References of "De Pauw, Edwin"
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See detailTravelling-wave ion mobility time-of-flight mass spectrometry as an alternative strategy for screening of multi-class pesticides in fruits and vegetables
Goscinny, Séverine ULg; Joly, Laure; De Pauw, Edwin ULg et al

in Journal of Chromatography. A (in press)

This paper reports a novel approach to screening multi-class pesticides by ion mobility timeof- flight mass spectrometry detection. Nitrogen was selected as mobility gas. After optimization of the ... [more ▼]

This paper reports a novel approach to screening multi-class pesticides by ion mobility timeof- flight mass spectrometry detection. Nitrogen was selected as mobility gas. After optimization of the different ion mobility parameters, determination of matrix effect on the drift times was conducted using different matrix extracts. The results showed that drift time values are not influenced by the matrix and also are independent of the concentration within the working range for 100 pesticides tested, making drift time a powerful additional identification tool. Based on statistics, 2% variation criteria provides a good fit for all the pesticides targeted, and could be considered as a maximum acceptable criteria associated with the drift time parameter for identification purpose. This 2% value is in agreement with already reported criteria, for instance, for GC or LC retention time in European documents. Finally, the well-known feature of mobility to separate complex mixtures was also tested to obtain purified extracted mass spectra of pesticides in fruit extract. [less ▲]

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See detailMass-spectrometry-based method for screening of new peptide ligands for G-protein-coupled receptors
Cologna Takeno, Camila; Gilles, Nicolas; Echterbille, Julien ULg et al

in Analytical and Bioanalytical Chemistry (in press)

G-protein-coupled receptors (GPCRs) constitute the largest family of transmembrane proteins. Although implicated in almost all physiological processes in the human body, most of them remain unexploited ... [more ▼]

G-protein-coupled receptors (GPCRs) constitute the largest family of transmembrane proteins. Although implicated in almost all physiological processes in the human body, most of them remain unexploited, mostly because of the lack of specific ligands. The objective of this work is to develop a new mass-spectrometry-based technique capable of identifying new peptide ligands for GPCRs. The strategy is based on the incubation of cellular membranes overexpressing GPCRs with a mixture of peptides that contains potential ligands. Peptide ligands bind to the receptors, whereas other peptides remain in the binding buffer. Bound peptides are eluted from membranes and directly detected, identified, and characterized by MALDI TOF–TOF. The results reveal the efficacy of the procedure for selecting a specific ligand of GPCRs in both simple and complex mixtures of peptides. This new approach may offer direct purification, identification, and characterization of the new ligand in a single workflow. The proposed method is labeling-free and, unlike radio-binding and other techniques, it does not require a previously known labeled ligand of the studied GPCR. All these properties greatly reduce the experimental constraints. Moreover, because it is not based on the principle of a competitive specific binding, this technique constitutes a new tool to discover new ligands not only for known GPCRs, but also for orphan GPCRs [less ▲]

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See detailCombined use of Ion Mobility and Collision-Induced Dissociation to investigate the opening of disulfide bridges by Electron-Transfer Dissociation in peptides bearing two disulfide bonds
Massonnet, Philippe ULg; Upert, Gregory; Smargiasso, Nicolas ULg et al

in Analytical Chemistry (in press)

Disulfide bonds are post-translational modifications (PTMs) often found in peptides and proteins. They increase their stability towards enzymatic degradations, and provide the structure and (consequently ... [more ▼]

Disulfide bonds are post-translational modifications (PTMs) often found in peptides and proteins. They increase their stability towards enzymatic degradations, and provide the structure and (consequently) the activity of such folded proteins. The characterization of disulfide patterns, i.e the cysteine connectivity, is crucial to achieve a global picture of the active conformation of the protein of interest. Electron Transfer Dissociation (ETD) constitutes a valuable tool to cleave the disulfide bonds in the gas phase, avoiding chemical reduction/alkylation in solution. To characterize the cysteine pairing, the present work proposes (i) to reduce by ETD one of the two disulfide bridges of model peptides , resulting in the opening of the cyclic structures, (ii) to separate the generated species by ion-mobility and, (iii) to characterize the species using CID. Results of this strategy applied to several peptides show different behaviors depending on the connectivity. The loss of SH• radical species, observed for all the peptides, confirms the cleavage of the disulfides during the ETD process. [less ▲]

