References of "Servais, Anne-Catherine"
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See detailCE-MS/MS for bottom-up proteomics: comparaison of two coupling interfaces with ion mobility qTOF
Gou, Marie-Jia ULiege; Nys, Gwenaël ULiege; COBRAIVILLE, Gaël ULiege et al

Conference (2019, May 20)

Untargeted bottom-up proteomic analysis aims to identify the highest number of peptides from complex protein mixtures. As the samples are of high complexity and that some proteins can be at very low ... [more ▼]

Untargeted bottom-up proteomic analysis aims to identify the highest number of peptides from complex protein mixtures. As the samples are of high complexity and that some proteins can be at very low concentrations, efficient and sensitive instruments have to be used in order to maximize peptide identification. Nowadays, capillary electrophoresis tandem mass spectrometry (CE-MS/MS) has gained interest in proteomic analysis as it is considered as complementary to the gold standard method, namely reverse phase liquid chromatography tandem mass spectrometry (RP-LC-MS/MS). However, the coupling of CE with MS is not straightforward. Indeed robust interface is needed in order to conserve the high-resolution in-capillary separation while ensuring a stable spray. Among the commercialized interfaces, the coaxial sheath liquid interface (« Triple tube », Agilent Technologies) and the nanoflow sheath liquid interface (« EMASS-II », CMP Scientific) have been tested for the analysis of BSA and E. coli proteome digests. Both interfaces were coupled with an IMS-qTOF-MS. Several parameters were optimized in order to maximize the sensitivity, such as the composition of the sheath liquid and different pre-concentration approaches (stacking, dynamic pH junction and transient isotachophoresis). In our study, transient isotachophoresis (tITP) was selected among other techniques and allowed the injection of large sample volumes without sacrificing separation efficiency. At the end, spray stability was found as the main strength of the triple tube interface, whereas the EMASS-II interface was found to provide higher sensitivity thanks to the reduced flow rate of the sheath liquid. [less ▲]

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See detailOptimisation of the separation of therapeutic phosphorothioate oligonucleotides by hydrophilic interaction liquid chromatography and capillary zone electrophoresis.
Demelenne, Alice ULiege; Gou, Marie-Jia ULiege; Parulski, Chloé et al

Conference (2019, May 20)

Introduction: Therapeutic oligonucleotides are short nucleic acids chemically synthetized that play a major role in gene regulation and the treatment of various diseases. They can target DNA, RNA ... [more ▼]

Introduction: Therapeutic oligonucleotides are short nucleic acids chemically synthetized that play a major role in gene regulation and the treatment of various diseases. They can target DNA, RNA, proteins, posttranslational protein modifications, carbohydrates, lipids or metabolites. Oligonucleotides are easily in-vivo degraded and need to be modified to improve their pharmacokinetic and pharmacodynamics properties. Phosphorothioate oligonucleotides (PS ON) is the most dominating modification where the oxygen atom of the phosphodiester bond is replaced by a sulfur atom. This result in enhanced resistance against nucleases degradation and thus increased half-life. Goals: Since many therapeutic oligonucleotides are arriving on the global market, there is an important need for appropriate analytical techniques to ensure their quality control. In this work, we optimized hydrophilic interaction liquid chromatography and capillary zone electrophoresis methods to detect PS ON impurities. Material and methods: Two complex mixtures were used to optimize the separation methods. Firstly, a mixture containing short PS ON of identical sequence varying by the position of the phosphodiester bonds will be analyzed. Secondly, a mixture of 20 PS ON of different lengths and thus different number of PS linkages will be used. Results and discussions: In hydrophilic interaction liquid chromatography, the stationary phase composition and the mobile phase composition and gradient were carefully optimized. In capillary zone electrophoresis, the pH and molarity of the background electrolyte will be studied. The final developed methods were compared in terms of peak efficiency, resolution and analysis time. The advantages and disadvantages related to each technique will be discussed. Conclusions: We demonstrated that hydrophilic interaction liquid chromatography and capillary electrophoresis are suitable techniques to differentiate closely related PS ON and could easily be applied for the quality control of those emerging medicines. [less ▲]

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See detailApplication of dual-cyclodextrin systems in capillary electrophoresis enantioseparations
Servais, Anne-Catherine ULiege; Fillet, Marianne ULiege

in Methods in Molecular biology: Chiral Separations (2019)

