References of "Servais, Anne-Catherine"
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See detailHyphenation of capillary zone electrophoresis with mass spectrometry for proteomic analysis: Optimization and comparison of two coupling interfaces
Gou, Marie-Jia ULiege; Nys, Gwenaël ULiege; COBRAIVILLE, Gaël ULiege et al

in Journal of Chromatography A (2020)

Capillary electrophoresis tandem mass spectrometry (CE–MS/MS) is an interesting tool for proteomic analysis as the separation principle is orthogonal to liquid chromatography tandem mass spectrometry ... [more ▼]

Capillary electrophoresis tandem mass spectrometry (CE–MS/MS) is an interesting tool for proteomic analysis as the separation principle is orthogonal to liquid chromatography tandem mass spectrometry (LC–MS/MS). The combination of both techniques can bring complementary information to enlarge proteome coverage. In this study, sample preconcentration techniques were investigated in order to improve sample loading and therefore sensitivity. Dynamic pH junction (DPJ) was found to be the most interesting approach by using 200 mM ammonium acetate (NH4Ac) adjusted to pH 10.0 as sample matrix. The use of DPJ allowed the identification of more peptides and proteins compared to conventional injections. Moreover, the sheath liquid (SL) composition was optimized in order to enhance signal intensity. A nanoflow SL interface (EMASS-II) was compared to the traditional coaxial SL interface (Triple tube) in terms of number of identified and proteins as well as detection sensitivity (peak area and peak height). MS acquisition was performed using both data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes. The results showed that the combined use of these two acquisition modes provided additional information in terms of identification. Moreover, the use of EMASS-II interface allowed the identification of approximately two times more peptides and proteins. Besides, there was an improvement in sensitivity using EMASS-II as peak height and peak area were improved by 4 and 6-fold, respectively, compared to the Triple tube. Altogether, by combining an efficient sample preconcentration method, a nanoflow CE–MS interface and a hybrid ion-mobility qTOF mass spectrometer, a satisfying sequence coverage was obtained by analyzing 1 µg of E. coli proteome digest. © 2020 [less ▲]

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See detailIon mobility high resolution mass spectrometry coupled to HILIC and CZE for the characterization of phosphodiester and phosphorothioate oligonucleotides
Demelenne, Alice ULiege; Gou, Marie-Jia ULiege; Nys, Gwenaël ULiege et al

Conference (2019, December 11)

Oligonucleotide-based medicines that can modulate gene expression have numerous potential applications in targeted therapies. Most of the commercialized therapeutic oligonucleotides are chemically ... [more ▼]

Oligonucleotide-based medicines that can modulate gene expression have numerous potential applications in targeted therapies. Most of the commercialized therapeutic oligonucleotides are chemically modified to increase their in vivo lifetime. With the emergence of oligonucleotides on the market, it is of increasing importance to develop analytical methods to study those modified oligonucleotides and their impurities. Chromatographic and electrophoretic approaches may be used for that purpose. In this work, poly-deoxy(thymidylic) acids (dT) and modified phosphorothioate oligonucleotides (PS) were studied. For the chromatographic approach, Hydrophilic Interaction Liquid Chromatography (HILIC) was chosen as it represents good alternative to commonly used ion-pair reversed-phase liquid chromatography for the analysis of polar compounds. Moreover, it avoids the use of ion pairing agents, which makes it more compatible with mass spectrometric detection. For the electrophoretic approaches, a capillary zone electrophoresis (CZE) with a MS-compatible background electrolyte was employed. Both techniques were coupled to a drift-tube ion-mobility quadrupole time-of-flight MS detector (DTIMS-QTOF) to assess the added value of this coupling for oligonucleotide characterization. Indeed, by using the measured collision cross section (CCS), the evaluation of the number of nucleotides was performed. Looking across the results, HILIC and CZE coupled to DTIMS-QTOF can be considered as promising tools for the quality control of oligonucleotides. [less ▲]

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See detailProteomic analysis using capillary electrophoresis hyphenated with high resolution mass spectrometry: comparison of two coupling interfaces
Gou, Marie-Jia ULiege; Nys, Gwenaël ULiege; COBRAIVILLE, Gaël ULiege et al

Poster (2019, December 11)

Capillary electrophoresis tandem mass spectrometry (CE-MS/MS) is an interesting tool for proteomic analysis as the separation principle is orthogonal to liquid chromatography tandem mass spectrometry (LC ... [more ▼]

