Publications of Edwin De Pauw
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See detailRadical-Pairing Interactions in a Molecular Switch Evidenced by Ion Mobility Spectrometry and Infrared Ion Spectroscopy
Hanozin, Emeline ULiege; Mignolet, Benoît ULiege; Martens, Jonathan et al

in Angewandte Chemie International Edition (in press)

The digital revolution sets a milestone in the progressive miniaturization of working devices and in the underlying advent of molecular machines. Foldamers involving mechanically entangled components with ... [more ▼]

The digital revolution sets a milestone in the progressive miniaturization of working devices and in the underlying advent of molecular machines. Foldamers involving mechanically entangled components with modular secondary structures are among the most promising designs for molecular switch-based applications. Characterizing the nature and dynamics of their intramolecular network following the application of a stimulus is the key to their performance. Here, we use non-dissociative electron transfers as a reductive stimulus in the gas phase and probe the consecutive co-conformational transitions of a donor-acceptor oligorotaxane foldamer using electrospray mass spectrometry interfaced with ion mobility and infrared ion spectroscopy. The comparison of collision cross section distributions for analogous closed-shell and radical molecular ions sheds light on their respective formation energetics while variations in their respective infrared absorption bands evidence different intramolecular organizations as the foldamer gets compact. These differences are compatible with the advent of radical-pairing interactions. [less ▲]

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See detailPotential Role of Epithelial Endoplasmic Reticulum Stress and Anterior Gradient Protein 2 Homolog in Crohn’s Disease Fibrosis
VIEUJEAN, Sophie ULiege; Hu, Shurong; Bequet, Emeline ULiege et al

in Journal of Crohn's and Colitis (2021)

Background and aims: Intestinal fibrosis is a common complication of Crohn’s disease (CD). It is characterised by an accumulation of fibroblasts differentiating into myofibroblasts secreting excessive ... [more ▼]

Background and aims: Intestinal fibrosis is a common complication of Crohn’s disease (CD). It is characterised by an accumulation of fibroblasts differentiating into myofibroblasts secreting excessive extracellular matrix. The potential role of the intestinal epithelium in this fibrotic process remains poorly defined. Methods: We performed a pilot proteomic study comparing the proteome of surface epithelium, isolated by laser-capture microdissection, in normal and fibrotic zones of resected ileal CD strictures (13 zones collected in 5 patients). Proteins of interests were validated by immunohistochemistry (IHC) in ileal and colonic samples of stricturing CD (n=44), pure inflammatory CD (n=29) and control (n=40) subjects. The pro-fibrotic role of one selected epithelial protein was investigated through in-vitro experiments using HT-29 epithelial cells and a CCD-18Co fibroblast to myofibroblast differentiation model. Results: Proteomic study revealed an endoplasmic reticulum (ER) stress proteins increase in the epithelium of CD ileal fibrotic strictures, including Anterior gradient protein 2 homolog (AGR2) and Binding-immunoglobulin protein (BiP). This was confirmed by IHC. In HT-29 cells, tunicamycin-induced ER stress triggered AGR2 intracellular expression and its secretion. Supernatant of these HT-29 cells, pre-conditioned by tunicamycin, led to a myofibroblastic differentiation when applied on CCD-18Co fibroblasts. By using recombinant protein and blocking agent for AGR2, we demonstrated that the secretion of this protein by epithelial cells can play a role in the myofibroblastic differentiation. Conclusions: The development of CD fibrotic strictures could involve epithelial ER stress and particularly the secretion of AGR2. [less ▲]

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See detailPotential Role of Epithelial Protein Disulphide Isomerases in Crohn’s Disease Fibrosis
Vieujean, Sophie ULiege; Hu, Shurong; Bequet, Emeline ULiege et al

in Acta Gastro-Enterologica Belgica (2021, March 03)

Background and aims: Intestinal fibrosis is a common complication of Crohn’s disease (CD) characterized by an accumulation of fibroblasts differentiating into activated myofibroblasts secreting excessive ... [more ▼]

