References of "De Pauw, Edwin"
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See detailNew Advances for In Situ Protein Identification by MALDI In-Source Decay FTMS Imaging
Calligaris, David ULg; Zimmerman, Tyler ULg; Debois, Delphine ULg et al

Poster (2012, April 18)

MALDI imaging mass spectrometry has proven to be effective for the discovery and the monitoring of disease-related proteins. With this technique a molecular analysis could be performed directly from ... [more ▼]

MALDI imaging mass spectrometry has proven to be effective for the discovery and the monitoring of disease-related proteins. With this technique a molecular analysis could be performed directly from tissue sections in the region of the diseased area. The use of in-source decay (ISD), allowing fast and reliable sequences assignments of proteins termini, has proven to be a crucial tool for proteins identification in solution and tissue slices. However, it is necessary to develop additional tools that allow unambiguous assignment of proteins sequences in complex tissue slices. The development of bioinformatic tools and the use of ultra-high mass resolution and high mass accuracy of Fourier transform ion-cyclotron (FTICR) mass spectrometry are ideal for this purpose. In this study, we show that FTICR mass spectrometry combined with data filtering with a software that subtracts matrix peaks aid protein identification. All measurements were carried out on a SolariX FTMS (9.4 Tesla) equipped with a Dual Source with a smartbeamTMII laser (Bruker Daltonics). Mouse brain tissue slices of 14 µm thickness were rinsed to obtain optimal sensitivity and high-quality ions. Before matrix application, a spot of myelin was deposited near mouse brain. 1,5-Diaminionaphtalene was sprayed using an ImagePrep (Bruker Daltonics). Results were interpreted using BioToolsTM 3.2 in combination with MascotTM (Matrix Science) for ISD spectra and FlexImagingTM 3.0 for MALDI-ISD imaging experiments. Matrix peaks were subtracted using an in-house written Java code that sequentially scans all peak lists from acquired spectra against the DAN mass list. Then, another Java code allows to create 2D ion images at selected m/z ratios. The studies were carried out by MALDI-ISD imaging to create interest on FTICR mass spectrometer for proteins identification in the field of biomarkers characterization. It is demonstrated that protein ISD leads to the same pattern of fragmentation observed during MALDI-TOF analyzes. Fragmentation generates cn- and zn-series ions of myelin in presence of DAN. The internal calibration of all the data provides a mass accuracy neighboring 2.5 ppm over the m/z range of interest (300-2500 Da) and a mass resolution of 70000 at m/z 400 Da. It allows the assignment of ISD fragments of proteins in the low mass range (m/z between 300 and 900) that is unambiguously validated by the “ISD signal” recorded from the spots of pure protein solution (myelin) near tissue slice. Moreover, the use of our software “cleans” MS imaging data by reducing/eliminating MALDI matrix peaks that are isobaric to an analyte peak. Novel aspect This study evidences the main input of FTICR mass spectrometer for pathologies diagnosis based on biomarkers localization and identification by MALDI-ISD imaging. [less ▲]

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See detailContribution of high mass resolution and accuracy of FTMS to molecular imaging
Debois, Delphine ULg; Calligaris, David ULg; Cimino, Jonathan ULg et al

Conference (2012, April 04)

Since its first implementation in 1997, MALDI Mass Spectrometry Imaging (MALDI MSI) has become an important tool in the proteomic arsenal, especially for biomarker hunting. First dedicated to high ... [more ▼]

Since its first implementation in 1997, MALDI Mass Spectrometry Imaging (MALDI MSI) has become an important tool in the proteomic arsenal, especially for biomarker hunting. First dedicated to high molecular weight, MALDI MSI is more and more used to map the distribution of small molecules too (lipids, drugs and metabolites,…). Last developments tend to improve the sample treatments to obtain the best spatial resolution as possible. From this perspective, great efforts have been made on the MALDI matrix deposition methods. Now, one of the remaining challenges for MALDI-MSI users consists of identification of detected molecules. For high molecular weight, methods inspired by classical proteomics techniques, are regularly used. Bottom-Up (PMF obtained after in situ trypsin digestion) and Top-Down (in situ In-Source Decay) approaches have been used directly from a tissue slice, leading to the identification of some of the most abundant proteins present at the surface of the tissue. When small molecules are analyzed, the identification is more straightforward. Indeed, tandem mass spectrometry can easily be used, leading to the fragmentation of the detected compounds which allows their unambiguous identification. This identification is even more reliable when high resolution exact mass measurements can be performed. In this talk, I will present how in our lab, we profit of the exceptional features of FT-ICR mass spectrometry for imaging and especially for identification purposes. The first example will deal with the benefit of high mass accuracy and high mass resolution for ISD-based protein identification. The mass accuracy and high mass resolution coupled with the use of a “cleaning” software allow unequivocal assignment of ISD fragments of proteins, in the low mass range (m/z between 300 and 900), whether from pure solutions or from tissue slices. The next examples will deal with the imaging of small molecules. The identification of drugs and their metabolites is facilitated with high mass accuracy. In our lab, we work on the localization of methadone and its first metabolite, EDDP in necrophagous fly larvae. In the mass range of these compounds (278-310 m/z), many matrix ion peaks are detected and the unique features of FT-ICR allows for unambiguous identification thanks to exact mass measurements. We also use MALDI Imaging to map the messenger molecules between plant roots and beneficial bacteria. The comparison of spectra recorded with a TOF/TOF instrument and with a FT-ICR demonstrates that high resolution allows for detecting molecules which could have been missed otherwise. It also allows to distinguish unknown compounds from alkali adducts of known molecules. [less ▲]