The enantioseparation of acidic and neutral compounds can be successfully achieved in capillary electrophoresis (CE) using dual-cyclodextrin (CD) systems. This chapter describes how to separate the ... [more ▼]

The enantioseparation of acidic and neutral compounds can be successfully achieved in capillary electrophoresis (CE) using dual-cyclodextrin (CD) systems. This chapter describes how to separate the enantiomers of acidic or neutral substances using dual-CD systems made up of a negatively charged CD derivative, i.e., sulfobutyl-β-CD or carboxymethyl-β-CD, in combination with a neutral one, namely heptakis(2,3,6-tri-O-methyl)-β-CD. An acidic compound (carprofen) and a weakly acidic drug (pentobarbital) were selected as model compounds. © Springer Science+Business Media, LLC, part of Springer Nature 2019. [less ▲]

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See detailEnantioseparations in nonaqueous capillary electrophoresis using charged cyclodextrins
Servais, Anne-Catherine ULiege; Fillet, Marianne ULiege

in Methods in Molecular biology: Chiral Separations (2019)

The enantioseparation of acidic and basic compounds can be successfully achieved in nonaqueous capillary electrophoresis (NACE) using single-isomer charged β-cyclodextrin (β-CD) derivatives of opposite ... [more ▼]

The enantioseparation of acidic and basic compounds can be successfully achieved in nonaqueous capillary electrophoresis (NACE) using single-isomer charged β-cyclodextrin (β-CD) derivatives of opposite charge to that of the analytes. This chapter describes how to separate the enantiomers of three basic substances selected as model compounds, i.e., alprenolol, bupranolol, and terbutaline, using the negatively charged heptakis(2,3-di-O-acetyl-6-O-sulfo)-β-CD (HDAS-β-CD). The enantiomers of three acidic drugs (tiaprofenic acid, suprofen, and flurbiprofen) are resolved using a monosubstituted amino β-CD derivative, namely 6-monodeoxy-6-mono(3-hydroxy)propylamino-β-CD (PA-β-CD). © Springer Science+Business Media, LLC, part of Springer Nature 2019. [less ▲]

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See detailInsulin aggregation assessment by capillary gel electrophoresis without sodium dodecyl sulfate: Comparison with size-exclusion chromatography
Demelenne, Alice ULiege; NAPP, Aurore ULiege; Bouillenne, Fabrice ULiege et al

in Talanta (2019), 199

Size-exclusion chromatography (SEC) is a method of choice for the analysis of protein aggregates in pharmaceuticals. The United States and European Pharmacopoeias currently use a SEC method with an acidic ... [more ▼]

Size-exclusion chromatography (SEC) is a method of choice for the analysis of protein aggregates in pharmaceuticals. The United States and European Pharmacopoeias currently use a SEC method with an acidic pH mobile phase to assess the content of aggregates in insulin formulations. In this article, we analyzed aggregated human insulin samples and demonstrated that both methods under neutral conditions, namely neutral pH SEC (nSEC) and capillary gel electrophoresis (CGE), yield to similar aggregate content contrary to SEC under acidic conditions (aSEC). aSEC showed polymeric complexes that were not observed in nSEC and CGE. During method development, the effect on SEC profiles of arginine and acetonitrile were highlighted. In CGE, the effect of SDS on disruption of non-covalent insulin aggregates was confirmed and the benefit of sodium deoxycholate addition in sieving gel was discussed. The three methods were applied to the analysis of an insulin formulation and similar results to those obtained for human insulin as raw material were observed. Finally, the CGE method was used to study the stability of human insulin under different storage conditions. In view of the obtained results one may question the relevance of the current pharmacopoeia method to study insulin aggregates by emphasizing the importance of the mobile phase composition and pH in SEC. The new CGE method developed is an easy method for studying non-covalent aggregates of insulin, which could be applied to other proteins. © 2019 Elsevier B.V. [less ▲]

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See detailHighly sensitive and selective separation of intact parathyroid hormone and variants by sheathless CE-ESI-MS/MS.
Nyssen, Laurent ULiege; Fillet, Marianne ULiege; Cavalier, Etienne ULiege et al

in Electrophoresis (2019)

Parathyroid hormone (PTH) is a common clinical marker whose quantification relies on immunoassays, giving variable results as batch, brand, or target epitope changes. Sheathless CE-ESI-MS, combining CE ... [more ▼]