Background and aims: Intestinal fibrosis is a common complication of Crohn’s disease (CD) characterized by an accumulation of fibroblasts differentiating into activated myofibroblasts secreting excessive extracellular matrix. In in-vitro experiments, this myofibroblastic differentiation is elicited by a whole series of factors among which transforming growth factor β1 (TGF-β1) seems to play a key role. The potential role of the intestinal epithelium in this fibrotic process remains poorly defined. Methods: We performed a pilot proteomic study comparing the proteome of surface epithelium isolated by laser-capture microdissection in normal and fibrotic zones of resected ileal CD strictures (13 zones collected in 5 patients). The pro-fibrotic role of selected epithelial proteins was investigated through in-vitro experiments using HT-29 epithelial cells and a CCD-18Co fibroblast to myofibroblast differentiation model. Results: Proteomic study revealed an endoplasmic reticulum (ER) stress proteins increase in the epithelium of CD ileal fibrotic strictures, including Anterior gradient protein 2 homolog (AGR2), Protein disulphide isomerase A6 (PDIA6) and Endoplasmic reticulum resident protein 44 (ERP44) which are 3 protein disulphide isomerases. In HT-29 cells, tunicamycin-induced ER stress triggered AGR2, PDIA6, ERP44 as well as TGF β1 intracellular expression and their secretion. Supernatant of these HT-29 cells, pre-conditioned by tunicamycin (Tm), led to a myofibroblastic differentiation when applied on CCD-18Co fibroblasts. The application of blocking agents for AGR2, PDIA6, ERP44 or TGF β1 in the supernatant of these Tm pre conditioned HT-29 cells, attenuated the myofibroblastic differentiation induced by this supernatant, suggesting a pro-fibrotic role of these secreted epithelial proteins. Conclusions: The development of CD fibrotic strictures may involve ER stress in epithelial cells, releasing a whole set of proteins into their environment, including AGR2, PDIA6, ERP44 as well as TGF-β1, which could exercise a pro-fibrotic role through a paracrine action. [less ▲]

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See detailMass shift in Mass Spectrometry Imaging: comprehensive analysis and practical corrective workflow
Mc Cann, Andréa ULiege; Rappe, Sophie ULiege; La Rocca, Raphaël ULiege et al

in Analytical and Bioanalytical Chemistry (2021)

tentative identification of compounds based on their m/z value. In typical MSI, a spectrum is taken at incremental 2D coordinates (pixels) across a sample surface. Single pixel mass spectra show the ... [more ▼]

tentative identification of compounds based on their m/z value. In typical MSI, a spectrum is taken at incremental 2D coordinates (pixels) across a sample surface. Single pixel mass spectra show the resolving power of the mass analyzer. Mass shift, i.e., variations of the m/z of the same ion(s), may occur from one pixel to another. The superposition of shifted masses from individual pixels peaks apparently degrades the resolution and the mass accuracy in the average spectrum. This leads to low confidence annotations and biased localization in the image. Besides the intrinsic performances of the analyzer, the sample properties (local composition, thickness, matrix deposition) and the calibration method are sources of mass shift. Here, we report a critical analysis and recommendations to mitigate these sources of mass shift. Mass shift 2D distributions were mapped to illustrate its effect and explore systematically its origin. Adapting the sample preparation, carefully selecting the data acquisition settings, and wisely applying post-processing methods (i.e., m/z realignment or individual m/z recalibration pixel by pixel) are key factors to lower the mass shift and to improve image quality and annotations. A recommended workflow, resulting from a comprehensive analysis, was successfully applied to several complex samples acquired on both MALDI ToF and MALDI FT-ICR instruments. [less ▲]

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See detailDual-polarity SALDI FT-ICR MS imaging and Kendrick mass defect data filtering for lipid analysis
Müller, Wendy ULiege; Verdin, Alexandre ULiege; Kune, Christopher ULiege et al

in Analytical and Bioanalytical Chemistry (2021), 413(10), 28212830

Lipids are biomolecules of crucial importance involved in critical biological functions. Yet, lipid content determination using mass spectrometry is still challenging due to their rich structural ... [more ▼]

Lipids are biomolecules of crucial importance involved in critical biological functions. Yet, lipid content determination using mass spectrometry is still challenging due to their rich structural diversity. Preferential ionisation of the different lipid species in the positive or negative polarity is common, especially when using soft ionisation mass spectrometry techniques. Here, we demonstrate the potency of a dual-polarity approach using surface-assisted laser desorption/ionisation coupled to Fourier transform-ion cyclotron resonance (SALDI FT-ICR) mass spectrometry imaging (MSI) combined with Kendrick mass defect data filtering to (i) identify the lipids detected in both polarities from the same tissue section and (ii) show the complementarity of the dual-polarity data, both regarding the lipid coverage and the spatial distributions of the various lipids. For this purpose, we imaged the same mouse brain section in the positive and negative ionisation modes, on alternate pixels, in a SALDI FT-ICR MS imaging approach using gold nanoparticles (AuNPs) as dual-polarity nanosubstrates. Our study demonstrates, for the first time, the feasibility of (i) a dual-polarity SALDI-MSI approach on the same tissue section, (ii) using AuNPs as nanosubstrates combined with a FT-ICR mass analyser and (iii) the Kendrick mass defect data filtering applied to SALDI-MSI data. In particular, we show the complementarity in the lipids detected both in a given ionisation mode and in the two different ionisation modes. [less ▲]

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See detailAdaptive Pixel Mass Recalibration for Mass Spectrometry Imaging Based on Locally Endogenous Biological Signals.
La Rocca, Raphaël ULiege; Kune, Christopher ULiege; Tiquet, Mathieu ULiege et al

in Analytical Chemistry (2021)

Mass spectrometry imaging (MSI) is a powerful and convenient method for revealing the spatial chemical composition of different biological samples. Molecular annotation of the detected signals is only ... [more ▼]

