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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 ▲]

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See detailContribution of Capillary Electrophoresis and Ion Mobility Spectrometry to probe conformational change during desolvation
Far, Johann ULg; Delvaux, Cédric ULg; Kune, Christopher ULg et al

Conference (2016, January 28)

The transfer of ions from the solution to the gas phase is a critical step to produce « native species ». Coming from a highly solvating medium, ionic species will tend to find a new equilibrium ... [more ▼]

The transfer of ions from the solution to the gas phase is a critical step to produce « native species ». Coming from a highly solvating medium, ionic species will tend to find a new equilibrium conformation in the gas phase. The pathway to reach the thermodynamically stable conformation involves crossing potential barriers of different heights. When these barriers are too high compared to the internal energy of the ions, it will result in “partial memories” (as structural preservation) of the conformation in solution. In order to evaluate the effect of the solvent evaporation and of the various collision processes encountered by the ions in the mass spectrometer, we developed two strategies: The first strategy consists in comparing in a single experiment the shape of the ions in solution and in the gas phase. Data are obtained by coupling Capillary Electrophoresis (CE) with Ion Mobility Mass Spectrometry (IMS). Drift times in solution and in the gas phase are directly compared. Deviations from their correlation points out changes in folding upon desolvatation. Preliminary results show that some of peptides issued from tryptic digest of BSA clearly change their conformation during desolvatation. The second strategy consists to monitor the shape of the different conformers observed for the intact cytochrome C during native runs in capillary electrophoresis and “native” ion mobility mass spectrometry, both using a physiological TRIS acetic pH7 buffer compared to a “MS friendly” ammonium acetate pH7 buffer. Finally as preliminary study, we proposed to use homopolymers for better understanding about the folding behavior in gas phase and the resulting shape of these ions using ion mobility spectrometry. [less ▲]

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See detailToxicokinetics of selenium in the slider turtle Trachemys scripta
Dyc, Christelle; Far, Johann ULg; Gandar, Frederic ULg et al

in Ecotoxicology (2016), 25

Selenium (Se) is an essential element that can be harmful for wildlife. However, its toxicity in poikilothermic amniotes, including turtles, remains poorly investigated. The present study aims at ... [more ▼]

Selenium (Se) is an essential element that can be harmful for wildlife. However, its toxicity in poikilothermic amniotes, including turtles, remains poorly investigated. The present study aims at identifying selenium toxicokinetics and toxicity in juvenile slider turtles (age: 7 months), Trachemys scripta, dietary exposed to selenium, as selenomethionine SeMet, for eight weeks. Non-destructive tissues (i.e. carapace, scutes, skin and blood) were further tested for their suitability to predict selenium levels in target tissues (i.e. kidney, liver and muscle) From conservation perspective. 130 juvenile yellow-bellied slider turtles were assigned in three groups of 42 individuals each (i.e. control, SeMet1 and SeMet2). These groups were subjected to a feeding trial including an eight-week supplementation period SP8 and a following four-week elimination period EP4. During the SP8, turtles fed on diet containing 1.1 ± 0.04, 22.1 ± 1.0 and 45.0 ± 2.0 µg.g-1 of selenium (control, SeMet1 and SeMet2, respectively). During the EP4, turtles fed on non-supplemented diet. At different time during the trial, six individuals per group were sacrificed and tissues collected (i.e. carapace, scutes, skin, blood, liver, kidney, muscle) for analyses. During the SP8 (Figure 1), both SeMet1 and SeMet2 turtles efficiently accumulated selenium from a SeMet dietary source. The more selenium was concentrated in the food, the more it was in the turtle body but the less it was removed from their tissues. Moreover, SeMet was found to be the more abundant selenium species in turtles’ tissues. Body condition (i.e. growth in mass and size, feeding behaviour and activity) and survival of the SeMet1 and SeMet2 turtles seemed to be unaffected by the selenium exposure. There were clear evidences that reptilian species are differently affected by and sensitive to selenium exposure but the lack of any adverse effects was quite unexpected. [less ▲]

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See detailStructural analysis of ruthenium–arene complexes using ion mobility mass spectrometry, collision-induced dissociation, and DFT
Cserwinska, Izabella; Far, Johann ULg; Kune, Christopher ULg et al

in Dalton Transactions (2016), 45

Ion mobility mass spectrometry (IM-MS) and collision-induced dissociation (CID) techniques were used to investigate the influence of the phosphine ligand on the physicochemical properties of [RuCl2(p ... [more ▼]

