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See detailIon Mobility-Mass Spectrometry: Going beyond the numbers
Haler, Jean ULg; Far, Johann ULg; Béchet, Eric ULg et al

Scientific conference (2017, July 03)

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See detailSeparation, identification and quantification of peptidoglycan fragments by zwitterionic hydrophilic interaction chromatography and capillary electrophoresis coupled to mass spectrometry
Boulanger, Madeleine ULg; Delvaux, Cédric ULg; Raymackers, Alice ULg et al

Poster (2017, June 05)

Bacterial peptidoglycan-derived muropeptides and peptides are soluble fragments acting as messengers in diverse cell-signalling events. As the peptidoglycan wall is a key target of antibiotics, bacteria ... [more ▼]

Bacterial peptidoglycan-derived muropeptides and peptides are soluble fragments acting as messengers in diverse cell-signalling events. As the peptidoglycan wall is a key target of antibiotics, bacteria have developed specific resistance mechanisms based on the detection of these fragments inside their cytoplasm. In our model strain, Bacillus licheniformis, the peptidoglycan dipeptide m-A2pm-D-Glu triggers a beta-lactamase induction. However, the nature and the concentration of cytoplasmic peptidoglycan fragments leading to the dipeptide formation are unknown. Additionally, the muropeptides sensing is involved in the innate immune response toward bacterial invasion and is therefore of considerable importance in eukaryotes self-defence functions. In this context, the development of reliable analytical methods aiming to identify and quantify those fragments in complex samples are of major interest. [less ▲]

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See detailN-Glycosylation of an IgG antibody secreted by Nicotiana tabacum BY-2 cells can be modulated through co-expression of human β-1,4-galactosyltransferase
Navarre, Catherine; Smargiasso, Nicolas ULg; Duvivier, Laurent et al

in Transgenic Research (2017), 26(3), 375-384

Nicotiana tabacum BY-2 suspension cells have several advantages that make them suitable for the production of full-size monoclonal antibodies which can be purified directly from the culture medium ... [more ▼]

Nicotiana tabacum BY-2 suspension cells have several advantages that make them suitable for the production of full-size monoclonal antibodies which can be purified directly from the culture medium. Carbohydrate characterization of an antibody (Lo-BM2) expressed in N. tabacum BY-2 cells showed that the purified Lo-BM2 displays N-glycan homogeneity with a high proportion (>70%) of the complex GnGnXF glycoform. The stable co-expression of a human β-1,4-galactosyltransferase targeted to different Golgi sub-compartments altered Lo-BM2N-glycosylation and resulted in the production of an antibody that exhibited either hybrid structures containing a low abundance of the plant epitopes (α-1,3-fucose and β-1,2-xylose), or a large amount of galactose-extended N-glycan structures. These results demonstrate the suitability of stable N-glycoengineered N. tabacum BY-2 cell lines for the production of human-like antibodies. © 2017, Springer International Publishing Switzerland. [less ▲]

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See detailComprehensive Ion Mobility Calibration Strategies based on Synthetic Polymers
Haler, Jean ULg; Kune, Christopher ULg; Chirot, Fabien et al

Conference (2017, May 09)

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See detailComprehensive Ion Mobility Calibration Strategies based on Synthetic Polymers
Haler, Jean ULg; Kune, Christopher ULg; Chirot, Fabien et al

Conference (2017, March 21)

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See detailPeptidoglycan fragments separation and identification by zwitterionic hydrophilic interaction chromatography and capillary electrophoresis coupled to mass spectrometry
Boulanger, Madeleine ULg; Raymackers, Alice ULg; Delvaux, Cédric ULg et al

Poster (2017, February 08)

Bacterial peptidoglycan-derived peptides and muropeptides are soluble unique fragments acting as messengers in diverse cell-signalling events. As the bacterial peptidoglycan wall is a major target of ... [more ▼]