Parathyroid hormone (PTH) is a common clinical marker whose quantification relies on immunoassays, giving variable results as batch, brand, or target epitope changes. Sheathless CE-ESI-MS, combining CE resolution power and low-flow ESI sensitivity, was applied to the analysis of PTH in its native conformation in the presence of related forms. Fused silica and neutral-coated capillaries were investigated, as well as preconcentration methods such as transient isotachophoresis (t-ITP), field-amplified sample injection (FASI) and electrokinetic supercharging (EKS). The method for the separation of PTH and its variants was first developed using fused-silica capillary with UV detection. An acidic background electrolyte (BGE) was used to separate 1-84 PTH (full length), 7-84 PTH and 1-34 PTH. Acetonitrile was added to the BGE to reduce peptide adsorption onto the capillary wall and t-ITP was used as analyte preconcentration method. The method was then transferred to a sheathless CE-ESI-MS instrument. When using a fused silica capillary, CE-MS was limited to mug/mL levels. The use of a neutral coating combined with FASI or EKS allowed a significant increase in sensitivity. Under these conditions, 1-84 PTH, 7-84 PTH and 1-34 PTH were detected at concentrations in the low ng/mL (FASI) or pg/mL (EKS) range. This article is protected by copyright. All rights reserved. [less ▲]

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See detailUltra-high-performance liquid chromatography-mass spectrometry method for neutrophil gelatinase-associated lipocalin as a predictive biomarker in acute kidney injury
Ion, Valentin ULiege; Nys, Gwenaël ULiege; COBRAIVILLE, Gaël ULiege et al

in Talanta (2019), 195

Neutrophil gelatinase associated lipocalin (NGAL) is a protein that was found to be overexpressed in acute kidney injury (AKI). The rise in NGAL concentration, both in urine or plasma, appears earlier ... [more ▼]

Neutrophil gelatinase associated lipocalin (NGAL) is a protein that was found to be overexpressed in acute kidney injury (AKI). The rise in NGAL concentration, both in urine or plasma, appears earlier than for other classical renal function markers such as serum creatinine, thus making it a suitable marker for early pathology detection. The aim of this study was to develop a method involving tryptic digestion, solid phase extraction and LC-MS/MS analysis to analyze NGAL in plasma medium using an isotope labeled surrogate protein, containing NGAL signature tags, as internal standard (QPrEST). The method was validated for the analysis of NGAL in an analytical range from 50 to 1250 ng/mL using two different proteotypic peptides. The method was further used to quantify the NGAL in human plasma samples for whom elevated NGAL values were expected. NGAL values were between 190.8 and 242.6 ng/mL for control group and between 228.1 and 3526.2 ng/mL for patient group. This study proved that the selection of the right internal standard is of utmost importance in targeted proteomics studies as the digestion steps might cause high variability. This study also confirmed that, although NGAL is highly resistant to proteases such as trypsin, the method could be fully validated according to FDA guidelines and subsequently used to assess NGAL levels in patient plasma with high analytical confidence. © 2018 [less ▲]

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See detailSelectivity evaluation of phenyl based stationary phases for the analysis of amino acid diastereomers by liquid chromatography coupled with mass spectrometry
Moldovan, R.-C.; Bodoki, E.; Servais, Anne-Catherine ULiege et al

in Journal of Chromatography A (2019)

D-amino acids (AA) analysis is becoming more and more relevant for metabolomics, therefore new analytical tools need to be developed. A common approach to achieve AA enantioseparation is chiral ... [more ▼]

D-amino acids (AA) analysis is becoming more and more relevant for metabolomics, therefore new analytical tools need to be developed. A common approach to achieve AA enantioseparation is chiral derivatization. Among the chiral derivatization reagents, (+) or (-)-1-(9-fluorenyl) ethyl chloroformate ((+) or (-)-FLEC) has proved to be one of the most versatile. Suitable chiral selectivity for FLEC derivatives of amino acids could be obtained in reversed-phase HPLC using nonpolar stationary phases (C4, C8 and C18) and tetrahydrofuran (THF) based mobile phases. This study is meant to provide alternatives to the use of THF as organic modifier by evaluating the selectivity obtained on two phenyl based stationary phases for 19 FLEC-DL-AA pairs of diastereomers using UHPLC-MS. Several mobile phases consisting of ammonium acetate and different common organic solvents (acetonitrile (ACN), methanol (MeOH), 2-propanol (IPA)) were tested using gradient elution. Experimental design was employed for the optimization of the separation conditions. In the optimized conditions, complete chiral separation can be achieved for 18 out of 19 FLEC-DL-AAs in less than 30 min. © 2019 Elsevier B.V. [less ▲]

