References of "De Pauw, Edwin"
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See detailA Venomics approach coupled to high-throughput toxin production strategies identifies the first venom-derived melanocortin receptor agonists.
Reynaud, Steve; Ciolek, Justyna; Degueldre, Michel ULiege et al

in Journal of medicinal chemistry (2020)

Animal venoms are rich in hundreds of toxins with extraordinary biological activities. Their exploitation is difficult due to their complexity and the small quantities of venom available from most ... [more ▼]

Animal venoms are rich in hundreds of toxins with extraordinary biological activities. Their exploitation is difficult due to their complexity and the small quantities of venom available from most venomous species. We developed a Venomics approach combining transcriptomic and proteomic characterization of 191 species and identified 20,206 venom toxin sequences. Two complementary production strategies based on solid-phase synthesis and recombinant expression in E. coli generated a physical bank of 3,597 toxins. Screened on hMC4R, this bank gave an incredible hit rate of 8%. Here, we focus on two novel toxins: N-TRTX-Preg1a, exhibiting an inhibitory cystine knot (ICK) motif, and N-BUTX-Ptr1a, a short scorpion-CSαβ structure. Neither N-TRTX-Preg1a nor N-BUTX-Ptr1a affect ion channels, the known targets of their toxin scaffolds, but bind to four melanocortin receptors with low micromolar affinities and activate the hMC1R/Gs pathway. Phylogenetically, these two toxins form new groups within their respective families and represent novel hMC1R agonists, structurally unrelated to the natural agonists. [less ▲]

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See detailUnravelling chemical mechanisms in microbial interactions by combining thin layer chromatography, ion mobility and MALDI imaging mass spectrometry
Mc Cann, Andréa ULiege; Kune, Christopher ULiege; La Rocca, Raphaël ULiege et al

Conference (2020, June 01)

Mass spectrometry (MS) is a method of choice in microbiology for untargeted detection and identification of bioactive compounds. Mass Spectrometry Imaging (MSI) has led to a growing interest in the in ... [more ▼]

Mass spectrometry (MS) is a method of choice in microbiology for untargeted detection and identification of bioactive compounds. Mass Spectrometry Imaging (MSI) has led to a growing interest in the in situ study of biomolecules produced when microorganisms interact with each other. However, in situ identification is still time-consuming and challenging due to the large chemical diversity contained in each pixel of the samples, resulting in very complex average MS spectra. Here, we propose to exploit the power of combining Kendrick Mass Defect (KMD) analysis and collision cross section (CCS) values using mobility for the characterization of families of related compounds in MSI. The identification of compounds was supported by rapid thin layer chromatography (TLC) separation coupled to MALDI-MS/MS detection. Bacillus velezensis GA1 and Pseudomonas sp. CMR12a were inoculated at different distances (0.5, 1 and 2 cm) on a semi-solid agar-based medium and incubated at 30°C. Regions of interest were cut directly from the Petri dish and transferred to the target ITO plate. This assembly was placed in a vacuum desiccator until completely dry and covered with HCCA matrix (Sunchrom sprayer). Ion mobility in imaging mode was performed using the timsTOF fleX (Bruker Daltonics, Bremen, Germany). TLC separation of ROI extracts is analyzed by MALDI-MS/MS imaging on the rapifleX instrument (Bruker Daltonics) for rapid screening of compounds and on the solariX instrument (Bruker Daltonics) for exact mass and isotopic distribution. The data were processed and integrated with in-house software. The coupling of MALDI-MSI with ion mobility separation brings additional structural features/information. It allows data to be filtered according to a range of CCS values and it improves the confidence level for the identification of detected analytes, in addition to exact mass determination. A relationship between CCS and mass was also performed. In combination with KMD analysis, various families of compounds, such as lipids or lipopeptides could be automatically detected and identified. Through this workflow, the comparison of the three different culture conditions was greatly simplified and highlighted the changes that occur in the metabolism of the bacteria. In particular, we were able to observe a variation within the lipid composition of Pseudomonas sp. CMR12a as a function of distance from the Bacillus velezensis GA1 colony. Finally, TLC separation was successfully optimized for lipopeptides and lipids and validated the identification of detected compounds by simplifying the spectra and allowing image analysis. TLC also enables high throughput, in part due to the parallel imaging of up to 6 traces on a single TLC run. TLC plate matrix coating strategies were compared to optimize the MALDI MS signal. This workflow will also be applied to time-lapse (or time-dependent) experiments, to monitor the bioactive compounds production and migration as a function of the co-culture interaction time. [less ▲]

