monitoring with immobilized enzymes.


Assuntos
Enzimas Imobilizadas/química , Núcleosídeo-Difosfato Quinase/química , Núcleosídeo-Fosfato Quinase/química , Timidina Quinase/química , Timidina/química , Análise de Injeção de Fluxo/métodos , Humanos , Cinética , Espectrometria de Massas/métodos , Nanopartículas/química , Fosforilação
7.
Anal Chim Acta ; 1020: 134-141, 2018 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-29655424

RESUMO

There have been many efforts to search for affordable and efficient cosmetic ingredients from natural sources and to evaluate their bioactivities using eco-responsible tools. Hyaluronidase, elastase and collagenase are responsible for the degradation of the main components of the extracellular matrix, namely the hyaluronic acid, elastin and collagen, respectively. The aim of this work was to develop a single capillary electrophoresis method to monitor simultaneously the activities of these three enzymes, without reactant immobilization or radioactivity use. The developed approach was used to evaluate the bioactivity of the red alga Jania rubens after microwave- or electrochemical-assisted extraction. For this purpose, the incubation time, the reactant concentrations, the separation buffer and the detection system were carefully chosen. CE with double detection system, LIF and HRMS connected in series, was used to ensure the simultaneous analysis of the substrates and products of the three enzymatic reactions. The optimized enzymatic conditions allowed the use of the same protocol to assess the 3 enzyme activities. These conditions consisted of 10 min pre-incubation of the enzyme (with alga extract) at 37 °C; 10 min incubation with the substrate at 37 °C and 10 min stop-time at 90 °C. 1.4 nL of each reaction mixture were co-injected into a 85 cm total length capillary using short-end injection. Ammonium acetate (50 mM, pH 9.0) was used for electrophoretic separation. All substrates and products were simultaneously detected in less than 10 min with good peak symmetry and efficiency, sufficient intra-day and inter-day repeatabilities (RSD < 4.5%; n = 3) and excellent LOQ (<5 nM). The results obtained using this multiple CE-based enzymatic assay showed the significant effect of Jania rubens ethanolic extracts on elastase, hyaluronidase and the metalloproteinase MMP-1.


Assuntos
Colagenases/metabolismo , Hialuronoglucosaminidase/metabolismo , Elastase Pancreática/metabolismo , Rodófitas/metabolismo , Colágeno/química , Colágeno/metabolismo , Elastina/química , Elastina/metabolismo , Técnicas Eletroquímicas , Eletroforese Capilar , Ácido Hialurônico/química , Ácido Hialurônico/metabolismo , Micro-Ondas , Estrutura Molecular , Rodófitas/química
8.
J Chromatogr A ; 1529: 1-28, 2017 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-29132826

RESUMO

Elastase, collagenase, hyaluronidase and tyrosinase, are very interesting enzymes due to their direct implication in skin aging and as therapeutic hits. Different techniques can be used to study these enzymes and to evaluate the influence of effectors on their kinetics. Nowadays, analytical techniques have become frequently used tools for miniaturizing enzyme assays. The main intention of this article is to review chromatographic and electrophoretic tools that study the four enzymes above mentioned. More specifically, the use of high-performance liquid chromatography and capillary electrophoresis and their derivative techniques for monitoring these enzymes will be investigated. The advantages and limitations of these assays will also be discussed. The original use of microscale thermophoresis and thin layer chromatography in this domain will also be covered.


Assuntos
Cromatografia , Eletroforese , Ensaios Enzimáticos/instrumentação , Enzimas/análise , Colagenases/análise , Colagenases/química , Colagenases/metabolismo , Enzimas/química , Enzimas/metabolismo , Hialuronoglucosaminidase/análise , Hialuronoglucosaminidase/química , Hialuronoglucosaminidase/metabolismo , Cinética , Monofenol Mono-Oxigenase/análise , Monofenol Mono-Oxigenase/química , Monofenol Mono-Oxigenase/metabolismo , Oxirredução , Elastase Pancreática/análise , Elastase Pancreática/química , Elastase Pancreática/metabolismo
9.
J Chromatogr A ; 1497: 19-27, 2017 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-28372836