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See detailEnergetics and Structural Characterization of Isomers Using Ion Mobility and Gas-phase H/D Exchange: Learning from Lasso Peptides
Hanozin, Emeline ULg; Morsa, Denis ULg; De Pauw, Edwin ULg

in Proteomics (2015), early view

State-of-the-art characterization of proteins using mass spectrometry namely relies on fragmentation methods which allows exploring featured dissociative reaction pathways. These pathways are often ... [more ▼]

State-of-the-art characterization of proteins using mass spectrometry namely relies on fragmentation methods which allows exploring featured dissociative reaction pathways. These pathways are often initiated by a series of potentially informative mass-constant conformational changes that are nonetheless frequently overlooked by lack of adequate investigation techniques. In the present study, we propose a methodology to readily address both structural and energetic aspects of stereoisomerization reactions using ion mobility coupled with mass spectrometry. To this end, a commercial spectrometer was used as a reactor comprising an energy resolved collisional activation step intended at promoting controlled conformational changes and a structural assignment step dedicated to the identification of the generated isomers. This identification relies on ion mobility and other on-line coupled techniques, namely an originally designed gas-phase H/D exchange experiment. We here apply this methodology to characterize the isomerization kinetics of capistruin, a 19-residue long lasso-folded peptide. We expect this approach to bring insights into the physical origin of global dissociation thresholds monitored in tandem mass spectrometry experiments and to set a promising basis for quantitative investigations of the stability of different molecular folds. [less ▲]

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See detailDe novo sequencing using MELD proteolysis coupled to a "sequence assembly" algorithm
Mazzucchelli, Gabriel ULg; Zimmerman, Tyler; Smargiasso, Nicolas ULg et al

in 63rd ASMS Conference Proceedings, May 30 - June 4 2015, St. Louis, MO (2015, June)

Introduction Protein de novo sequencing requires a method that combines extensive MSMS fragmentation and an appropriate data processing. This can be applied either on intact protein or on its proteolytic ... [more ▼]

Introduction Protein de novo sequencing requires a method that combines extensive MSMS fragmentation and an appropriate data processing. This can be applied either on intact protein or on its proteolytic peptides. Peptide analysis has the advantage to be compatible with well-known workflows. Nevertheless the connectivity between peptides is lost. Taking these considerations into account, a specific digestion method and a sequence assembly software were developed. The MELD method relies on a combination of MultiEnzymatic AND Limited proteolytic Digestions. The MELD generates in a single experiment numerous different peptides with miss-cleavages that overlap when aligned on the matching protein sequence. The two major benefices are an increased probability to obtain the entire protein sequence and a redundancy in the protein sequence matches. Methods The MELD consists in two parallel 2h digestions both using an optimized protease mixture. The mixtures are composed of the same proteases but in different relative quantities. Analyses were performed by UPLC-Orbitrap (IClass, Waters, QExactive, Thermo). Data were processed with PEAKS (BSI) to generate de novo sequence candidates. After data importation into our software, a seed sequence is set or can be found automatically. The software extends the seed sequence in both directions with moving windows of three amino acids, plus a fourth one to be added. The added amino acid is validated in several ways, including by the total frequency of occurrence, by larger windows of aa, by the local spectrum-derived confidence, and by combinations of these factors. Preliminary Data The MELD protocol was first validated by applying a traditional database search workflow on several commercial proteins with an inter-day and inter-individual procedure. These experiments showed 100% sequence coverage for each protein analyzed, involving information on peptide identity and modifications localization. Strong confident identification was obtained due to multiple overlapping peptides matches with the given sequence and with a high number of overlapping peptides assignments. The analysis of the four proteins provided the following results: HSA, Myoglobin and Lysozyme were identified with 100% sequence coverage with respectively 890, 300 and 120 unique peptides (CV<10%, peptide FDR<0.1%). The variable region of each heavy and light chains of Adalimumab antibody were identified with 100% sequence coverage. With the MELD, an average of 10 different peptides covering each sequence stretch of the protein could be obtained. The combinatory effect of the multiple enzymes used and the limited digestions leads to an increased robustness, very high confidence identifications and allows clear localization of PTMs. The MELD protocol as presented here and tested on several pure proteins digested in solution, certainly improves the general "bottom-up" strategy applied for highly confident protein identification and would allow better protein characterization, even for those having PTMs. In addition, in our analysis, each fragment position of the entire protein sequence was evidenced either by a "y" or a "b" fragment ion. This high and confident amount of information enables extensive de novo sequencing using PEAKS software, followed by application of our “sequence assembly” algorithm. The first version of our assembling tool on MELD experimental data generated long sequence tags, up to 90 amino acids long. [less ▲]