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See detailQuality control of therapeutic oligonucleotides using HILIC-UV and CZE-UV
Gou, Marie-Jia ULiege; Demelenne, Alice ULiege; Parulski, Chloé et al

Poster (2018, December 19)

Detailed reference viewed: 16 (4 ULiège)
See detailQuality control and aggregation follow-up of insulin formulations
Demelenne, Alice ULiege; Napp, Aurore ULiege; Radermecker, Régis ULiege et al

Conference (2018, December 19)

The prevalence of diabetes is increasing every year making insulin formulations widely prescribed medicines. Fast and efficient methods to assess the quality of such biopharmaceutical products are thus ... [more ▼]

The prevalence of diabetes is increasing every year making insulin formulations widely prescribed medicines. Fast and efficient methods to assess the quality of such biopharmaceutical products are thus required, more particularly methods to assess the content of API and potential aggregates. We started our study on insulin aggregation using the European Pharmacopeia method. Since methods to assess aggregate content are often contradictory, we also developed original and orthogonal methods using size-exclusion chromatography (SEC) and capillary gel electrophoresis (CGE). It was demonstrated that methods under neutral conditions (SEC and CGE) yield to similar aggregate content contrary to pharmacopeia SEC method that works under acidic conditions. Ion-mobility Q-TOF mass spectrometer was also used to confirm the presence and the identity of insulin dimers. Then, we applied the three methods to the analysis of an insulin formulation and similar results to those obtained for human insulin as raw material were observed. We also used the CGE method to study the stability of human insulin under different storage conditions. Finally, we used UHPLC and mass spectrometry to quantify insulin formulation from different supply chains. We demonstrated that all the analyzed formulations had a potency between 95.0 % and 105.0 % of the potency stated on the label. This was useful to dispel doubts regarding issues in insulin cold chain supply recently described in the litterature. [less ▲]

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See detailStrategies for the study of plant virus-based virus-like particles (VLP) displaying human papillomavirus (HPV) L2 epitope
Yazdani, Razieh; Shams-Bakhsh, Masoud; Thelen, Nicolas ULiege et al

Poster (2018, December 19)

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See detailDevelopment and optimization of a derivatization protocol for phenethylamine compounds using FITC-CE-LIF method
Emonts, Paul ULiege; Dispas, Amandine ULiege; Ninane, Caroline et al

Poster (2018, December 19)

In the framework of the development of an innovative microfluidic system based on capillary electrophoresis separation, we optimized a fluorescein isothiocyanate (FITC) derivatization protocol for Laser ... [more ▼]

In the framework of the development of an innovative microfluidic system based on capillary electrophoresis separation, we optimized a fluorescein isothiocyanate (FITC) derivatization protocol for Laser Induced Fluorescence (LIF) detection. The microfluidic device will present a high sensitivity thanks to the fluorescence detection and an innovative hydrodynamic injection approach. To highlight the analytical performances of this microfluidic system, we will use synthetic cathinones (and some derivatives) as targeted compounds. They present a panel of closely related structures and isobaric compounds. In addition to analytical challenges, this category of drugs is a current topic regarding to public health. Indeed synthetic cathinones were described as the second most frequently seized group of new psychoactive substances in EU in 2016. Moreover some of them are currently sell on internet as bath salts without any legislation. In this context, fast, easy and reliable analytical tools are required to track and quantify these compounds. In the present study, we initiated the optimization of the derivatization of model compounds (phenethylamine family) using FITC. The panel of model analytes includes primary and secondary amino groups to be representative of future real samples. Design of experiments strategy was used to optimize the derivatization protocol in order to reach an easy sample preparation and to maximize the derivatization efficiency. Finally, the CE developed method will be transferred to the microfluidic system. Then separation performances comparison of traditional CE-LIF vs µCE-LIF will allow to highlight the advantages of microfluidics such as the lower amount of sample required, the gain in time and money, the significant decrease of solvent and its compact size. [less ▲]

