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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 detailThe use of Ion Mobility Mass Spectrometry for isomer composition determination extracted from Se-rich yeast
Far, Johann ULg; Delvaux, Cédric ULg; Kune, Christopher ULg et al

in Analytical Chemistry (2014), just accepted

The isomer ratio determination of a selenium-containing metabolite produced by Se-rich yeast was performed. Electrospray Ionization and Ion Mobility Mass Spectrometry (IM-MS) were unsuccessfully used in ... [more ▼]

The isomer ratio determination of a selenium-containing metabolite produced by Se-rich yeast was performed. Electrospray Ionization and Ion Mobility Mass Spectrometry (IM-MS) were unsuccessfully used in order to resolve the isomers according to their Collisional Cross Section (CCS) difference. The isomer ratio determination of 2,3-dihydroxypropionylselenocystathionine was performed after multidimensional liquid chromatography preconcentration from a water extract of Se-rich yeast using preparative size exclusion, anion exchange and capillary reverse phase columns coupled to IM-MS. 4’-nitrobenzo-15-crown-5 ether, a Selective Shift Reagent (SSR), was added after the last chromatographic dimension in order to specifically increase the CCS of one of the isomers by the formation of a stable host-guest system with the crown-ether . Both isomers were consequently fully resolved by IM-MS and the relative ratio of the isomers was determined: 11-13% and 87-89%. The present data compared favorably with literature to support the analytical strategy despite the lack of authentic standard for method validation. In addition, computational chemistry methods were successfully applied to design the SSR and to support the experimental data. [less ▲]

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

Poster (2014, June 24)

Routine analyses using Mass Spectrometry (MS) detection became a very popular due to the low limit of detection, great sensitivity, selectivity and specificity. Moreover MS enables structural elucidation ... [more ▼]

Routine analyses using Mass Spectrometry (MS) detection became a very popular due to the low limit of detection, great sensitivity, selectivity and specificity. Moreover MS enables structural elucidation and physical (or physicochemical) properties determination using low amount of not especially pure samples. One of these properties is substantially the stoichiometry determination of non-covalent complexes such as Ligand receptor systems (e.g. DNA-drugs, Hormones/drugs-receptor, quaternary structure arrangement of polypeptides, …). MS determination in native condition (Native MS) of this stoichiometry allows large scale screening of potential drugs candidates in pharmaceutical context. The addition of Ion-Mobility (IM) to mass spectrometry (IM-MS) with computational chemistry support allows the structural conformation monitoring (as Collisional Cross Section or CCS) and the elucidation of interactions of the non-covalent complexes. Nonetheless such determinations suppose/assume that desolvation steps and gas phase transfer during MS detection do not modify the tridimensional structure of these systems or the magnitude order of the involved interactions (dipole-dipole, dipole-ion, ion-ion, hydrophobic interactions). Further studies (Circular Dichroism, Nuclear Magnetic resonance, UV-Visible spectra …) are generally required to confirm the MS data but there are unfortunately time consuming. This project proposes the coupling of Capillary Electrophoresis (CE) to IM-MS in order to obtain in one injection the quantitative and qualitative data of non-covalent complexes and the monitoring of tridimensional conformation modification between the liquid and gas phase. Indeed CE allows the determination of numerous physicochemical properties (dissociation constant, pK values, hydrodynamic radius determination …). The structural data (i.e. hydrodynamic radius and CCS) are compared to the data obtained by IM-MS as a proof of concept using tryptic digest of Bovine Serum Albumine (BSA). [less ▲]

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See detailContribution of ion mobility for structural analysis and analytical chemistry: Use of selective IMS shift reagents (SSR)
Kune, Christopher ULg; Far, Johann ULg; Delvaux, Cédric ULg et al

Poster (2014, June 19)

Ion mobility is a gas phase separation technique based on the Collisional Cross Section (CCS) of ions. It discriminates isobaric and isomeric ions provided their CCS difference is larger than the ... [more ▼]

Ion mobility is a gas phase separation technique based on the Collisional Cross Section (CCS) of ions. It discriminates isobaric and isomeric ions provided their CCS difference is larger than the instrumental resolution. This work proposes a new method to overcome this limitation while providing additional structural information. A Selective Shift Reagent (SSR) is a ligand specifically modifying the CCS of ions. Indeed specific non-covalent complexes can be form with a suitable SSR to reach the required selectivity and the CCS induced shift. A CID dissociation of the complex may be used after IMS separation to produce specific MS/MS spectra of the targeted analyte. This concept paves the way for new analytical strategies by ion mobility based on non-covalent complex formation. [less ▲]

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See detailDesign of a sprayer that makes CE-ESI-MS easy and robust
Far, Johann ULg; De Pauw, Edwin ULg; Falmagne, Jean-Bernard et al

Conference (2014, May 28)

Presentation of a CEMS interface design for easy and robust coupling of Capillary Electrophoresis with Mass Spectrometry equipped with a nanospray source. Principles and specification were presented.