Bacterial peptidoglycan-derived peptides and muropeptides are soluble unique fragments acting as messengers in diverse cell-signalling events. As the bacterial peptidoglycan wall is a major target of antibiotics, bacteria have developed specific resistance mechanisms based on the detection of such fragments. In addition, the muropeptides sensing is involved in the innate immune response toward bacterial invasion and is therefore of major importance in the eukaryotes self-defence functions. In Bacillus licheniformis 749/I, the peptidoglycan dipeptide m-A2pm-D-Glu triggers beta-lactam resistance via the induction of a beta-lactamase, BlaP. This induction process relies on a complex regulation system for which the nature and the concentration of peptidoglycan fragments leading to the formation of dipeptide moiety inside the cytoplasm are unknown. In this context, the development and the validation of a reliable method to identify and quantify those cytoplasmic fragments is of major interest. Conventionally, the peptidoglycan is first digested by mutanolysin in order to generate muropeptides which are subsequently analyzed by reversed-phase liquid chromatography (RP-LC, C18). However, this technique is not effective enough to separate the peptides that, as a result, are eluted in the flow through . In this work, we developed two novel analytical separation methods, namely capillary electrophoresis (CE) and zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) both coupled to mass spectrometry (MS), aiming at overcoming the drawbacks encountered in traditional separation techniques. Both methods show great results in the identification of peptidoglycan fragments in complex samples. CE analysis lead to muropeptides and peptides separation whereas ZIC-HILIC only retains peptides. Nevertheless, the latter has been optimized and validated for the cytoplasmic peptidoglycan peptides identification and quantification. Althogether, ZIC-HILIC-MS and CE-MS have proved to be powerful analytical tools for the identification and quantification of peptidoglycan fragments in complex matrix samples. Further optimizations are still ongoing for the analysis of muropeptides, which hopefully will lead to the identification and quantification of cytoplasmic peptidoglycan fragments composition during the Bacillus licheniformis 749/I BlaP beta-lactamase induction process. [less ▲]

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See detailAccurate drift time determination by traveling wave ion mobility spectrometry: The concept of the diffusion calibration
Kune, Christopher ULg; Far, Johann ULg; De Pauw, Edwin ULg

in Analytical Chemistry (2016), 88

Ion mobility spectrometry (IMS) is a gas phase separation technique which relies on differences in collision cross section (CCS) of ions. Ionic clouds of unresolved conformers overlap if the CCS ... [more ▼]

Ion mobility spectrometry (IMS) is a gas phase separation technique which relies on differences in collision cross section (CCS) of ions. Ionic clouds of unresolved conformers overlap if the CCS difference is below the instrumental resolution expressed as CCS/ΔCCS. The experimental arrival time distribution (ATD) peak is then a superimposition of the various contributions weighted by their relative intensities. This paper introduces a strategy for accurate drift time determination using traveling wave ion mobility spectrometry (TWIMS) of poorly resolved or unresolved conformers. This method implements through a calibration procedure the link between the peak full width at half maximum (FWHM) and the drift time of model compounds for wide range of settings for wave heights and velocities. We modified a Gaussian equation which achieves the deconvolution of ATD peaks where the FWHM is fixed according to our calibration procedure. The new fitting Gaussian equation only depends on two parameters: The apex of the peak (A) and the mean drift time value (μ). The standard deviation parameter (correlated to FWHM) becomes a function of the drift time. This correlation function between μ and FWHM is obtained using the TWIMS calibration procedure which determines the maximum instrumental ion beam diffusion under limited and controlled space charge effect using ionic compounds which are detected as single conformers in the gas phase. This deconvolution process has been used to highlight the presence of poorly resolved conformers of crown ether complexes and peptides leading to more accurate CCS determinations in better agreement with quantum chemistry predictions [less ▲]

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See detailSupramolecular influence on cis–trans isomerization probed by ion mobility spectrometry
Czerwinska, Izabella; Kulesza, Alexander; Choi, Changmin et al

in Physical Chemistry Chemical Physics [=PCCP] (2016), 18

We used tandem ion mobility spectrometry measurements to investigate how the photo-isomerization of a chromophore (a methylpyridinium derivative) is affected by the complexation with a crown ether. A ... [more ▼]

We used tandem ion mobility spectrometry measurements to investigate how the photo-isomerization of a chromophore (a methylpyridinium derivative) is affected by the complexation with a crown ether. A dramatic blue-shift of the photo-isomerization spectrum was observed upon complexation, which could be well reproduced by ab initio calculations. Our results support that the observed changes in the photo-physical properties of the chromophore originate from the charge-solvation of its pyridinium moiety by the host cage. [less ▲]

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See detailAccurate drift time determination by traveling wave ion mobility spectrometry: The concept of the diffusion calibration
Kune, Christopher ULg; Far, Johann ULg; De Pauw, Edwin ULg

Conference (2016, October 18)

Ion mobility spectrometry (IMS) is a gas phase separation technique which relies on differences in the collisional cross section (CCS) of ions. Ionic clouds of unresolved conformers overlapped if the CCS ... [more ▼]