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See detailCESI-MS Workflow for Protein Quantification
Nyssen, Laurent ULiege; Fillet, Marianne ULiege; CAVALIER, Etienne ULiege et al

Poster (2018, October)

Introduction: Sheathless CE-MS interfaces allow increase in sensitivity by coupling low-flow electrospray ionization and tandem MS detection. Peak intensity will depend on spray voltage as well as ... [more ▼]

Introduction: Sheathless CE-MS interfaces allow increase in sensitivity by coupling low-flow electrospray ionization and tandem MS detection. Peak intensity will depend on spray voltage as well as migration and injection conditions. Nevertheless, these parameters influence each other and require methodical optimization to get the most of each instrument. In the present work we share our experience with sheathless CE-MS and neutral coating to analyze peptide and protein samples. Methods: Experiments were conducted on a CESI 8000 capillary electrophoresis holding a neutral coated OptiMS cartridge and coupled to a QT 6500 mass spectrometer. Separation buffer and voltage, curtain gas and source temperature were conserved through experiments. Separation pressure and source voltage were optimized while applying voltage and pressure on separation buffer spiked with the peptide used (pI 9.5 marker from the Advance cIEF starter kit by Sciex). A daily reference run was used to compare modifications to the injection despite variable capillary performance. Finally, shifts in spray voltage due to injection parameters were determined using sequences of runs with different spray voltages. Preliminary results: Decreasing separation pressure from 5 to 1.5 psi increased peptide intensity; electrokinetic injection (EKI) increased peak intensity compared to hydrodynamic injection (HDI); the HDI of a water plug before the EKI increased peak intensity further, as well as a high percentage of acetonitrile in the sample medium. Finally, we compared our initial and our final conditions. In both cases, a positive Q1 scan of 1000 Da/s for m/z 300 to 1000 was acquired, and the electropherograms display the extracted ion current for a 1 m/z interval centered on the m/z of the doubly charged peptide. In the initial method, the peptide was diluted in BGE and was introduced by HDI (1 % of total length); 5 psi pressure were applied to both inlet and outlet; source voltage was 1800 V. When analyzing a 1:160 (v/v) dilution of the peptide, the intensity recorded for [M+2H]2+ was 9.3e7 counts. In the final method, the peptide was diluted in 75:25 acetonitrile:water (v/v) and was introduced by EKI (+ 10 kV 100s). Before the EKI, a HDI of water (0.5 % of total length) was performed, and after the EKI, separation buffer was introduced by HDI (0.5 % of total length). The separation pressure was changed to 1.5 psi and the spray voltage adjusted to 1600 V. When analyzing a 1:160000 (v/v) dilution of the peptide, the recorded intensity of [M+2H]2+ was 9e8 counts. Therefore, following these guidelines, we were able to increase intensity by a 10000 factor. Novel aspect: Frequent monitoring of spray voltage and peak intensity in similar conditions allows good inter-run comparison and troubleshooting. [less ▲]

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See detailCombination of analytical approaches for the characterization of plant virus-based VLP displaying HPV L2 epitope
Yazdani, Razieh; Shams-Bakhsh, Masoud; Hassani-Mehraban, Afshin et al

Conference (2018, September 12)

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See detailInsulin aggregation assessment by size-exclusion chromatography and capillary gel electrophoresis
Demelenne, Alice ULiege; Napp, Aurore ULiege; Servais, Anne-Catherine ULiege et al

Poster (2018, September 10)

Size-exclusion chromatography (SEC) is the method of choice for the analysis of protein aggregates in biopharmaceuticals. Aggregates could be formed during production and storage of formulations and may ... [more ▼]