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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 (2014, April 08)

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. The 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. 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 ▲]

Detailed reference viewed: 39 (12 ULg)
See detailInnovative analytical strategies for small molecules analysis by ion-mobility mass spectrometry
Eppe, Gauthier ULg; Goscinny, Séverine; Far, Johann ULg et al

Conference (2014, January)

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See detailInnovative analytical strategy using ion-mobility shifting additive for isobaric selenium compound identification in selenomethionine standards by IMS
Kune, Christopher ULg; Far, Johann ULg; Eppe, Gauthier ULg et al

Conference (2013, July 09)

Selenium (Se) is a trace element which is both essential and toxic depending on its concentration and its chemical form. Selenomethionine (SeMet) is one of the widely used selenium standard during ... [more ▼]

Selenium (Se) is a trace element which is both essential and toxic depending on its concentration and its chemical form. Selenomethionine (SeMet) is one of the widely used selenium standard during Selenium speciation studies. This work was focused on the elaboration of an analytical strategy for the detection and the structural elucidation of an isobaric Se interference, which is found in standard solutions of SeMet by high resolution mass spectrometry (Rm/Δm > 20.000). The structural elucidation of these compounds requires the isolation of the respective parent ion. Nevertheless, the mass difference between SeMet and its interference is less than 0.02Da which is well below the window selection of conventional techniques in mass spectrometry (Quadripole, ion trap). The empirical formula and double bound equivalent (DBE) of these ions suggest different tridimensional structures which lead to a discrimination depending on the ion mobility. This separation is observed, both in gaseous and liquid phase, by Ion Mobility Spectrometry (IMS), Capillary Electrophoresis (CE) and Liquid Chromatography (LC) which are hyphenated to mass spectrometry as detector. The separation efficiency of these ions by IMS and CE is improved by using specific shifting agents (18-Crown-6 Ether) selective to only one of these ions. This strategy has successfully separated the two isobaric ions present and leads to the structural elucidation of the isobar contaminant of SeMet. [less ▲]

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See detailInnovative analytical strategy using ion-mobility for structural or functional selenium isomers identification by ion mobility spectrometry
Far, Johann ULg; Kune, Christopher ULg; Lobinski, Ryszard et al

Poster (2013, July)

Selenium (Se) is a trace element which is both essential and toxic depending on its concentration and its chemical form. Se-rich yeast is one of the most popular Se source for supplementation. The ... [more ▼]

Selenium (Se) is a trace element which is both essential and toxic depending on its concentration and its chemical form. Se-rich yeast is one of the most popular Se source for supplementation. The classical method of speciation is related to multidimensional liquid chromatography (LC) hyphenated to mass spectrometry (MS) Recent advances in Se speciation led to greatly improve the Se speciation in these samples but isomers identification and quantification remain challenging. This work focuses on the elaboration of an innovative analytical strategy for the detection and the structural elucidation of isobaric selenium compounds present in Se-rich yeast. A specific complex formation agent acts as a chemical probe for the detection of chemical function. The addition of a complexing agent can improve the discrimination between structural or functional Se isomers using ion mobility techniques as Ion Mobility Spectrometry (IMS) by increasing the molecular weight (i.e. the m/z ratio in MS) and the collision cross section of a target ion after selective complexation. This Ion Mobility orthogonal separation improves the structural elucidation. Crown ethers used as shifting agents can specifically form complexes with primary amines. The addition of crown ether to different low molecular weight fractions obtained by multidimensional LC of a water extract from Se-rich yeast permitted to detect Se isomers and confirmed their structure using IMS. [less ▲]

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See detailDevelopment of an analytical strategy to measure major selenium-containing species in juvenile turtles (Trachemys scripta scripta) by SAX-HPLC-ICP MS
Far, Johann ULg; Dyc, Christelle; Das, Krishna ULg et al

Poster (2013, July)

Sea turtles are exposed to many environmental elements such as selenium (Se). Sea turtles are listed under the Red List of threatened species by the International Union for Conservation of Nature. It is ... [more ▼]

Sea turtles are exposed to many environmental elements such as selenium (Se). Sea turtles are listed under the Red List of threatened species by the International Union for Conservation of Nature. It is thus mandatory to use low-invasive tissue collection (skin, carapace, blood ) for estimating Se exposure in these highly protected turtles. For this purpose, a biological modal Trachemys scripta scripta (or slider turtle) was selected. For two months, juvenile turtles were dietary exposed to Se by spiking the food with Selenomethionine (SeMet) or Methionine as control groups. Individuals were sacrificed after different time of exposure and tissues (skin, liver, muscle, carapace and blood) collected to perform Se speciation and determine some biological endpoints. An analytical strategy was developed to cope with the very low amount of available sample. It is briefly consisting by reduction, alkylation and proteolysis of the entire freeze-dried tissues followed by sample clean-up using ultra-filtration membrane. Then anion exchange HPLC using salt and pH gradient was developed to prevent the introduction of organic solvents, which cause severe fooling of ICP MS and avoid ultra-trace analyses of sea water in routine analysis. This method successfully achieved the detection and quantification at ppm level of expected species (i.e SeMet, selenocysteine, inorganic Se) and also unknown species but their relative amounts were time and tissues dependent. [less ▲]

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