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See detailSolvent Adducts in Ion Mobility Spectrometry: Toward an Alternative Reaction Probe for Thermometer Ions
Morsa, Denis ULiege; Hanozin, Emeline ULiege; Eppe, Gauthier ULiege et al

in Journal of the American Society for Mass Spectrometry (2020)

The fragmentation of benzylpyridinium "thermometer" ions is widely used to quantify the energetics of ions studied by mass spectrometry and other hyphenated techniques such as ion mobility. The reaction ... [more ▼]

The fragmentation of benzylpyridinium "thermometer" ions is widely used to quantify the energetics of ions studied by mass spectrometry and other hyphenated techniques such as ion mobility. The reaction pathway leads to a benzylium cation with the release of a neutral pyridine. Using trapped ion mobility spectrometry, we noticed that the addition of acetonitrile, present in the electrosprayed solvent mixture, could occur on some electrophilic benzylium cations. This process results in the formation of adducts and in the appearance of a supplementary mobility peak. We here demonstrate that the addition takes place both in the electrospray source and inside the mobility analyzer, thereby evidencing possible outflow of solvent vapors downstream the instrument. By further characterizing the initial kinetics and the resulting equilibrium linked with the addition reaction, we presently discuss these as alternative probes to calibrate ion temperature in the framework of ion mobility. [less ▲]

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See detailHuman Liver-Derived Extracellular Matrix for the Culture of Distinct Human Primary Liver Cells
Alevra Sarika, Niki; Payen, Valéry; Fleron, Maximilien ULiege et al

in Cells (2020), 9(6)

The lack of robust methods to preserve, purify and in vitro maintain the phenotype of the human liver’s highly specialized parenchymal and non-parenchymal cell types importantly hampers their exploitation ... [more ▼]

The lack of robust methods to preserve, purify and in vitro maintain the phenotype of the human liver’s highly specialized parenchymal and non-parenchymal cell types importantly hampers their exploitation for the development of research and clinical applications. There is in this regard a growing interest in the use of tissue-specific extracellular matrix (ECM) to provide cells with an in vitro environment that more closely resembles that of the native tissue. In the present study, we have developed a method that allows for the isolation and downstream application of the human liver’s main cell types from cryopreserved material. We also isolated and solubilized human liver ECM (HL-ECM), analyzed its peptidomic and proteomic composition by mass spectrometry and evaluated its interest for the culture of distinct primary human liver cells. Our analysis of the HL-ECM revealed proteomic diversity, type 1 collagen abundance and partial loss of integrity following solubilization. Solubilized HL-ECM was evaluated either as a coating or as a medium supplement for the culture of human primary hepatocytes, hepatic stellate cells and liver sinusoidal endothelial cells. Whereas the solubilized HL-ECM was suitable for cell culture, its impact on the phenotype and/or functionality of the human liver cells was limited. Our study provides a first detailed characterization of solubilized HL-ECM and a first report of its influence on the culture of distinct human primary liver cells. [less ▲]

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See detailIncreased Endoplasmic Reticulum stress specific chaperones characterise CD fibrosis epithelium tissues and participates to in vitro induction of intestinal fibroblasts differentiation
Vieujean, Sophie ULiege; Hu, Shurong; Bequet, Emeline ULiege et al

Poster (2020, March 05)

Background: Intestinal fibrosis is a complication of Crohn’s disease (CD) characterized by myofibroblasts and extracellular matrix accumulation within the submucosa and smooth muscles, leading to bowel ... [more ▼]