Capillary electrophoresis tandem mass spectrometry (CE-MS/MS) is an interesting tool for proteomic analysis as the separation principle is orthogonal to liquid chromatography tandem mass spectrometry (LC-MS/MS). The combination of both techniques can bring complementary information to enlarge proteome coverage. In this study, sample preconcentration techniques were investigated in order to improve sample loading and therefore sensitivity. The use of dynamic pH junction allowed the identification of more peptides and proteins compared to conventional injections. The sheath liquid composition was also optimized in order to enhance signal intensity. A nanoflow sheath liquid interface (EMASS-II) was compared to the traditional coaxial sheath liquid interface (Triple tube) in terms of number of identified peptides and proteins as well as in terms of sensitivity (peak area and peak height). The use of EMASS-II interface allowed the identification of approximately two times more peptides and proteins. Besides, there was an improvement in sensitivity using EMASS-II as peak height and peak area were improved by a factor of 4 and 6-fold, respectively, compared to the Triple tube. [less ▲]

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See detailDevelopment of a sensitive CE-LIF method for the analysis of synthetic cathinones.
Emonts, Paul ULiege; Dispas, Amandine ULiege; Servais, Anne-Catherine ULiege et al

Poster (2019, December 11)

Synthetic cathinones (SCs) are phenylalkylamine compounds related to natural cathinone from Catha Edulis leaves. Given their structural similarities with amphetamines, these compounds are mainly drugs of ... [more ▼]

Synthetic cathinones (SCs) are phenylalkylamine compounds related to natural cathinone from Catha Edulis leaves. Given their structural similarities with amphetamines, these compounds are mainly drugs of abuse. Indeed these substances constitute the second most frequently seized group of new psychoactive substances (NPS) and counted more than 130 compounds in Europe (EMCDDA 2016). In this context, reliable analytical tools are required to track these substances. In this study, our goal was to develop a capillary electrophoresis separation method coupled to laser induced fluorescence detection (CE-LIF) to analyze SCs. CE separation was optimized by means of BGE composition study. Various additives have been investigated to achieve separation of these closely related compounds. Due to their lack of native fluorescence, analytes were derivatized using fluorescein isothiocyanate isomer I (FITC). A protocol adapted to small basic compounds was previously optimized using DoE strategy. The CE-LIF optimized method could be proposed as a generic method for the screening of SCs. [less ▲]

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See detailTargeted proteomics reveals serum amyloid A variants and alarmins S100A8-S100A9 as key plasma biomarkers of rheumatoid arthritis
Nys, Gwenaël ULiege; COBRAIVILLE, Gaël ULiege; Servais, Anne-Catherine ULiege et al

in Talanta (2019)

Serum amyloid A (SAA) and S100 (S100A8, S100A9 and S100A12) proteins were previously identified as biomarkers of interest for rheumatoid arthritis (RA). Among SAA family, two closely related isoforms (SAA ... [more ▼]

Serum amyloid A (SAA) and S100 (S100A8, S100A9 and S100A12) proteins were previously identified as biomarkers of interest for rheumatoid arthritis (RA). Among SAA family, two closely related isoforms (SAA-1 and SAA-2) are linked to the acute-phase of inflammation. They respectively exist under the form of three (α, β, and γ) and two (α and β) allelic variants. We developed a single run quantitative method for these protein variants and investigated their clinical relevance in the context of RA. The method was developed and validated according to regulations before being applied on plasma coming from RA patients (n = 46), other related inflammatory pathologies (n = 116) and controls (n = 62). Depending on the activity score of RA, SAA1 isoforms (mainly of SAA1α and SAA1β subtypes) were found to be differentially present in plasma revealing their dual role during the development of RA. In addition, the weight of SAA1α in the total SAA response varied from 32 to 80% depending on the pathology studied. A negative correlation between SAA1α and SAA1β was also highlighted for RA early-onset (r = −0.41). SAA2 and S100A8/S100A9 proteins were significantly overexpressed compared to control samples regardless of RA stage. The pathophysiological relevance of these quantitative and qualitative characteristics of the SAA response remains unknown. However, the significant negative correlation observed between SAA1α and SAA1β levels in RA early-onset suggests the existence of still unknown regulatory mechanisms in these diseases. [less ▲]

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See detailSeparation of Intact Parathyroid Hormone and Variants Using a Highly Sensitive Sheathless CE-ESI-MS/MS Method
Nyssen, Laurent ULiege; Fillet, Marianne ULiege; CAVALIER, Etienne ULiege et al

Poster (2019, September 26)

INTRODUCTION Parathyroid hormone (PTH) is a common clinical marker whose quantification relies on immunoassays, giving variable results as batch, brand, or target epitope changes. Moreover, immunoassays ... [more ▼]