RESUMO

Hyaluronidase degrades hyaluronic acid, the principal component of the extracellular matrix. Inhibition of this enzyme is thus expected to hinder skin aging. Brown alga Padina pavonica activity toward hyaluronidase was evaluated using capillary electrophoresis (CE)-based enzymatic assays. This green technique allows evaluation of the biological activity of the natural material in an economic manner. Pressurized liquid extraction (PLE), microwave assisted extraction (MAE), supercritical fluid extraction and electroporation extraction techniques were used. Extraction conditions were optimized to obtain cosmetically acceptable Padina pavonica extracts with the best inhibition activity. CE-based assays were conducted using only a few nanoliters of reactants, a capillary of 60cm total length and of 50µm internal diameter, +20kV voltage for separation in 50mM ammonium acetate buffer (pH 9.0) and 200nm wavelength for detection. The reaction mixture was incubated for 1h and CE analysis time was about 11min. A novel online CE-assay using transverse diffusion of laminar flow profiles for in-capillary reactant mixing allowed efficient monitoring of hyaluronidase kinetics with Km and Vmax equal to 0.46±0.04mgmL-1 and 137.1±0.3nMs-1 (r2=0.99; n=3), respectively. These values compared well with literature, which validates the assay. Water extracts obtained by PLE (60°C; 2 cycles) and MAE (60°C; 1000W; 2min) presented the highest anti-hyaluronidase activity. The half maximal effective concentration (IC50) of water PLE extract was 0.04±0.01mgmL-1 (r2=0.99; n=3). This value is comparable to the one obtained for Einsenia bicyclis phlorotannin fractions (IC50=0.03mgmL-1), which makes Padina pavonica bioactivity very promising.


Assuntos
Eletroforese Capilar/métodos , Hialuronoglucosaminidase/antagonistas & inibidores , Extração Líquido-Líquido/métodos , Micro-Ondas , Feófitas/química , Pressão , Alga Marinha/química , Água/química , Cromatografia com Fluido Supercrítico , Difusão , Eletroforese Capilar/normas , Eletroporação , Concentração Inibidora 50 , Cinética , Reprodutibilidade dos Testes
10.
Environ Sci Pollut Res Int ; 24(13): 12293-12300, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28357792

RESUMO

A commercial molecularly imprinted polymer (MIP) dedicated to glyphosate (GLY) and its main metabolite, aminomethylphosphonic acid (AMPA), was lately assessed as "POCIS-like" sampler on mineral water. The obtained results were encouraging with 111 and 122 mL day-1 as sampling rates for GLY and AMPA, respectively. Therefore, before applying this passive sampler to environmental waters, the commercial phase was tested on different water matrices as a solid-phase extraction (SPE) device. The SPE protocol was carried on 250 mg of MIP with the following three steps: conditioning by Milli-Q water, loading of the sample (15 mL), and elution of the analytes by 4 mL 0.1 M HCl that were evaporated to dryness and recovered in 15 mL of the suitable solvent for analysis. This protocol was first applied to mineral water spiked by GLY and AMPA at environmental concentration levels (25-750 ng L-1). Analyses were carried out by ultra-performance liquid chromatography hyphenated to tandem mass after derivatization of GLY and AMPA by 9-fluorenylmethylchloroformate. The linear correlation between concentrations measured with and without SPE on MIP was proved.Furthermore, other extractions showed that high concentrations of metal ion interferents (lead(II), cadmium(II), and zinc(II)) in groundwaters did not reduce SPE performance of the MIP.Then, concentration assays were undertaken and brought noteworthy results, such as the recovery of 80% GLY and AMPA from groundwater spiked at 10 ng L-1 and concentrated 100 times. For this purpose, ion exclusion chromatography hyphenated to mass was applied without previous derivatization of the analytes. The same concentration factor and analytical method were applied to 100 ng L-1 spiked sea water with recoveries of 96% for GLY and 121% for AMPA.


Assuntos
Polímeros/química , Espectrometria de Massas em Tandem , Fluorenos , Glicina/análogos & derivados , Impressão Molecular , Extração em Fase Sólida
11.
J Sep Sci ; 40(2): 558-566, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27813309

RESUMO

Amino acids play a key role in food analysis, clinical diagnostics, and biochemical research. Capillary electrophoresis with laser-induced fluorescence detection was used for the analysis of several amino acids. Amino acid labeling with fluorescein isothiocyanate was conducted using microwave-assisted derivatization at 80°C (680 W) during only 150 s. Good electrophoretic resolution was obtained using a background electrolyte composed of sodium tetraborate buffer (100 mM; pH 9.4) and ß-cyclodextrin (10 mM), and the limits of quantification were 3-30 nM. The developed capillary electrophoresis with laser-induced fluorescence method was used to analyze amino acids in Dunaliella salina green algae grown under different conditions. A simple extraction technique based on electroporation of the cell membrane was introduced. A home-made apparatus allowed the application of direct and alternating voltages across the electrochemical compartment containing a suspension of microalgae in distilled water at 2.5 g/L. A direct voltage of 12 V applied for 4 min gave the optimum extraction yield. Results were comparable to those obtained with accelerated-solvent extraction. The efficiency of electroporation in destroying microalgae membranes was shown by examining the algae surface morphology using scanning electron microscopy. Stress conditions were found to induce the production of amino acids in Dunaliella salina cells.