Background: Intestinal fibrosis is a complication of Crohn’s disease (CD) characterized by myofibroblasts and extracellular matrix accumulation within the submucosa and smooth muscles, leading to bowel strictures. No medical treatment exists to treat or reverse intestinal fibrosis leading often to surgical resection. The potential role of intestinal epithelium in the fibrotic process remains poorly defined. Methods: We performed a pilot study on ileal fibrostricturing CD surgical samples (n=5), comparing the proteome of surface epithelium isolated by laser capture microdissection in normal and fibrotic zones. Confirmation of the specific protein increases was obtained by immunohistochemistry in colonic and ileal samples of CD (n=44) compared to healthy subjects (n=40), as well as in intestinal epithelial cell line under induced Endoplasmic Reticulum (ER) stress. A model of fibroblast to myofibroblast differentiation induction was also challenged using preconditioned media of intestinal epithelial cells after a pulsed ER stress. Results: Label free proteomics revealed high ER stress in the epithelium surrounding fibrotic bowel wall, involving Anterior gradient protein 2 homolog (AGR2) and 78kDA glucose regulated protein (BiP). Confirmation of both proteins increase was obtained by immunohistochemistry. ER stress induction in intestinal epithelial cells was associated with an intracellular increase of AGR2, BiP and ER stress markers as sXPB1 and CHOP. AGR2 was also detected in the culture medium of these epithelial cells and myofibroblast differentiation was obtained using this culture medium. Conclusions: The increase of ER stress proteins observed in fibrostenosing tissues together with These preliminary evidences of fibroblast to myofibrobast differentiation obtained by paracrine action of intestinal epithelial cell preconditioned to ER stress induction, suggest a role of epithelial ER stress in Crohn’s disease intestinal fibrosis. [less ▲]

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See detailSLC12A2 as a potential histological marker of ulcerative colitis associated colorectal dysplasia
Merli, Angela-Maria ULiege; Vieujean, Sophie ULiege; Massot, Charlotte et al

Conference (2020, March 04)

Introduction: Patients suffering from ulcerative colitis (UC) are at increased risk of developing dysplasia (DAI) and colorectal cancer (CAC). Differentiating DAI from inflammation remains difficult for ... [more ▼]

Introduction: Patients suffering from ulcerative colitis (UC) are at increased risk of developing dysplasia (DAI) and colorectal cancer (CAC). Differentiating DAI from inflammation remains difficult for both endoscopists and anatomopathologists due to macro and microscopic features shared by these lesions. Aim: The aim of our work was to confirm, by histological evaluation, a potential proteomic biomarker discriminating early DAI lesions from chronic inflamed and normal tissues in UC. Methods: We included 15 paired tissues from UC patients (n=5) presenting low-grade DAI. Epithelial cells were isolated by laser capture microdissection and analyzed by label-free proteomics. We selected one protein differentially distributed between DAI, inflamed (I) and normal (N) tissues for confirmation by immunochemistry (IHC). IHC characterization was performed using both the staining intensity score (0 to 4) and the staining pattern: “gradient” (staining intensity increasing from the epithelium lumen to the bottom of the crypts) or “no gradient” (homogenous staining). UC patients with DAI (n=28), dysplastic lesion in non-inflammatory colon (DSp) (n=9), CAC (n=14) and at high risk of CAC (>10 years of UC duration) but free of dysplasia or cancer (n=23) were included. We further studied this potential marker tissue distribution in the mouse model of CAC (AOM/DSS treated mice) to trace its presentation at different evolution stages and assessed low (n=51), high-grade DAI (n=35) and CAC (n=38), as well as relevant paired control tissues. This potential tissue marker was finally evaluated in sporadic precancerous colorectal lesions of UC-free patients with low (n=19) and highgrade (n=16) adenomas and cancerous lesions (CRC): pT1 to pT4 (n=82) and compared to paired normal tissues when available. Results: Proteomics identified 1070 proteins among which 19 showed a differential distribution between DAI and I or N. The sodium chloride co-transporter SLC12A2 was only identified in DAI. SLC12A2 IHC “no gradient” staining pattern was associated to DAI and DSp compared to I or N (with p <0.0001 and 0.0002 respectively). The IHC score was also higher for DAI, DSp and CAC compared to paired I and N (p<0.0001 and 0.0084 respectively). These results were confirmed from low-grade dysplasia to more advanced lesions in the AOM/DSS mice model. The “no gradient” pattern was also significantly associated to low and high-grade adenomas, and CRC of UC-free patients compared to normal control tissues. The sensitivity and specificity of SLC12A2 histological pattern reached 89% and 95% for DAI versusI; 90% and 93% for CAC and/or DAI versus I. In addition, the sensitivity and specificity reached 99% and 87% for all precancerous and cancerous lesions (DAI, DSp, CAC and CRC) versus N and I (including also non-progressing UC patients). Conclusions: A specific histological pattern for SLC12A2 is associated to precancerous and cancerous colorectal lesions, and is able to be discriminate these lesions from inflammation and normal tissue in UC. The continuous upregulation of SLC12A2 in advanced colorectal lesionsin the CAC mice model also suggests a role of this protein in the pathophysiology of inflammation-associated colon neoplasia. [less ▲]