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See detailMultiple analyses of microbial communities applied to the gut of the wood-feeding termite Reticulitermes flavipes fed on artificial diets
Tarayre, Cédric ULg; Bauwens, Julien ULg; Mattéotti, Christel et al

in Symbiosis (2015)

The purpose of this work was the observation of the differences between the microbial communities living in the gut of the termite Reticulitermes flavipes fed on different diets. The termites were fed on ... [more ▼]

The purpose of this work was the observation of the differences between the microbial communities living in the gut of the termite Reticulitermes flavipes fed on different diets. The termites were fed on poplar wood (original diet) and artificial diets consisting of crystalline cellulose (with and without lignin), α-cellulose (with and without lignin) and xylan. The termites were then dissected and the protist communities were analyzed through microscopy, leading to the conclusion that protist species are strongly influenced by diets. BIOLOG ECO Microplates® were used to assess the metabolic properties of the different types of consortia, highlighting strong differences on the basis of principal component analysis and calculation of similarity rates. The microorganisms were cultivated in liquid media corresponding to the artificial diets before being characterized through a metagenetic analysis of gut microbiota (16S ribosomal DNA). This analysis identified several phyla: Acidobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Fibrobacteres, Firmicutes, Nitrospirae, OP9, Planctomycetes, Proteobacteria, Spirochaetes, TM6, Tenericutes, Verrucomicrobia and WS3. The OTUs were also determined and confirmed the abundance of Proteobacteria, Bacteroidetes, Firmicutes and Verrucomicrobia. It was possible to isolate several strains from the liquid media, and one bacterium and several fungi were found to produce interesting enzymatic activities. The bacterium Chryseobacterium sp. XAvLW produced α-amylase, β-glucosidase, endo-1,4-β-D-glucanase, endo-1,4-β-D-xylanase and filter paper-cellulase, while the fungi Sarocladium kiliense CTGxxyl and Trichoderma virens CTGxAviL generated the same activities added with endo-1,3-β-D-glucanase. [less ▲]

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See detailStudy of N-linked glycosylation in different life stages of the red flour beetle (Tribolium castaneum)
Smargiasso, Nicolas ULg; Walski, Tomasz; Van Damme, Els et al

Poster (2015, June)

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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 detailProteome variations in Macrobrachium rosenbergii exposed to chlordecone: a gel-free proteomic approach
Lafontaine, Anne ULg; De Pauw, Edwin ULg; Forget-Leray, Joelle et al

Poster (2015, May 06)

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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 detailMetabolomic, proteomic and preclinical imaging of patient-derived tumor xenografts for improving treatment of liver metastases patients
Perez Palacios, A; Blomme, A; Boutry, S et al

in Acta Gastro-Enterologica Belgica (2015, March), 78(1), 134

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See detailComparison of early stages of colorectal cancer by label free proteomics
QUESADA CALVO, Florence ULg; MEUWIS, Marie-Alice ULg; Bertrand, Virginie ULg et al

in Acta Gastroenterologica (2015, February 27)

Introduction and objectives: Colorectal cancer (CRC) is the second most frequent cancer in women and the third in men. Identification of the mechanisms of progression in these early CRC stages is ... [more ▼]