INTRODUCTION Parathyroid hormone (PTH) is a common clinical marker whose quantification relies on immunoassays, giving variable results as batch, brand, or target epitope changes. Moreover, immunoassays may cross-react with PTH variants such as C-terminal fragments stemming from PTH catabolism. These issues make it difficult to compare results obtained in different laboratories. A reference quantification method is necessary to harmonize PTH assays, both sensitive and selective enough to detect PTH at low concentrations among a variety of closely related compounds. OBJECTIVES In this study, our main goal was to reach a very high sensitivity (pg/mL range) for the analysis of PTH and its variants. Two variants were selected, namely 7-84 PTH as C-terminal fragment and 1-34 PTH as related peptide, but also as potential internal standard for future works. METHODS To achieve our goal, we developed a sheathless CE-ESI-MS method for the separation of 1-34 PTH, 7-84 PTH, and 1-84 PTH. 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). RESULTS The method for the separation of PTH and its variants was first developed using fused-silica capillary with UV detection. 1-84 PTH (full length), 7-84 PTH and 1-34 PTH were separated using an acidic background electrolyte containing acetonitrile to reduce peptide adsorption onto the capillary wall. Ammonium acetate was used as sample medium to improve sensitivity through t-ITP. The method was then transferred to a sheathless CE-ESI-MS instrument. CE-MS on fused silica capillary was limited to µg/mL levels. Indeed, despite the MS detection, only samples containing at least 10 µg/mL of 1-84 PTH, 7-84 PTH, and 1-34 PTH could be analyzed. 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 100 ng/mL using FASI while 1-84 PTH and 1-34 PTH were detected at 100 pg/mL using EKS. The estimated LODs (S/N = 3) for the EKS method were 25 pg/mL for 1-84 PTH and 10 pg/mL for 1-34 PTH, while there was no signal anymore for 7-84 PTH at these levels. CONCLUSION The developed sheathless CE-ESI-MS method has the potential to reach the low pg/mL range in biological samples after the optimization of the sample preparation method. [less ▲]

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See detailQuality control of therapeutic phosphorothioate oligonucleotides by hydrophilic interaction liquid chromatography coupled with ION-MOBILITY QTOF
Demelenne, Alice ULiege; Gou, Marie-Jia ULiege; Nys, Gwenaël ULiege et al

Conference (2019, September 17)

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

Therapeutic oligonucleotides are short nucleic acids chemically synthetized. They play a major role in gene regulation and the treatment of various diseases. They 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 pharmacodynamic properties. Phosphorothioate oligonucleotides 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. Currently, the FDA has approved 6 drugs but more than 180 are being tested in clinical trials. There is thus an important need for appropriate analytical techniques to ensure their quality control. In this study, we compared the selectivity obtained with several hydrophilic interaction liquid chromatography (HILIC with diol, amide or phosphorylcholine functional groups) stationary phases on various oligonucleotides mixtures. Indeed, HILIC represents good alternative to commonly used ion-pair reversed-phase liquid chromatography for the analysis of polar compounds. Moreover, it avoids the use of ion pairing agents, which makes it more compatible with mass spectrometric detection. In this project, we investigated the coupling of HILIC with ion-mobility quadrupole time-of-flight MS detector (IM-QTOF). Ion-mobility provides a third separation dimension to mass spectrometry, as the ions will be separated based on their shape and size. Chromatographic and IMS performances were considered to assess the multidimensional efficacy of each tested system. As conclusion we can say that HILIC-IMS approach brings new potentiality in the quality control of emerging oligonucleotides. [less ▲]

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See detailComparison of two capillary electrophoresis-mass spectrometry interfaces for proteomic analysis
Gou, Marie-Jia ULiege; Nys, Gwenaël ULiege; Demelenne, Alice ULiege et al

Poster (2019, September 17)

Untargeted bottom-up proteomic analysis aims to identify the highest number of peptides from complex protein digests. The application of this strategy to real sample might lead to the discovery of new ... [more ▼]