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See detailIncreased Endoplasmic Reticulum stress specific chaperones characterise CD fibrosis epithelium tissues and participate to in vitro induction of intestinal fibroblasts differentiation
Vieujean, Sophie ULiege; Hu, Shurong; Bequet, Emeline et al

Poster (2020, February 14)

Background: Intestinal fibrosis is a complication of Crohn’s disease (CD) characterized by myofibroblasts and extracellular matrix accumulation within the submucosa and smooth muscles, leading to bowel ... [more ▼]

Background: Intestinal fibrosis is a complication of Crohn’s disease (CD) characterized by myofibroblasts and extracellular matrix accumulation within the submucosa and smooth muscles, leading to bowel strictures. No medical treatment exists to treat or reverse intestinal fibrosis leading often to surgical resection. The potential role of intestinal epithelium in the fibrotic process remains poorly defined. Methods: We performed a pilot study on ileal fibrostricturing CD surgical samples (n=5), comparing the proteome of surface epithelium isolated by laser capture microdissection in normal and fibrotic zones. Confirmation of the specific protein increases was obtained by immunohistochemistry in colonic and ileal samples of CD (n=44) compared to healthy subjects (n=40), as well as in intestinal epithelial cell line under induced Endoplasmic Reticulum (ER) stress. A model of fibroblast to myofibroblast differentiation induction was also challenged using preconditioned media of intestinal epithelial cells after a pulsed ER stress. Results: Label free proteomics revealed high ER stress in the epithelium surrounding fibrotic bowel wall, involving Anterior gradient protein 2 homolog (AGR2) and 78kDA glucose regulated protein (BiP). Confirmation of both proteins increase was obtained by immunohistochemistry. ER stress induction in intestinal epithelial cells was associated with an intracellular increase of AGR2, BiP and ER stress markers as sXPB1 and CHOP. AGR2 was also detected in the culture medium of these epithelial cells and myofibroblast differentiation was obtained using this culture medium. Conclusions: The increase of ER stress proteins observed in fibrostenosing tissues together with These preliminary evidences of fibroblast to myofibrobast differentiation obtained by paracrine action of intestinal epithelial cell preconditioned to ER stress induction, suggest a role of epithelial ER stress in Crohn’s disease intestinal fibrosis. [less ▲]

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See detailData treatment for mass spectrometry
La Rocca, Raphaël ULiege; Quinton, Loïc ULiege; De Pauw, Edwin ULiege

Conference (2020, January 28)

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See detailData processing and interpretation of mass spectrometry imaging using high resolving mass power
Mc Cann, Andréa ULiege; La Rocca, Raphaël ULiege; Kune, Christopher ULiege et al

Poster (2020, January 28)

1 Introduction Mass Spectrometry Imaging (MSI) is a powerful analytical tool allowing untargeted investigation of spatial distribution of molecular species in a large variety of samples, by recording 2D ... [more ▼]

1 Introduction Mass Spectrometry Imaging (MSI) is a powerful analytical tool allowing untargeted investigation of spatial distribution of molecular species in a large variety of samples, by recording 2D distribution of all the detectable compounds in the sample. Over the years, the application of MSI has become increasingly diverse, from bacteria-bacteria interaction understanding to biomarker discovery. To this end, it is necessary to combine high spectral resolution with efficient data processing, in order to maximise the extraction of the information contained in large datasets. Moreover, in addition to deal with thousands of features, pixel-to-pixel mass shift must be taken into account before applying any data-mining tool. For that reason, we propose the combination of a post-acquisition label-free recalibration method and an algorithm to cluster features based on their Kendrick mass defect (KMD) in MSI. 2 Theory KMD consists in a change of basis from the IUPAC mass scale to a Kendrick mass based on the nominal mass of a defined Kendrick mass unit, such as CH2 (here, nominal mass is set at 14 a.u.)1. The KMD is obtained by subtracting the nominal Kendrick mass to the exact Kendrick mass. This mathematical transformation enables to map the detected molecules in mass spectrometry based on their chemical composition. 3 Material and methods MALDIFT-ICR-MS (SolariX XR 9.4T) images were first converted into imzML open format. Each spectrum from the imzML file obtained for an MSI are individually peak picked. Then, our algorithm creates, for each pixel, a linear model linking m/z error and m/z. The model is creating by finding database hit with similar mass shift. Finally, a new imzML file is created by recalibrating each pixel with their estimated linear model. Then, we developed a software to filter features (m/z peaks) based on their KMD from an imzML file. This software calculates first a KMD for each m/z values detected in the mean mass spectrum of the image. The MSI data is then filtered based on KMD value to conserve only ions whose KMD is included in the user-defined KMD range. The reduced ion lists were then divided into different compounds families, based on the repetition of the KMD. Finally, an image is generated for each compounds family. Thereby reducing the number of features of an image2. 4 Results and discussion The KMD filtering method applied on a mouse brain tissue analysis by MSI appears to be biologically relevant since it enabled to map in a single step all members of important families of biomolecules. Among the detected families of biomolecules, lipids shows different distributions and localisations. Some of these lipids belonged to the glycerophosphocholines (GPCs), the hexosylceramides (HexCers), lysophoshocholins (LPCs) and the sphingomyelins (SMs) class. Moreover, The KMD analysis highlighted that some of the detected GPCs on the brain tissue sections from mouse were differentially co-localized, depending on their unsaturation degree. All these results suggested that the KMD could be considered instead of the mass-to-charge (m/z) values classically used for the analysis of images by MSI. The use of our in-house developed software for KMD analysis enables an automated, faster and efficient data analysis of MSI images. 5 Conclusion The combination of recalibration before using Kendrick mass defect mass filter is an essential step to be able to interpret the image reconstruction of chemically-related compounds. This methods speed up the identification process and facilitates the data analysis without losing data information. [less ▲]