Ion mobility spectrometry (IMS) is a gas phase separation technique which relies on differences in the collisional cross section (CCS) of ions. Ionic clouds of unresolved conformers overlapped if the CCS difference is below the instrumental resolution expressed as Ω/ΔΩ. The experimental arrival time distribution (ATD) peak is then a superimposition of the various contributions weighted by their relative intensities. We have developed a strategy for accurate drift time determination using traveling wave ion mobility spectrometry (TWIMS) of poorly and unresolved conformers. This method implements through a calibration procedure the link between the peak full width at half maximum (FWHM) and the drift time of model compounds for wide range of settings for wave heights and velocities. We modified a Gaussian equation which achieves the deconvolution of ATD peaks where the FWHM is fixed according to our calibration procedure. The new fitting Gaussian equation only depends on two parameters: The apex of the peak (A) and the mean drift time value (µ). The standard deviation parameter (correlated to FWHM) becomes a function of the drift time. This correlation function between µ and FWHM is obtained using the TWIMS calibration procedure which determines the maximum instrumental ion beam diffusion using ionic compounds which are detected as single conformers in the gas phase. This deconvolution process has been used to highlight the presence of poorly resolved conformers for couples of crown ethers and peptides leading to CCS determination in better agreement with quantum chemistry predictions. [less ▲]

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See detailThe concept of the diffusion calibration for accurate drift time measurement by traveling wave ion mobilty
Kune, Christopher ULg; Far, Johann ULg; De Pauw, Edwin ULg

Poster (2016, October 13)

Ion mobility spectrometry (IMS) is a gas phase separation technique which relies on differences in the collisional cross section (CCS) of ions. Ionic clouds of unresolved conformers overlapped if the CCS ... [more ▼]

Ion mobility spectrometry (IMS) is a gas phase separation technique which relies on differences in the collisional cross section (CCS) of ions. Ionic clouds of unresolved conformers overlapped if the CCS difference is below the instrumental resolution expressed as Ω/ΔΩ. The experimental arrival time distribution (ATD) peak is then a superimposition of the various contributions weighted by their relative intensities. We have developed a strategy for accurate drift time determination using traveling wave ion mobility spectrometry (TWIMS) of poorly and unresolved conformers. This method implements through a calibration procedure the link between the peak full width at half maximum (FWHM) and the drift time of model compounds for wide range of settings for wave heights and velocities. We modified a Gaussian equation which achieves the deconvolution of ATD peaks where the FWHM is fixed according to our calibration procedure. The new fitting Gaussian equation only depends on two parameters: The apex of the peak (A) and the mean drift time value (µ). The standard deviation parameter (correlated to FWHM) becomes a function of the drift time. This correlation function between µ and FWHM is obtained using the TWIMS calibration procedure which determines the maximum instrumental ion beam diffusion using ionic compounds which are detected as single conformers in the gas phase. This deconvolution process has been used to highlight the presence of poorly resolved conformers for couples of crown ethers and peptides leading to CCS determination in better agreement with quantum chemistry predictions. [less ▲]

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See detailFirst Results Using TIMS on Systems Requiring High IMS Resolution
Haler, Jean ULg; Massonnet, Philippe ULg; Morsa, Denis ULg et al

Conference (2016, June 05)

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See detailPeptidoglycan fragments separation by CE/LC-MS
Boulanger, Madeleine ULg; Delvaux, Cédric ULg; Far, Johann ULg et al

Poster (2016, May 24)

In Bacillus licheniformis 749/I, the induction of BlaP beta-lactamase relies on a complex regulation system. During this process, the intracellular repressor BlaI is inactivated by a dipeptide coming from ... [more ▼]

In Bacillus licheniformis 749/I, the induction of BlaP beta-lactamase relies on a complex regulation system. During this process, the intracellular repressor BlaI is inactivated by a dipeptide coming from the peptidoglycan (PG) degradation via an “AND Gate” regulation. This regulation involves the cellular stress induced by the beta-lactam, the membrane receptor BlaR1 and the PG turnover. Briefly, the induction occurs when the extracellular domain of BlaR1 is acylated by the antibiotic which leads to a reorganization of the transmembrane segments and the receptor autocleavage. Simultaneously, the beta-lactam partially inhibits the penicillin-binding protein 1 (PBP1), triggering increased PG turnover and accumulation of PG fragments. Some of these fragments could enter in the cytoplasm and undergo enzymatic degradations which lead to the formation of the pro co-activator (tripeptide L-Ala-D-Glu-m-A2pm). This pro co-activator generates the co-activator, the dipeptide D-Glu-m-A2pm. Nowadays the nature and the concentration of PG fragments inside the cytoplasm are unknown. Therefore, the development of different analytical methods is required in order to identify those cytoplasmic fragments. In this poster, three different ways to separate PG fragments are discussed. [less ▲]

Detailed reference viewed: 105 (13 ULg)