Untargeted bottom-up proteomic analysis aims to identify the highest number of peptides from complex protein digests. The application of this strategy to real sample might lead to the discovery of new proteic entities of biological interest. As the samples are of high complexity and that some proteins could be present 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. For this purpose, optimization of basic parameters such as BGE composition was first carried out using a simple peptide mix. Several parameters were then optimized in order to maximize the sensitivity, such as the composition of the sheath liquid, the interface position and different pre-concentration approaches (stacking, dynamic pH junction and transient isotachophoresis). Finally, transient isotachophoresis (tITP) was selected among other techniques and allowed the injection of large sample volumes without sacrificing separation efficiency. In our study, two commercialized interfaces were compared by analysing E. coli proteome digest. The coaxial sheath liquid interface (« Triple tube », Agilent Technologies) and the nanoflow sheath liquid interface (« EMASS-II », CMP Scientific) were both coupled with an IMS-qTOF-MS. Eventually, spray stability was found to be 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 detailCapillary electrophoresis tandem mass spectrometry for proteomic analysis: optimization and comparison of two coupling interfaces
Gou, Marie-Jia ULiege; Nys, Gwenaël ULiege; Demelenne, Alice ULiege et al

Poster (2019, September 01)

Untargeted bottom-up proteomic analysis aims to identify the highest number of peptides from complex protein digests. The application of this strategy to real sample might lead to the discovery of new ... [more ▼]

Untargeted bottom-up proteomic analysis aims to identify the highest number of peptides from complex protein digests. The application of this strategy to real sample might lead to the discovery of new proteic entities. As the samples are of high complexity and that some proteins could be present 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. For this purpose, optimization of basic parameters such as BGE composition was first carried out using a simple peptide mix. Several parameters were then optimized in order to maximize the sensitivity, such as the composition of the sheath liquid, the interface position and different pre-concentration approaches (stacking, dynamic pH junction and transient isotachophoresis). Finally, transient isotachophoresis (tITP) was selected among other techniques and allowed the injection of large sample volumes without sacrificing separation efficiency. In our study, two commercialized interfaces were compared by analysing E. coli proteome digest. The coaxial sheath liquid interface (« Triple tube », Agilent Technologies) and the nanoflow sheath liquid interface (« EMASS-II », CMP Scientific) were both coupled with an IMS-qTOF-MS. Eventually, spray stability was found to be 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 detailDetermination of iohexol by capillary blood microsampling and UHPLC-MS/MS
Ion, Valentin ULiege; Le Goff, Caroline ULiege; Cavalier, Etienne ULiege et al

in Journal of Pharmaceutical Analysis (2019), 9(4), 259-265

One of the most important tools used to evaluate kidney function in the context of chronic kidney disease or other renal function related pathologies is the exploration of glomerular filtration rate (GFR ... [more ▼]

One of the most important tools used to evaluate kidney function in the context of chronic kidney disease or other renal function related pathologies is the exploration of glomerular filtration rate (GFR). Iohexol is up to this moment a good candidate molecule for the GFR assessment since it exhibits minimum protein binding rates and minimum extra-renal clearance, being neither secreted nor reabsorbed at the tubular level. This study proposes and evaluates a new LC-MS/MS method for the iohexol determination from capillary blood, prelevated using volumetric absorbative microsampling (VAMS) systems. As an alternative to VAMS, a brand new HemaPEN® device for micro-prelevation was also tested. A new high throughput sample preparation protocol adapted for iohexol quantification from whole blood VAMS samples was developed. The medium term stability study of iohexol in dried whole blood VAMS samples that was conducted showed a good stability of this molecule for up to 12 days. By collecting only 10 μL of blood, iohexol can be analyzed from dried whole blood VAMS samples for concentration ranges between 1 and 250 μg/mL. Due to the analyte stability in VAMS for up to 12 days, this approach might be successfully applied for GFR assessment for clinical cases allowing minimum invasiveness and even delayed analysis. © 2019 Xi'an Jiaotong University [less ▲]

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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 detailCharacterization of plant virus-based virus-like particles displaying human papillomavirus L2 epitope
Yazdani, Razieh; Shams-Bakhsh, Masoud; Hassani-Mehraban, Afshin et al

Conference (2019)

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See detailProduction and characterization of virus-like particles of grapevine fanleaf virus presenting L2 epitope of human papillomavirus minor capsid protein.
Yazdani, Razieh; Shams-Bakhsh, Masoud; Hassani-Mehraban, Afshin et al

in BMC Biotechnology (2019), 19(1), 81

BACKGROUND: Virus-like particle (VLP) platform represents a promising approach for the generation of efficient and immunogenic subunit vaccines. Here, the feasibility of using grapevine fanleaf virus ... [more ▼]