Ion mobility mass spectrometry (IM-MS) and collision-induced dissociation (CID) techniques were used to investigate the influence of the phosphine ligand on the physicochemical properties of [RuCl2(p-cymene)(PCy3)] (1), [RuCl2(p-cymene)-(PPh3)] (2), and [RuCl2(p-cymene)(PTA)] (3) in the gas phase (PTA is 1,3,5-triaza-7-phosphaadamantane). Electrospray ionization of complexes 1 and 2 led to the corresponding [RuCl(p-cymene)(PR3)]+ ions via the dissociation of a chlorido ligand, whereas RAPTA-C (3) afforded two molecular ions by in-source oxidation ([RuIIICl2(p-cymene)(PTA)]+) or protonation ([RuCl2(p-cymene)(PTA+H)]+). Control experiments showed that the balance between these two ionization paths was strongly influenced by the nature of the solvent used for infusion. Collision cross sections (CCSs) of the four molecular ions accurately reflected the variations of steric bulk inferred from the Tolman steric parameters (θ) of the phosphine ligands. Moreover, DFT calculations combined with a model based on the kinetic theory of gases (the trajectory method of the IMoS software) afforded reliable CCS predictions. The almost two times higher dipole moment of [RuCl2(p-cymene)-(PTA+H)]+ (μ = 13.75 D) compared to [RuIIICl2(p-cymene)(PTA)]+ (μ = 7.18 D) was held responsible for increased ion-induced dipole interactions with a polarizable drift gas such as N2. Further experiments with He and CO2 confirmed that increasing the polarizability of the buffer gas improved the separation between the two molecular ions derived from complex 3. The fragmentation patterns of complexes 1–3 were determined by CID. The sequence of collision voltages at which 50% of a precursor ion dissociates (V50) recorded for the molecular ions derived from compounds 1–3 was in good agreement with simple electronic considerations based on the donor strength of the phosphine ligand. Thus, the CCS and V50 parameters used to determine the shape and stability of ionic species in the gas phase are complementary to the Tolman steric and electronic parameters (θ and TEP) commonly used by organometallic chemists in condensed phases. [less ▲]

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See detailInterlaboratory study to evaluate the robustness of capillary electrophoresis-mass spectrometry for peptide mapping
Wenz, Christian; Barbas, Coral; Lopez-Gonzalvez, Angeles et al

in Journal of Separation Science (2015)

A collaborative study on the robustness and portability of a capillary electrophoresis-mass spectrometry method for peptide mapping was performed by an international team, consisting of 13 independent ... [more ▼]

A collaborative study on the robustness and portability of a capillary electrophoresis-mass spectrometry method for peptide mapping was performed by an international team, consisting of 13 independent laboratories from academia and industry. All participants used the same batch of samples, reagents and coated capillaries to run their assays, whereas they utilized the capillary electrophoresis-mass spectrometry equipment available in their laboratories. The equipment used varied in model, type and instrument manufacturer. Furthermore, different types of sheath-flow capillary electrophoresis–mass spectrometry interfaces were used. Migration time, peak height and peak area of ten representative target peptides of trypsin-digested bovine serum albumin were determined by every laboratory on two consecutive days. The data were critically evaluated to identify outliers and final values for means, repeatability (precision within a laboratory) and reproducibility (precision between laboratories) were established. For relative migration time the repeatability was between 0.05 and 0.18% RSD and the reproducibility between 0.14 and 1.3% RSD. For relative peak area repeatability and reproducibility values obtained were 3–12 and 9–29% RSD, respectively. These results demonstrate that capillary electrophoresis-mass spectrometry is robust enough to allow a method transfer across multiple laboratories and should promote a more widespread use of peptide mapping and other capillary electrophoresis-mass spectrometry applications in biopharmaceutical analysis and related fields. [less ▲]

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See detailProbing the conformational changes during desolvation of ions using orthogonal mobility methods (CE-IMS)
Far, Johann ULg; Kune, Christopher ULg; Delvaux, Cédric ULg et al

Poster (2015, July 29)

The transfer of ions from the solution to the gas phase is a critical step to produce « native species ». Coming from a highly solvating medium, ionic species will tend to find a new equilibrium ... [more ▼]

The transfer of ions from the solution to the gas phase is a critical step to produce « native species ». Coming from a highly solvating medium, ionic species will tend to find a new equilibrium conformation in the gas phase. The pathway to reach the thermodynamically stable conformation involves crossing potential barriers of different heights. When these barriers are too high compared to the internal energy of the ions, it will result in “partial memories” (as structural preservation) of the conformation in solution. In order to evaluate the effect of the solvent evaporation and of the various collision processes encountered by the ions in the mass spectrometer, we developed two strategies: The first strategy consists in comparing in a single experiment the shape of the ions in solution and in the gas phase. Data are obtained by coupling capillary electrophoresis with Ion Mobility Mass Spectrometry. Drift times in solution and in the gas phase are directly compared. Deviations from their correlation points out changes in folding upon desolvatation. Preliminary results show that among peptides issued from tryptic digest of BSA some of them clearly change their conformation during desolvatation. The second strategy consists in probing changes of conformation once the ions are in the gas phase. The ions are rapidly heating by collisions ions during their transfer to the IMS. The heating is obtained by increasing their collision energy, rapidly followed by thermalisation in the IMS cell. The ions may be kinetically trapped in their new conformations. This allows comparing barriers between different ions geometries. In summary this work intends to evaluate the extent of conformational “memory” of the ions of different nature for best experimental condition allowing “native mass spectrometry” [less ▲]