Assuntos
Aminoácidos/análise , Aminoácidos/isolamento & purificação , Técnicas de Química Analítica/métodos , Eletroforese Capilar , Eletroporação , Lasers , Microalgas/química , Clorófitas/química , Clorófitas/metabolismo , Fluorescência
12.
Anal Chim Acta ; 951: 140-150, 2017 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-27998482

RESUMO

The biology of hyaluronidase activity on age related turnover of the hyaluronic acid (HA) in skin dermis and epidermis has not been established. Elucidation of this phenomenon enables discovery of novel compounds for skin health. As a simple and green technique, capillary electrophoresis (CE) was used for the first time for the determination of the kinetic constants (Km, Vmax and IC50) of the enzymatic degradation of HA. Reaction products were identified using CE/high-resolution mass spectrometry (HRMS) after appropriate optimization. Best results in terms of signal sensitivity were obtained using 10 mM ammonium acetate (pH 9.0) BGE, a sheath liquid composed of methanol-water (80:20, v/v) with 0.02% (v/v) formic acid at 10 µL min-1 and an ESI voltage at -4 kV. Km and Vmax were determined (n = 3) using CE/UV at 200 nm as 0.24 ± 0.02 mg mL-1 and 150.4 ± 0.1 nM s-1, respectively. They were also successfully obtained by CE/HRMS (n = 3) with Km of 0.49 ± 0.02 mg mL-1 and Vmax of 155.7 ± 0.2 nM s-1. IC50 of a standard natural inhibitor, epigallocatechin gallate, was also determined by CE-UV/HRMS. Kinetic constant values obtained by CE compared well with literature which validated the developed CE-based assay. In addition, the activity of homemade tetrasaccharides of biotinylated chondroitin sulfate CS-A or CS-C (4- or 6- sulfated in a homogeneous or heterogeneous way) on the hydrolysis reaction of hyaluronidase was evaluated. Hyaluronidase was mostly dose-dependently inhibited by CS-A tetrasaccharides sulfated in a homogeneous way. Two trisaccharides from truncated linkage region of proteoglycans were also tested as inhibitors or activators. CE-based assay showed that even a small modification of one hydroxyl group changes the influence on hyaluronidase activity. CE-based assay can be used for the screening of natural and synthetic inhibitors of hyaluronidase activity for cosmetic and therapeutic applications.


Assuntos
Eletroforese Capilar , Hialuronoglucosaminidase/química , Espectrometria de Massas , Cinética
13.
J Chromatogr A ; 1431: 215-223, 2016 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-26777089

RESUMO

Capillary electrophoresis-laser induced fluorescence (CZE-LIF) and microscale thermophoresis (MST) were used for the first time to study the inhibition of human neutrophil elastase (HNE). We recently studied HNE kinetics (Km and Vmax) by developing an in-capillary CZE-LIF assay based on transverse diffusion of laminar flow profiles (TDLFP) for reactant mixing. In this work, the former assay was adapted to monitor HNE inhibition. Two natural well known HNE inhibitors from the triterpene family, ursolic acid and oleanolic acid, were tested to validate the developed assay. Since the solubility of pentacyclic triterpenes in aqueous media where the enzymatic reaction will take place is limited, the effect of DMSO and ethanol on HNE was studied using microscale thermophoresis (MST). An agglomeration of the enzyme was revealed when preparing the inhibitor in 5% (v/v) DMSO. This phenomenon did not occur in the presence of ethanol. Therefore, ethanol was used as inhibitor solvent, at a limited percentage of 20% (v/v). In these conditions and after optimization of the TDLFP approach, the repeatability (RSD on migration times and peak-areas inferior to 2.2%) of the CZE-LIF assay and the sensitivity (LOQ of few nM) were found to be satisfactory for conducting inhibition assays. IC50 values for ursolic and oleanolic acid were successfully determined. They were respectively equal to 5.62±0.10µM (r(2)=0.9807; n=3) and to 8.21±0.23µM (r(2)=0.9887; n=3). Excellent agreement was found between the results obtained by CE and those reported in literature which validates the developed method. Particularly, the CE-based assay is able to rank HNE inhibitors relative to each other. Furthermore, MST technique was used for evaluating HNE interaction with the ursolic acid. Up to 16 capillaries were automatically processed to obtain in one titration experiment the dissociation constant for the HNE-ursolic acid complex. Ki was found to be 2.72±0.66µM (n=3) which is in excellent agreement with the value determined by CE enzyme inhibition studies (Ki=2.81µM) confirming the reliability of the developed CE assay and the competitive inhibition mode of ursolic acid.