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See detailEffective Temperature and Structural Rearrangement in Trapped Ion Mobility Spectrometry
Morsa, Denis ULiege; Hanozin, Emeline ULiege; Eppe, Gauthier ULiege et al

in Analytical Chemistry (2020)

Modern ion mobility instrumentations are typically operated above the low field limit, which may activate the ions and cause structural rearrangement or fragmentation during the analysis. Here, we ... [more ▼]

Modern ion mobility instrumentations are typically operated above the low field limit, which may activate the ions and cause structural rearrangement or fragmentation during the analysis. Here, we quantitatively assessed the internal heating experienced by ions during trapped ion mobility spectrometry (TIMS) experiments. To this end, the fragmentation yields of fragile benzylpyridinium “thermometer” ions were monitored during both the accumulation and analysis steps inside the TIMS tunnel. The corresponding fragmentation rate constants were translated into a vibrational effective temperature Teff,vib. Our results demonstrate significant fragmentation upstream and inside the TIMS tunnel that corresponds to Teff,vib ≈ 510 K during both the accumulation and analysis steps. Broadening our scope to cytochrome c and lysozyme, we showed that although compact “native” folds can be preserved, the collision cross section distributions are highly sensitive to the transmission voltages and the analysis timescale. Our results are discussed with regard to Teff,vib data previously acquired on traveling-wave (TWIMS) ion mobility in the context of native mass spectrometry and conformational landscape exploration. [less ▲]

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See detailIntertwined metal homeostasis, oxidative and biotic stress responses in the Arabidopsis frd3 mutant
Scheepers, Maxime ULiege; Spielmann, Julien ULiege; Boulanger, Madeleine ULiege et al

in Plant Journal (2020), 102

FRD3 (FERRIC REDUCTASE DEFECTIVE 3) plays a major role in iron (Fe) and zinc (Zn) homeostasis in Arabidopsis. It transports citrate, which enables metal distribution in the plant. A frd3 mutant is dwarf ... [more ▼]

FRD3 (FERRIC REDUCTASE DEFECTIVE 3) plays a major role in iron (Fe) and zinc (Zn) homeostasis in Arabidopsis. It transports citrate, which enables metal distribution in the plant. A frd3 mutant is dwarf and chlorotic and displays constitutive Fe deficiency response and strongly altered metal distribution in tissues. Here, we have examined the interaction between Fe and Zn homeostasis in a frd3 mutant exposed to varying Zn supply. Detailed phenotyping using transcriptomic, ionomic, histochemical and spectroscopic approaches revealed the full complexity of the frd3 mutant phenotype, which resulted from altered transition metal homeostasis, manganese toxicity, oxidative and biotic stress responses. The cell wall played a key role in these processes, as a site for Fe and hydrogen peroxide accumulation, and displayed modified structure in the mutant. Finally, we showed that Zn excess interfered with these mechanisms and partially restored root growth of the mutant, without reverting the Fe deficiency response. In conclusion, the frd3 mutant molecular phenotype is more complex than previously described, and illustrates how the response to metal imbalance depends on multiple signaling pathways. [less ▲]