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See detailCONTRIBUTION OF ION MOBILITY FOR STRUCTURAL ANALYSIS AND ANALYTICAL CHEMISTRY: THE USE OF PROBE LIGANDS AND SELECTIVE IMS SHIFT REAGENTS
Kune, Christopher ULg; Far, Johann ULg; Delvaux, Cédric ULg et al

Conference (2015, July 28)

Ion mobility is a gas phase separation technique sensitive to the Collisional Cross Section (CCS) difference of ions (as CCS/ΔCCS). It discriminates isobaric and isomeric ions when CCS difference is ... [more ▼]

Ion mobility is a gas phase separation technique sensitive to the Collisional Cross Section (CCS) difference of ions (as CCS/ΔCCS). It discriminates isobaric and isomeric ions when CCS difference is larger than the instrumental resolution (roughly 50). To overcome the usual resolution of ion mobility (IM), it is necessary to use new strategies in addition to the optimization of the ion mobility parameters. This work proposes a new method to bypass this limitation while providing additional structural information by the use of Selective Shift Reagents (SSR). A SSR can specifically bind with a target ion depending of their physicochemical properties like chemical groups, steric hindrance, polarity, space charge effects… In this strategy, the choice of SSR is fundamental. SSR could be empirically selected or assisted and designed by computational chemistry prediction. SSR can be used as a chemical probe which can support physicochemical properties and help or confirm hypotheses for structural elucidation. They can also drastically change the CCS of a target ion present in a complex mixture (e.g. biological origin sample) as shifting reagent for e.g. quantification purpose. Models used for the proof of concept have been selected in order to lead to an expected or predictive result. Firstly crown ethers have been used as SSR in IMS to shift the protonated valine drift time from the protonated proline drift time according to their chemical groups and especially amino groups. The selectivity of SSR leads to an improved separation between valine and proline. Secondly three structural isomers of diaminonaphthalene were investigated experimentally and theoretically using computational chemistry support after the addition of different crown ethers or β-cyclodextrin as SSR to improve the separation of these isomers by IMS. Finally, the concept of SSR was successfully applied to biological origin samples to elucidate structure and allows the quantification of selenium (Se) containing compounds present in an aqueous extract of Se rich yeast. [less ▲]

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See detailIs the natural shape of ions in the gas phase spherical ? The allegory of the cave (Plato) applied in mass spectrometry
Far, Johann ULg; Haler, Jean ULg; Kune, Christopher ULg et al

Conference (2015, May 13)

The transfer of ions from solution to gas phase is a critical step to produce « native species ». Coming from a highly solvating medium, ionic species will tend to find new equilibrium conformations in ... [more ▼]

The transfer of ions from solution to gas phase is a critical step to produce « native species ». Coming from a highly solvating medium, ionic species will tend to find new equilibrium conformations in the gas phase. The pathway to reach the thermodynamically stable conformation(s) involves crossing potential barriers according to the type of interactions involved. When these barriers are too high compared to the internal energy of the ions, it may result in a “partial memory” (as structural preservation) of the conformation in solution. In order to evaluate the effect of the solvent evaporation and of the various collision processes encountered by the ions in the mass spectrometer we based our strategy on the determination of deviations from a spherical shape, supposed to be the natural state of ions in the gas phase. [less ▲]

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See detailCharacterization of Venom Peptides using Microfluidic Separation Techniques coupled to Mass Spectrometry (LC-MS and CE-MS)
Degueldre, Michel ULg; Delvaux, Cédric ULg; Far, Johann ULg et al

Poster (2015, March)

More than the half of the principal sub-kingdoms of the animal world contains species that produce venom whose purposes are to immobilize, kill and pre-digest the preys. These venoms represent an ... [more ▼]

More than the half of the principal sub-kingdoms of the animal world contains species that produce venom whose purposes are to immobilize, kill and pre-digest the preys. These venoms represent an exceptionally rich source of various biologically active peptides, both in their structures and their effects, which are more and more useful for human being1. Yet, the total characterization of such complex samples require advanced analytical techniques mainly due to the complexity of the sample (hundreds of compounds), the limited quantities usually available and the presence of numerous PTMs, especially disulfide bridges and specific folding. Here we present a method that combines LC and CE separation techniques coupled to mass spectrometry (MS) to characterize the peptide composition of the snake venom Naja atra. The characterization will not only focus on the toxin sequencing (LC-MS and LC-MS/MS), but will also aim at analyzing the folding of the toxins (CE-MS). To this end, native and reduced/alkylated toxins will be analyzed by both techniques. Final result targets the determination of the global hydrophobic pattern and native tridimensional folding of these strongly reticulated peptides. (1) Richard J. Lewis & Maria L. Garcia, Therapeutic potential of venom peptides. Nature Reviews Drug Discovery 2003, 2, 790-802. [less ▲]

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See detailContribution of Ion Mobilities and Mass Spectrometry for structural elucidation and conformational studies
Far, Johann ULg

Scientific conference (2014, December 02)

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