Assuntos
Técnicas de Química Analítica/métodos , Eletroforese Capilar , Elastase de Leucócito/antagonistas & inibidores , Serpinas/metabolismo , Difusão , Ativação Enzimática/efeitos dos fármacos , Fluorescência , Humanos , Cinética , Lasers , Ácido Oleanólico/farmacologia , Triterpenos Pentacíclicos/química , Reprodutibilidade dos Testes , Triterpenos/química , Triterpenos/farmacologia
14.
J Chromatogr A ; 1419: 116-24, 2015 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-26454788

RESUMO

Skin aging is a progressive process determining the ultimate skin appearance. Human neutrophil elastase (HNE) has been shown to play an important role in the degradation of the extracellular matrix. In order to assay HNE kinetics, a novel online capillary zone electrophoresis (CZE) assay has been developed in this study for the determination of the maximum velocity (Vmax) and of the Michaelis-Menten constant (Km) of HNE regarding several potential substrates. These assays are based on short-end injection to shorten analysis time, on transverse diffusion of laminar flow profiles (TDLFP) for in-capillary reactant mixing, and on UV or laser-induced fluorescence (LIF) detection. Kinetic constants for a referenced peptidic substrate were determined using not only online assays but also offline (pre-capillary) mode. The results obtained were cross compared and compared to the literature in order to validate the developed assays. The hydrolysis of three new potential fluorogenic substrates by HNE was also monitored. Two new peptidic substrates for HNE were identified through this study. Km values of these novel substrates were successfully determined using online CZE assay (Km ∼0.07mM). This value was in the same order of magnitude of that of the referenced substrate despite the presence of the labeling group 5-carboxyfluorescein (5-FAM). HNE activity has never been assessed using online CZE-based assay, neither with UV nor with LIF detection. The developed assay conducted with the new labeled substrates is particularly sensitive (LOQ of few nM), does not require the presence of micelles in the BGE (which is the case for the reference substrate) and only necessitates few nanoliters of reactants making it particularly adapted for screening studies.


Assuntos
Elastase de Leucócito/análise , Difusão , Eletroforese Capilar/métodos , Fluorescência , Corantes Fluorescentes/análise , Humanos , Hidrólise , Cinética , Lasers , Peptídeos/análise , Especificidade por Substrato , Raios Ultravioleta
15.
Electrophoresis ; 36(21-22): 2768-2797, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26256543

RESUMO

CE has become a frequently used tool for miniaturizing enzyme assays due particularly to its well-recognized low sample consumption. CE-based enzyme assays cover all aspects of kinetic analysis including the evaluation of enzyme activity, substrate and modulator characterization, and identification. These assays are performed to conduct high-quality primary (hit finding) and secondary (confirmation by determining the half maximal inhibitory concentration, IC50 ) screening. Nowadays, the kinase family is among the most actively studied pharmaceutical targets in oncology, and in neurodegenerative and inflammation diseases. In this article, we review the fundamentals of the different approaches that may be employed for assaying kinase kinetics. Their advantages and limitations will also be discussed by covering the literature of CE-based assays for purified kinases as well as for kinases in living cells. The last section will be devoted to perspectives by showing some applications of multiplexed and of microchip devices for high-throughput screening kinase assays.