Mass spectrometry imaging (MSI) is a powerful and convenient method for revealing the spatial chemical composition of different biological samples. Molecular annotation of the detected signals is only possible if a high mass accuracy is maintained over the entire image and the m/z range. However, the change in the number of ions from pixel-to-pixel of the biological samples could lead to small fluctuations in the detected m/z-values, called mass shift. The use of internal calibration is known to offer the best solution to avoid, or at least to reduce, mass shifts. Their “a priori” selection for a global MSI acquisition is prone to false positive detection and therefore to poor recalibration. To fill this gap, this work describes an algorithm that recalibrates each spectrum individually by estimating its mass shift with the help of a list of pixel-specific internal calibrating ions, automatically generated in a data-adaptive manner (https://github.com/LaRoccaRaphael/MSI_recalibration). Through a practical example, we applied the methodology to a zebrafish whole-body section acquired at a high mass resolution to demonstrate the impact of mass shift on data analysis and the capability of our algorithm to recalibrate MSI data. In addition, we illustrate the broad applicability of the method by recalibrating 31 different public MSI data sets from METASPACE from various samples and types of MSI and show that our recalibration significantly increases the numbers of METASPACE annotations (gaining from 20 up to 400 additional annotations), particularly the high-confidence annotations with a low false discovery rate. [less ▲]

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See detailUV-screening pigment enabling ancient photosynthesis
Lara, Yannick ULiege; Mc Cann, Andréa ULiege; Malherbe, Cédric ULiege et al

Conference (2021)

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See detailSurface‐assisted laser desorption/ionization mass spectrometry imaging: A review
Müller, Wendy ULiege; Verdin, Alexandre ULiege; De Pauw, Edwin ULiege et al

in Mass Spectrometry Reviews (2020)

In the last decades, surface‐assisted laser desorption/ionization mass spectrometry (SALDI‐MS) has attracted increasing interest due to its unique capabilities, achievable through the nanostructured ... [more ▼]

In the last decades, surface‐assisted laser desorption/ionization mass spectrometry (SALDI‐MS) has attracted increasing interest due to its unique capabilities, achievable through the nanostructured substrates used to promote the analyte desorption/ionization. While the most widely recognized asset of SALDI‐MS is the untargeted analysis of small molecules, this technique also offers the possibility of targeted approaches. In particular, the implementation of SALDI‐MS imaging (SALDI‐MSI), which is the focus of this review, opens up new opportunities. After a brief discussion of the nomenclature and the fundamental mechanisms associated with this technique, which are still highly controversial, the analytical strategies to perform SALDI‐MSI are extensively discussed. Emphasis is placed on the sample preparation but also on the selection of the nanosubstrate (in terms of chemical composition and morphology) as well as its functionalization possibilities for the selective analysis of specific compounds in targeted approaches. Subsequently, some selected applications of SALDI‐MSI in various fields (i.e., biomedical, biological, environmental, and forensic) are presented. The strengths and the remaining limitations of SALDI‐MSI are finally summarized in the conclusion and some perspectives of this technique, which has a bright future, are proposed in this section. [less ▲]

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See detailResponse to Comment on Effective Temperature and Structural Rearrangement in Trapped Ion Mobility Spectrometry
Morsa, Denis ULiege; Hanozin, Emeline ULiege; Gabelica, Valérie et al

in Analytical Chemistry (2020), 92(24), 633416337

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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 ULiege 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 ULiege 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 detailOn the Risks of Phylogeny-Based Strain Prioritization for Drug Discovery: Streptomyces lunaelactis as a Case Study
Martinet, Loïc ULiege; Naômé, A.; Baiwir, Dominique ULiege et al

in Biomolecules (2020), 10(7),

Strain prioritization for drug discovery aims at excluding redundant strains of a collection in order to limit the repetitive identification of the same molecules. In this work, we wanted to estimate what ... [more ▼]

Strain prioritization for drug discovery aims at excluding redundant strains of a collection in order to limit the repetitive identification of the same molecules. In this work, we wanted to estimate what can be unexploited in terms of the amount, diversity, and novelty of compounds if the search is focused on only one single representative strain of a species, taking Streptomyces lunaelactis as a model. For this purpose, we selected 18 S. lunaelactis strains taxonomically clustered with the archetype strain S. lunaelactis MM109T. Genome mining of all S. lunaelactis isolated from the same cave revealed that 54% of the 42 biosynthetic gene clusters (BGCs) are strain specific, and five BGCs are not present in the reference strain MM109T. In addition, even when a BGC is conserved in all strains such as the bag/fev cluster involved in bagremycin and ferroverdin production, the compounds produced highly differ between the strains and previously unreported compounds are not produced by the archetype MM109T. Moreover, metabolomic pattern analysis uncovered important profile heterogeneity, confirming that identical BGC predisposition between two strains does not automatically imply chemical uniformity. In conclusion, trying to avoid strain redundancy based on phylogeny and genome mining information alone can compromise the discovery of new natural products and might prevent the exploitation of the best naturally engineered producers of specific molecules. [less ▲]

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