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See detailCharacterization of New Fengycin Cyclic Lipopeptide Variants Produced by Bacillus amyloliquefaciens (ET) Originating from a Salt Lake of Eastern Algeria.
Ait Kaki, Asma ULiege; Smargiasso, Nicolas ULiege; Ongena, Marc ULiege et al

in Current Microbiology (2020)

Fengycin antibiotic displays a strong antifungal activity and inhibits the growth of a wide range of plant pathogens especially filamentous fungi. The main objective of the present study is to ... [more ▼]

Fengycin antibiotic displays a strong antifungal activity and inhibits the growth of a wide range of plant pathogens especially filamentous fungi. The main objective of the present study is to characterize fengycin variants produced by B. amyloliquefaciens strain (ET). LC-MS analysis of fengycin extracts has shown several molecular ion peaks corresponding to conventional fengycin homologues (MH + : m/z 1463.9; 1491.9; 1506) and some new ones (MH + : m/z 1433; 1447; 1461; and 1477). Further characterization of these precursor ions was carried out by LC-MS.MS analysis. Reporter fragment ions were observed (named A and B), they correspond to the cleavage of Orn2-Tyr3 (A), Glu1-Orn2 (B), and used for identifying fengycin variants. The reporter fragment couple ions [A/B] at [m/z 966.5/1080.5] and [m/z 994.4 /1108.5] represent fengycin A and B, respectively. The diagnostic ions at ([m/z 980/1094]) may correspond to fengycin C3, D, S or B2. Interestingly, unknown diagnostic product ions at [m/z 951/1065] and [m/z 979/1093] were detected for the first time in this study which prove that they correspond to new fengycin variants, named fengycin X and fengycin Y, respectively. The fengycin X results from a substitution of the glutamine amino acid (Q), at position 8 of the fengycin A peptide part, by an isoleucine (I) or a leucine (L) residue. This mutation should be the same in fengycin Y but compared to fengycin B. [less ▲]

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See detailCombination of Capillary Zone Electrophoresis-Mass Spectrometry, Ion Mobility-Mass Spectrometry, and Theoretical Calculations for Cysteine Connectivity Identification in Peptides Bearing Two Intramolecular Disulfide Bonds
Delvaux, Cédric ULiege; Massonnet, Philippe ULiege; Kune, Christopher ULiege et al

in Analytical Chemistry (2019)

Disulfide bonds between cysteine residues are commonly involved in the stability of numerous peptides and proteins and are crucial for providing biological activities. In such peptides, the appropriate ... [more ▼]

Disulfide bonds between cysteine residues are commonly involved in the stability of numerous peptides and proteins and are crucial for providing biological activities. In such peptides, the appropriate cysteine connectivity ensures the proper conformation allowing an efficient binding to their molecular targets. Disulfide bond connectivity characterization is still challenging and is a critical issue in the analysis of structured peptides/proteins targeting pharmaceutical or pharmacological utilizations. This study describes the development of new and fast gas-phase and in-solution electrophoretic methods coupled to mass spectrometry to characterize the cysteine connectivity of disulfide bonds. For this purpose, disulfide isomers of three peptides bearing two intramolecular disulfide bonds but different cysteine connectivity have been investigated. Capillary zone electrophoresis and ion mobility both coupled to mass spectrometry were used to perform the separation in both aqueous and gas phases, respectively. The separation efficiency of each technique has been critically evaluated and compared. Finally, theoretical calculations were performed to support and explain the experimental data based on the predicted physicochemical properties of the different peptides. [less ▲]

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See detailRapid identification of chemically-related compounds produced by bacteria by Kendrick mass defect filtering applied to high resolution imaging mass spectrometry
Mc Cann, Andréa ULiege; Kune, Christopher ULiege; La Rocca, Raphaël ULiege et al

Poster (2019, October 30)

Introduction Over the last years, lots of progress have been done in the development of mass spectrometry imaging, making the technique more and more accessible for various applications, such as ... [more ▼]