16.
Anal Bioanal Chem ; 407(10): 2821-8, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25711986

RESUMO

The hyperphosphorylation of tau protein is associated with the development of the neuronal pathology of Alzheimer's disease. As most conventional methods study only particular phosphorylation sites of tau, it is necessary to develop a simple and reliable assay to determine the phosphorylation of tau at multiple sites. Capillary electrophoresis (CE)-based enzymatic assays are not yet used to monitor tau phosphorylation. The present work aims to develop CE-based assays to evaluate tau phosphorylation by the glycogen synthase kinase 3-ß (GSK3ß). A novel pre-capillary CE assay was first developed. An in-capillary CE-based enzymatic assay was also used since this approach is known to be time- and cost- effective. The enzymatic reaction was monitored by quantifying the product adenosine 5'- diphosphate (ADP). The influence of two classes of glycosaminoglycan (GAG), namely heparin and heparan sulfate, on the phosphorylation reaction was also assessed. Results obtained by both CE approaches were comparable and in excellent agreement with those reported in the literature using conventional radiometric and immunoblotting methods. In fact, CE results confirmed the inductory effect of the sulfated sugars heparin and heparan sulfate on tau hyperphosphorylation, probably because of the exposition of new sites phosphorylatable by GSK3ß. This study shows that simple (no-labeling), rapid (less than 30 min per assay), and eco-friendly (no-radioactivity) CE-based kinase assays can give insight into the abnormal phosphorylation of tau. They can be extended to screen different modulators of tau phosphorylation to highlight their function and to develop effective drugs for neurodegenerative disease treatments.


Assuntos
Eletroforese Capilar/métodos , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas tau/metabolismo , Difosfato de Adenosina/análise , Difosfato de Adenosina/metabolismo , Eletroforese Capilar/instrumentação , Glicogênio Sintase Quinase 3 beta , Heparina/química , Heparina/metabolismo , Heparitina Sulfato/química , Heparitina Sulfato/metabolismo , Humanos , Limite de Detecção , Fosforilação
17.
J Chromatogr A ; 1367: 161-6, 2014 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-25280874

RESUMO

Sulfonylureas (SUs) are one of the most widely used herbicides to control weeds in crops. Herein, capillary electrophoresis (CE) was used to determine four sulfonylureas in natural waters, namely chlorsulfuron (CS), iodosulfuron methyl (IM), metsulfuron methyl (MSM) and mesosulfuron methyl (MSS). First of all, a bare silica capillary was chosen with 10mM of 1-butyl-3-methylimidazolium tetrafluoroborate (bminBF4) as electrophoretic buffer (pH 9.6) containing 2 mg L(-1) of surfactant-coated single-wall carbon nanotubes (SC-SWCNTs). A dramatic deviation in migration times was observed. Therefore, a poly(diallyldimethylammonium) chloride (PDADMAC) statically coated cationic capillary was used to improve repeatability and to alter the selectivity of the separation. The electroosmotic flow (EOF) measurement revealed that the SC-SWCNTs were strongly adsorbed at the surface of the PDADMAC coating even in the absence of the surfactant-coated nanotubes in the electrolyte buffer. Consequently, a stable strong cathodic EOF and excellent repeatabilities were obtained with relative standard deviations (RSDs) on migration times and on corrected peak areas below 0.9 and 1.5%, respectively. The separation of the SUs was conducted in only 6 min. No regeneration of the coating between analyses was necessary, and high peak efficiencies up to 173,000 theoretical plates were obtained. The bi-layer coating was subsequently used to analyze sulfonylureas in tap water, in several mineral waters as well as in underground waters spiked with SUs and directly injected into the CE capillary.


Assuntos
Eletroforese Capilar/métodos , Nanotubos de Carbono/química , Dodecilsulfato de Sódio/química , Ureia/análise , Compostos Alílicos/química , Eletrólitos/química , Eletro-Osmose , Imidazóis/química , Polietilenos/química , Compostos de Amônio Quaternário/química , Tensoativos/química
18.
Anal Bioanal Chem ; 406(15): 3743-54, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24817345