Size-exclusion chromatography (SEC) is the method of choice for the analysis of protein aggregates in biopharmaceuticals. Aggregates could be formed during production and storage of formulations and may lead to complications in insulin therapy. For that reason, they are important parameters to investigate for insulin quality control. The United States and European Pharmacopoeias currently use a SEC method with acidic mobile phase for the quantification of aggregates in insulin formulations. However, changes in aggregates assessment have been reported when the mobile phase composition differs from the sample dissolution medium. [1] To investigate the impact of mobile phase on human insulin aggregates analysis, the aggregated human insulin samples were analyzed by SEC using neutral (nSEC) or acidic mobile phases (aSEC). The aggregated samples were obtained by dissolving human insulin in acidic media, followed by agitation for 8, 16, 24, 32, 40 and 48 hours respectively. During SEC method development, the impact of arginine and acetonitrile addition to the mobile phase was pointed out. Additionally, an orthogonal capillary-gel electrophoresis method (CGE) for the assessment of insulin aggregates was developed. The optimal pH 8.1 CGE buffer was formulated without SDS in order to preserve the non-covalent aggregates. After the optimization of the methods, human insulin and aggregated samples were analyzed using nSEC, aSEC and CGE. A similar increase of dimers percentage with incubation time was noticed by both nSEC and CGE, while no significant increase of dimers content was observed by aSEC. However, an insulin polymeric complex was detectable for some samples with aSEC. The three methods were used to analyze an insulin formulation and a similar tendency was observed. The results obtained emphasize the importance of mobile phase choice in SEC. The good correlation between dimers percentage obtained by nSEC and CGE suggests that these technics most probably enable the detection of the species initially present in the sample and do not change the composition of the sample during analysis. The developed CGE method is a fast and reliable tool for the study of the complex process of insulin aggregation. Furthermore, the CGE method could be easily applied to other proteins since it proved to be highly reproducible and has the advantages of low sample consumption, and no expensive column or organic solvents are required. [less ▲]

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See detailPartial filling capillary electrophoretic mobility shift competition assay: a versatile and reliable tool for the assessment of weak biomolecular interactions
Farcas, Elena ULiege; Servais, Anne-Catherine ULiege; Hanson, Julien ULiege et al

Conference (2018, September 10)

Fragment-based drug discovery (FBDD) proved its efficacy in the past 20 years, due to its ability to perform efficient and fruitful optimization campaigns, and is now a well recognized strategy for both ... [more ▼]

Fragment-based drug discovery (FBDD) proved its efficacy in the past 20 years, due to its ability to perform efficient and fruitful optimization campaigns, and is now a well recognized strategy for both academia and pharmaceutical industry. FBDD detects low molecular-weight (MW) ligands (fragments) that bind to biologically important targets, then a structure-guided fragment growing or merging approach is performed giving rise to potent molecules with drug-like properties. However, the analytical arsenal able to point out weak interactions is rather expensive, time consuming or unable to reflect the physiological environment. In this framework, we developed a generic, fully automated, microscale electrophoretic mobility shift competition assay that can be used for primary screening of weak biomolecular interactions between fragments and the target of interest. The affinity capillary electrophoresis (ACE) competitive approach is based on the monitoring of the competition of fragments with a known target inhibitor (PL) for the same active site. The consequence of the competition is a modification of PL electrophoretic mobility, modification that can be measured and used for ligand screening and/or IC50 determination. To achieve our goal, particular attention has been paid to the optimization of the binding environment parameters: an optimal buffer was used for the binding measurements, a partial filling approach was considered to gain sensitivity and to reduce protein consumption and a neutral dynamic coating was performed to reduce protein adsorption to the capillary wall. Moreover, the binding partners concentrations and the electrophoretic conditions were carefully optimized. It is noteworthy that the interactions occur in solution, using the protein in its native form, thus mimicking the physiological environment.The accuracy and reliability of the proposed method was demonstrated by monitoring the competition of two known fragments inhibiting thrombin, namely benzamidine and p-aminobenzamidine and a relatively weak inhibitor, nafamostat with a known thrombin inhibitor, pefabloc (PEFA). The measured IC50 were found to be in good accordance with the previously reported ones. Additionally, a small chemical library was built to evaluate the performance of the newly developed screening-bioassay. The optimized method proved to be remarkably reproducible (migration time RSDs < 1.2%) and selective. The results prove the high discriminatory potency of the method and its ability to screen neutral, negatively or positively charged molecules, as well as molecules that have no or low UV-VIS absorbance, significantly expanding the applicability of the assay compared to a direct approach [1]. Finally, the ability of this approach to discriminate between competitive and irreversible thrombin binders was also demonstrated.References [1] E. Farcas, C. Bouckaert, A.-C. Servais, J. Hanson, L. Pochet, M. Fillet, Analytica Chimica Acta, 2017, 984, 211-222 [less ▲]

Detailed reference viewed: 61 (8 ULiège)