Introduction Over the last years, lots of progress have been done in the development of mass spectrometry imaging, making the technique more and more accessible for various applications, such as biomarkers discovery or bioactive compounds identification. However, the progresses made in terms of spatial and instrumental resolution has for consequences the dramatic increase of dataset size, shifting the burden from data production to data analysis and compounds identification. We propose here to use a semi-targeted method based on Kendrick mass defect (KMD) analysis to immediately identify the chemistry-related compounds in mass spectrometry imaging applied to microbiology samples. Thanks to a software developed in-house, we are now able to better understand the bacteria-bacteria interactions. Materials and methods Bacteria strains were inoculated on a semi-solid agar-based medium and incubated at 30°C. Region of interest was cut directly from the petri dish and transferred to the target ITO plate, previously covered with double sided conductive carbon tape. This assembly was then put in a vacuum desiccator until complete drying (overnight), and covered with HCCA matrix. Mass spectrometry images were obtained using a FT-ICR mass spectrometer (9.4T SolariX, Bruker Daltonics, Bremen, Germany). Data analysis was performed on an in-house software. Results & Discussion KMD filtering for mass spectrometry imaging enabled the rapid identification of chemically-related compounds such as lipopeptides or lipids, independently of the signal intensity and without the need of an extensive database search. For each detected family of compounds, an image was generated, enabling to link the chemically-related compounds identified with their spatial localization. The analysis of the bacteria-bacteria interaction was greatly simplified by our in-house software, and we were able to have a better understanding of the underlying chemical mechanisms involved. [less ▲]

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See detailAu@Ag nanoparticles as joint substrate for SERS and SALDI-MS multimodal imaging of cancerous tissues
Verdin, Alexandre ULiege; Malherbe, Cédric ULiege; Bertrand, Virginie ULiege et al

Conference (2019, October 28)

Metallic nanoparticles (NPs) are used in mass spectrometry imaging (MSI) as substrate for desorption/ionisation of analytes (SALDI-MS). They provide untargeted information about the small molecules ... [more ▼]

Metallic nanoparticles (NPs) are used in mass spectrometry imaging (MSI) as substrate for desorption/ionisation of analytes (SALDI-MS). They provide untargeted information about the small molecules content of tumors which can unreveal its subtype, or predicts its response to therapy. NPs can be used in Raman spectroscopy to design SERS nanoprobes (NP covered by a Raman-active molecule, and functionalized by a targeting molecule): combined with Raman micro-imaging, they can localize and/or quantify a biological target such as a cellular membrane receptor to monitor its expression in tissues (linked to tumor evolution and survival). Developing a multimodal imaging methodology based on SERS nanoprobes and untargeted SALDI-MS, and using metallic NPs as joint substrate, could provide local information both on the receptor status and on the tumor chemical composition. We synthesized gold@silver core@shell NPs (Au@Ag) which enable a fine optical properties tuning by controlling the silver shell thickness, leading to simultaneous high UV absorption needed for SALDI and high Raman enhancement factors in the visible range needed for SERS. We used the NPs to synthesize Folate-targeted nanoprobes in the SERS imaging approach to evaluate the expression of Folate Receptor alpha (FRα), an indicator of patient survival, in cancerous tissues. The same bare NPs were used in SALDI-MS imaging to investigate the small molecules content of adjacent tissue slides. SERS imaging. Following the SERS intensity on the tissue slide allowed us to highlight higher SERS intensities in the tumor region from the ovary samples. We could perform imaging experiments at the tissue scale down to the cellular scale owing to the high spatial resolution offered by Raman Spectroscopy. SALDI-MS imaging. Discriminative m/z analysis was performed and ROC curves were calculated to highlight m/z values that discriminate the tumor and healthy areas. 11 specific ions are more intense in the tumor than in the healthy part and 4 ions were found more intense in the healthy than in the tumor area. By combining the two techniques, complementary information about ovarian tumors could be obtained. [less ▲]

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See detailA Mass Spectrometry Imaging approach for the fundamental study of Surface-Assisted Laser Desorption/Ionisation mechanisms
Müller, Wendy ULiege; Malherbe, Cédric ULiege; De Pauw, Edwin ULiege et al

Poster (2019, October)

Surface-Assisted Laser Desorption/Ionisation Mass Spectrometry (SALDI-MS), which employs inorganic nanosubstrates to promote desorption/ionisation of analytes, is a promising technique for the analysis of ... [more ▼]