RESUMO

Human kinases are one of the most promising targets for cancer therapy. Methods able to measure the effects of drugs on these cell agents remain crucial for biologists and medicinal chemists. The current work therefore sought to develop an in-capillary enzymatic assay based on capillary electrophoresis (CE) to evaluate the inhibition of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt), and the mammalian target of rapamycin (mTOR). These kinases belong to the same signaling pathway PI3K/Akt/mTOR. For this proposal, the capillary was used as a nanoreactor in which a few nanoliters of the kinase, its substrate, adenosine triphosphate (ATP), and the potent inhibitor were separately injected. A transverse diffusion of laminar flow profiles (TDLFP) approach was employed to mix the reactants. Adenosine diphosphate (ADP ) was detected online at 254 nm. The CE assay was first developed on the α isoform of PI3K. It was compared to five commercial kits frequently used to assess kinase inhibition, based on time-resolved fluorescence resonance energy transfer (TR-FRET) and bioluminescence. Each assay was evaluated in terms of sensitivity (S/B), reproducibility (Z'), and variability (r (2)). This CE method was easily extended to assay the inhibition of the ß, γ, and δ isoforms of PI3K, and of the other kinases of the pathway, Akt1 and mTOR, since it is based on in-capillary mixing by TDLFP and on ADP quantification by simple UV absorption. This work shows for the first time the evaluation of inhibitors of the kinases of the PI3K/Akt/mTOR pathway using a common in-capillary CE assay. Several inhibitors with a wide range of affinity toward these enzymes were tested.


Assuntos
Eletroforese Capilar/métodos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Androstadienos/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Transferência Ressonante de Energia de Fluorescência , Humanos , Concentração Inibidora 50 , Luminescência , Inibidores de Proteínas Quinases/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Wortmanina
19.
Anal Chim Acta ; 807: 153-8, 2014 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-24356232

RESUMO

Myrosinase is a unique enzyme that catalyzes the hydrolysis of glucosinolates (GLS) to isothiocyanate (ITC), glucose and sulfate. Isothiocyanates display a diversified very interesting biological activity. In this study, capillary electrophoresis (CE) was used for the first time for evaluating myrosinase kinetics (maximum velocity Vmax and Michaelis-Menten constant Km) and to assess the affinity of a variety of substrates toward this enzyme. The pre-capillary approach was chosen since it is very simple to conduct. For this, the enzymatic reaction was performed in a micro-vial. The reaction mixture volume was of only 100 µL and the incubation lasted only 5 min at 37±1°C. Short-end injection of few tens of nanoliters (~25 nL) of the reaction mixture was performed which decreased analysis time without using any electroosmotic modifier. The sulfate produced was detected and quantified with a contactless capacitively coupled conductivity detector (C(4)D) allowing the evaluation of myrosinase kinetics. This study shows, that capillary electrophoresis with contactless conductivity detection can be very useful for monitoring myrosinase activity. Comparing to the conventional spectrophotometric method (1982), the CE method developed here is simple, automated, economic, rapid (incubation for few minutes) and robust. Results compared very well with those reported in literature using the conventional method. Moreover, the affinity of a variety of natural and synthetic glucosinolates toward this enzyme has been assessed for the first time.


Assuntos
Eletroforese Capilar , Glucosinolatos/análise , Glicosídeo Hidrolases/metabolismo , Condutividade Elétrica , Glucosinolatos/metabolismo , Isotiocianatos/análise , Cinética , Espectrofotometria , Especificidade por Substrato , Sulfatos/análise
20.
J Chromatogr A ; 1314: 298-305, 2013 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-24075461

RESUMO

A capillary electrophoresis (CE)-based enzyme assay method has been developed to screen protein kinase inhibitors. Four human kinases GSK3ß, DYRK1A, CDK5/p25 and CDK1/cyclin B were chosen to test this novel method. These enzymes have been identified as very promising targets to develop treatments against cancer and neurodegenerative diseases. The efficiency of drugs against these relevant biological targets has never been carried out by CE. For this proposal, the capillary was used as a nanoreactor in which four reactants (the enzyme, its two substrates and its potential inhibitor) were successively injected, mixed by using transverse diffusion of laminar flow profiles and incubated. The adenosine 5'-diphosphate (ADP) formed during the enzymatic reaction was detected by UV and quantified. The efficiency of the developed CE method was validated by determining the IC50 values of a wide variety of inhibitors covering a large domain of affinity toward kinases and containing representative and chemically divergent skeletons. Excellent agreement was found between the results obtained by CE and those reported in the literature when using conventional radiometric enzyme assays. Moreover, CE was successfully used to determine the inhibitory effect of several potential inhibitors that was not yet assessed by conventional methods and is crucial for structure activity relation studies. This novel CE method is simple, rapid, very economic (few tens of nanoliters per IC50) and eco-friendly since no radioactivity was required. It could be extended to high-throughput screening of kinase inhibitors, which is of great interest for biomedical and pharmaceutical research fields.


Assuntos
Eletroforese Capilar/métodos , Inibidores de Proteínas Quinases/análise , Misturas Complexas , Humanos , Espectrofotometria Ultravioleta
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