Introduction and objectives: Colorectal cancer (CRC) is the second most frequent cancer in women and the third in men. Identification of the mechanisms of progression in these early CRC stages is important to develop new diagnostic and therapeutic tools. Formalin-Fixed Paraffin-Embedded (FFPE) specimens are materials that enable proteomic clinical research. Hence our aim was to address the comparison of FFPE samples from early CRC stages patients using shotgun proteomic analysis. Methods: We performed a retrospective study on 36 CRC tissue samples (pT1N0M0, n=16 and pT2N0M0, n=20) compared together and with 40 control tissue samples (20 patients with diverticulitis, using paired inflamed (DI) and healthy tissue (DH)). Each tissue slice was macrodissected to enrich in epithelial cells. We used FFPE-FASP kit (Expedeon) for sample preparation and protein digests were analyzed using 2D-nanoAquity UPLC separation online with Q-Tof Synapt HDMSTM G2 using ion mobility as additional separation. We performed protein identification and differential analysis using Progenesis QI for proteomics (Nonlinear Dynamics). Results and discussion: We selected 149 proteins differentially distributed between T1 and T2 CRC stages which were not significantly different between CRC and DH or DI. Only 30 proteins were significantly more abundant in T1 versus T2 and 119 were distributed inversely, with a minimum fold ratio of 2. Among those, ATP synthase subunit beta, Aspartate-tRNA ligase, Haptoglobin and Kininogen were identified. . Moreover, we validated Kininogen and 3 others proteins with a significant differential distribution between pT1N0M0 and pT2N0M0 stages by immunohistochemistry. Conclusion: This FFPE retrospective study comparing T1 and T2 CRC highlighted proteins already previously identified as potential CRC biomarkers. These proteins may reflect important early changes in cancer development and may help understanding early tumor progression. [less ▲]

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See detailPotential Proteomic Biomarkers Associated with Mucosal Healing and Relapse Prediction after IFX Withdrawals in Crohn’s Disease
MEUWIS, Marie-Alice ULg; QUESADA CALVO, Florence ULg; Baiwir, Dominique ULg et al

in Acta gastroenterologica (2015, February 26)

Introduction and objectives: In Crohn's disease (CD), there is a discrepancy between clinical activity of the disease (symptoms) and intestinal healing. However absence of tissue healing is associated ... [more ▼]

Introduction and objectives: In Crohn's disease (CD), there is a discrepancy between clinical activity of the disease (symptoms) and intestinal healing. However absence of tissue healing is associated with the risk of relapse and tissue damage progression. Endoscopy is costly and invasive. Hence biomarkers correlating with intestinal healing could improve disease management and potentially decrease the number of endoscopy when patients are in clinical remission. Aim: We aimed to identify potential biomarkers associated to CD mucosal healing and relapse after IFX withdrawals by a shotgun label-free proteomic study. Methods: We used the STORI1 clinical trial cohort (n=103) aiming at identifying markers associated to relapse prediction after Infliximab treatment withdrawals. We used serum samples of patients in clinical remission (at base line). We grouped these according to the degree of intestinal healing seen at endoscopy or according to relapse occurrence during the 28 month follow-up and composed pooled samples. We performed depletion of the 20 most abundant plasma proteins on each serum pools and ran a proteomic label-free differential analysis using 2D-nanoUPLC-MSE HDMS Synapt G2 for data acquisition. We performed different statistical analysis. Moreover, a Gene Ontology annotation was also performed for the potential biomarkers highlighted. Results and Discussion: We identified analysing these depleted serum pools 430 different proteins and 188 proteins common to all samples. Among these, 40 were found with a significant differential abundance in the groups compared. We selected some among the most significant one (ratio>1.3) or being by nature consistent with the context of this study (sample origin and clinical question addressed). For example, the C-reactive protein (CRP) was found with a significant Ratio of 2 between Relapsers and Non Relapsers. The other potential biomarkers associated to mucosal healing or to relapse prediction, were selected for further validation by Western Blot analysis (WB), routine laboratory tests and also by a Mass Spectrometry based technology: multiplexed selected reaction monitoring (SRM). Multiplexed SRM will enable quantitative analysis of these candidates in each individual patient as well as WB tests. Conclusions: This research strategy and the validation results on potential biomarkers associated to mucosal healing or relapse after treatment cessation in this cohort of CD patients, as well as tests done on other CD patients, might provide new opportunities for patient follow-up test developments. The next step is to perform SRM validation on the STORI cohort and design signatures using these potential biomarkers SRM data for prognosis power evaluation. 1. Louis E, Mary JY, Vernier-Massouille G, et al. Maintenance of remission among patients with Crohn's disease on antimetabolite therapy after infliximab therapy is stopped. Gastroenterology 2012;142:63-70 e5; quiz e31. [less ▲]

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