BACKGROUND: Virus-like particle (VLP) platform represents a promising approach for the generation of efficient and immunogenic subunit vaccines. Here, the feasibility of using grapevine fanleaf virus (GFLV) VLPs as a new carrier for the presentation of human papillomavirus (HPV) L2 epitope was studied. To achieve this goal, a model of the HPV L2 epitope secondary structure was predicted and its insertion within 5 external loops in the GFLV capsid protein (CP) was evaluated. RESULTS: The epitope sequence was genetically inserted in the alphaB-alphaB(") domain C of the GFLV CP, which was then over-expressed in Pichia pastoris and Escherichia coli. The highest expression yield was obtained in E. coli. Using this system, VLP formation requires a denaturation-refolding step, whereas VLPs with lower production yield were directly formed using P. pastoris, as confirmed by electron microscopy and immunostaining electron microscopy. Since the GFLV L2 VLPs were found to interact with the HPV L2 antibody under native conditions in capillary electrophoresis and in ELISA, it can be assumed that the inserted epitope is located at the VLP surface with its proper ternary structure. CONCLUSIONS: The results demonstrate that GFLV VLPs constitute a potential scaffold for surface display of the epitope of interest. [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 detailEvaluation of Hydrophilic Interaction Liquid Chromatography, Capillary Zone Electrophoresis and Drift Tube Ion-Mobility Quadrupole Time Of Flight mass spectrometry for the characterization of phosphodiester and phosphorothioate oligonucleotides
Demelenne, Alice ULiege; Gou, Marie-Jia ULiege; Nys, Gwenaël ULiege et al

in Journal of Chromatography. A (2019)

Oligonucleotide-based medicines that can modulate gene expression have numerous potential applications in targeted therapies. Most of the commercialized therapeutic oligonucleotides are chemically ... [more ▼]

Oligonucleotide-based medicines that can modulate gene expression have numerous potential applications in targeted therapies. Most of the commercialized therapeutic oligonucleotides are chemically modified to increase their in vivo lifetime. In this work, we studied poly-deoxy(thymidylic) acids (dT) and modified phosphorothioate oligonucleotides (PS). Several analytical techniques, including ion-pair reverse phase liquid chromatography, are described in the literature to assess their quality but most of them present significant drawbacks. In the present study, dT and PS mixtures were analyzed by hydrophilic interaction liquid chromatography (HILIC) and capillary zone electrophoresis (CZE) coupled to ultraviolet detection. In HILIC, the selectivities of three types of stationary phases (dihydroxypropane, phosphorylcholine and amide) were compared. Optimal conditions were determined and consisted of an amide stationary phase with a mobile phase made up of water, acetonitrile and 15 mM ammonium acetate (pH 5.5). In those conditions, high resolving power and good repeatability were achieved. In CZE, the effect of the background electrolyte (BGE), its pH and concentration were evaluated. A BGE made up of 300 mM ammonium acetate adjusted to pH 6.0 was selected. Finally, the two techniques were compared in terms of selectivity, repeatability and peak efficiency. In the second part of the study, HILIC and CZE were both coupled to a drift-tube ion-mobility quadrupole time-of-flight MS detector (DTIMS-QTOF) to assess the added value of this coupling for oligonucleotide characterization. Indeed, by using the measured collision cross section (CCS), the evaluation of the number of nucleotides was performed. Looking across the results, HILIC and CZE coupled to DTIMS-QTOF can be considered as promising tools for the quality control of oligonucleotides. [less ▲]

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See detailCapillary electrophoresis, high-performance liquid chromatography, and thin-layer chromatography analyses of phenolic compounds from rapeseed plants and evaluation of their antioxidant activity.
Huang, Yang; Jansen, Olivia ULiege; Frederich, Michel ULiege et al

in Journal of Separation Science (2019)

Rapeseed plants, known for oil production, are also known to contain phenolic compounds such as phenolic acids and flavonoids, with potential antioxidant and anticancer activities. The separation and ... [more ▼]

Rapeseed plants, known for oil production, are also known to contain phenolic compounds such as phenolic acids and flavonoids, with potential antioxidant and anticancer activities. The separation and identification of 11 phenolic acids in rapeseed extracts (including leaves, flowers, Chinese seeds, Belgian seeds, and cake) by capillary electrophoresis were investigated. The results were compared with those obtained with high-performance liquid chromatography and thin-layer chromatography and showed that the capillary electrophoresis technique offers several advantages for the identification of phenolic compounds in various rapeseed extracts. The antioxidant activity of rapeseed extracts and reference compounds was evaluated using four different approaches, namely, 2,2'-azinobis- (3-ethylbenzohiazoline-6-sulfonic acid assay, free radical 2,2-diphenyl-1-picrylhydrazyl assay, electron paramagnetic resonance spectroscopy and the measurement of the total polyphenol content. The contents of total polyphenols in the tested extracts were ranging between 5.4 and 21.1% m/m and ranked as follows: Chinese seeds > Belgian seeds > Flowers > Cake > Leaves. [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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