Surface-Assisted Laser Desorption/Ionisation Mass Spectrometry (SALDI-MS), which employs inorganic nanosubstrates to promote desorption/ionisation of analytes, is a promising technique for the analysis of small molecules. Indeed, SALDI-MS does not require any organic matrix, reducing the desorption/ionisation of interfering organic ions (in low m/z region) and requiring no co-crystallisation, which simplifies the sample preparation. However, while most papers are focused on the development of new nanosubstrates, the fundamental aspects of SALDI-MS have not been studied in detail yet. Indeed, SALDI-MS involves complicated processes, which make the understanding of SALDI-MS desorption/ionisation mechanisms less straightforward. Our study aims at investigating the SALDI-MS processes for various nanosubstrates. We observed that the ionisation of the metallic nanosubstrates occurs during SALDI-MS experiments and that each nanomaterial is increasingly ionised from a different energy threshold. Moreover, the desorption/ionisation and fragmentation of model molecules (benzylpyridinium salts) seem to be correlated with the appearance of the metallic nanosubstrates ions, demonstrating that the nanosubstrate destruction/restructuring may be involved in the desorption/ionisation processes. [less ▲]

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See detailImpact of penicillin stress on peptidoglycan peptides recycling in Bacillus spp.
Dauvin, Marjorie ULiege; Delvaux, Cédric ULiege; Joris, Bernard ULiege et al

Poster (2019, September 25)

In Gram-positive bacteria, the recycling of peptides resulting from peptidoglycan (PG) degradation occurring during cell growth or penicillin stress is not yet known in details. Furthermore, in B ... [more ▼]

In Gram-positive bacteria, the recycling of peptides resulting from peptidoglycan (PG) degradation occurring during cell growth or penicillin stress is not yet known in details. Furthermore, in B. licheniformis, cytoplasmic PG peptides accumulation have been shown to be involved in penicillin resistance1. However, the mechanism that regulates this accumulation remains unknown. To shed light upon this regulation pathway, we deleted in B. subtilis 168 and B. licheniformis MW3 ykfABCD genes postulated to be involved in PG peptide recycling1. For both strains we have quantified the cytoplasmic PG peptides during cell growth in absence or in presence of β-lactam. Penicillin stress in our case is generated by the addition of cephalosporin C. On the other hand, we studied the expression of dppABCDE-ykfABCD by following the expression of yfp reporter gene under the control of Pdpp promoter. [less ▲]

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See detailA Mechanistic Study of Protonated Aniline to Protonated Phenol Substitution Considering Tautomerization by Ion Mobility Mass Spectrometry and Tandem Mass Spectrometry
Kune, Christopher ULiege; Delvaux, Cédric ULiege; Haler, Jean ULiege et al

in Journal of the American Chemical Society (2019), 30

We report the use of ion mobility mass spectrometry (IMMS) and energy-resolved collisional activation to investigate gas-phase reactions of protonated aniline and protonated phenol. Protonated aniline ... [more ▼]

We report the use of ion mobility mass spectrometry (IMMS) and energy-resolved collisional activation to investigate gas-phase reactions of protonated aniline and protonated phenol. Protonated aniline prototropic tautomerization and nucleophilic substitution (SN1) to produce phenol with traces of water in the IMMS cell are reported. Tautomerization of protonated phenol and its ability to form protonated aniline in presence of ammonia in the gas phase are also observed. These results are supported by energy landscapes obtained from computational chemistry. These structure modifications in the IMMS cell affected the measured collision cross section (CCS). A thorough understanding of the gas-phase reactions occurring in IMMS appears mandatory before using the experimental CCS as a robust descriptor which is stated by the recent literature. [less ▲]

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See detailRapid Visualization of Chemically Related Compounds Using Kendrick Mass Defect As a Filter in Mass Spectrometry Imaging
Kune, Christopher ULiege; Mc Cann, Andréa ULiege; La Rocca, Raphaël ULiege et al

in Analytical Chemistry (2019), 91(20), 13112-13118

Kendrick mass defect (KMD) analysis is widely used for helping the detection and identification of chemically related compounds based on exact mass measurements. We report here the use of KMD as a ... [more ▼]

Kendrick mass defect (KMD) analysis is widely used for helping the detection and identification of chemically related compounds based on exact mass measurements. We report here the use of KMD as a criterion for filtering complex mass spectrometry data set. The method allow automated, easy and efficient data processing, enabling the reconstruction of 2D distributions of families of homologous compounds from MSI images. We show that KMD filtering, based on in-house software, is suitable and robust for high resolution (full width at half-maximum, fwhm, at m/z 410 of 20 000) and very high-resolution (fwhm, at m/z 410 of 160 000) MSI data. This method has been successfully applied to two different types of samples, bacteria cocultures, and brain tissue sections. [less ▲]

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