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See detailAdvances in proteomics for the FP7 Venomics project
Degueldre, Michel ULg; Quinton, Loïc ULg; De Pauw, Edwin ULg

Scientific conference (2012, April)

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See detailDisulfide bond scrambling in partially reduced and alkylated peptides revealed by Ion Mobility Mass Spectrometry
Echterbille, Julien ULg; Quinton, Loïc ULg; De Pauw, Edwin ULg

Poster (2012, March 29)

Animal venoms are mainly composed of peptide toxins, which are highly structured by many disulfide bridges. In these toxins, disulfides play different major roles such as increasing the toxins efficiency ... [more ▼]

Animal venoms are mainly composed of peptide toxins, which are highly structured by many disulfide bridges. In these toxins, disulfides play different major roles such as increasing the toxins efficiency by lowering their immunogenicity or providing the adequate conformation to efficiently bind to the biological receptor. Peptide sequencing followed by determination of the cysteine pairings is still challenging and, therefore, an important step in structural analysis. This work was, in its beginning, focused on the development of ion mobility (IMS) based methodology used to assign disulfides. The strategy relies on the analysis of partially reduced/alkylated disulfide containing peptides. The resulting mixture is analyzed by ion mobility, followed by MS/MS acquisition on each mobility resolved species. Surprisingly, first investigations revealed, after partial reduction, a disulfide rearrangement phenomenon. Indeed, some of the cystein pairings were not those expected to be. These experiments were conducted on ¿-CnI and ¿-GI toxins purified from the venoms of Conus consors and Conus geographus marine snails, respectively. Each toxin contains four cysteines linked together with two disulfide bridges. Peptides were partially reduced by an excess of dithiothreitol and then alkylated by a large excess of iodoacetamide. The resulting mixture was purified on a microcolumn before being analyzed by nanoESI-Synapt-G2. Fragmentation was performed after the mobility cell, to obtain specific fragments of each species. Each toxin partially reduced/alkylated results, theoretically, in a mixture of fully oxidized (two disulfides oxidized), fully reduced (two disulfides reduced) and partially reduced forms (one of the two disulfides reduced). Thanks to the mass shift created by the alkylation, an isolation of the species which m/z ratio corresponds to one disulfide reduced and alkylated has been done in the quadrupole before the mobility separation. The arrival time distribution of triply charged ions reveals the presence of different species (4 in the case of ¿-GI and 2 for ¿-CnI), characterized by different relative cross sections in the gas-phase. As ion mobility resolved species give characteristic fragments upon fragmentation (after IMS), we were able to identify a scrambling of the disulfides (isomerization). In simple words, other disulfide bonds than expected ones were characterized. We suppose that the scrambling phenomenon occurs in solution,during the reduction step, since the alkylation cannot avoid rearrangement. The method is now being applied to more complex systems containing 3 or 4 disulfide bridges. The influence of the charge state on the mobility separation is systematically analyzed in terms of structural implications. [less ▲]

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See detailThe usefulness of Ion Mobility-Mass Spectrometry for Small Molecules Analysis
Far, Johann ULg; Goscinny, Séverine ULg; Joly, Laure et al

Conference (2012, March)

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See detailRisk assessment of Belgian adults for furan contamination through the food chain
Scholl, Georges ULg; Humblet, Marie-France ULg; Scippo, Marie-Louise ULg et al

in Food Additives & Contaminants (2012), 29(3), 345-353

Risk assessment is an interdisciplinary process used to quantify the risk linked to a hazard. In the present paper it is applied to quantify the risk linked to furan ingestion through the food chain for ... [more ▼]

Risk assessment is an interdisciplinary process used to quantify the risk linked to a hazard. In the present paper it is applied to quantify the risk linked to furan ingestion through the food chain for the Belgian adult population. Two approaches, deterministic and probabilistic, were carried out in parallel. The deterministic method relied on a case study, whereas the probabilistic approach involved statistical distributions of contamination and consumption data to calculate a statistical distribution of the daily intake. First, the deterministic method revealed a low estimated daily intake (EDI) for the average population (380 ng*(kgbw*day)–1) and a huge contribution of coffee consumption to the EDI (55%). Increasing or decreasing the daily coffee consumption by one cup can affect the EDI by about 22%. Afterwards, the probabilistic approach showed that the average population has a low EDI (494 ng*(kgbw*day) 1), and that high contamination levels were only registered in a small proportion of the population. Finally, a comparison of the RfDchronic oral showed that less than 10% of the Belgian population had an EDI above the reference dose proposed by the USEPA; the majority of the population had an EDI 20% below the reference dose. The margin of exposure (MoE) approach indicated that the level of risk related to furan intake through ingestion is low, with a MoE>10,000 for more than 10% of the population and no result < 100. [less ▲]

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See detailDifferential proteomic analysis of a human breast tumor and its matched bone metastasis identifies cell membrane and extracellular proteins associated with bone metastasis
Dumont, Bruno ULg; Castronovo, Vincenzo ULg; Peulen, Olivier ULg et al

in Journal of Proteome Research (2012)

The classical fate of metastasizing breast cancer cells is to seed and form secondary colonies in bones. The molecules closely associated with these processes are predominantly present at the cell surface ... [more ▼]

The classical fate of metastasizing breast cancer cells is to seed and form secondary colonies in bones. The molecules closely associated with these processes are predominantly present at the cell surface and in the extracellular space, establishing the first contacts with the target tissue. In this study, we had the rare opportunity to analyze a bone metastatic lesion and its corresponding breast primary tumor obtained simultaneously from the same patient. Using mass spectrometry, we undertook a proteomic study on cell surface and extracellular protein-enriched material. We provide a repertoire of significantly modulated proteins, some with yet unknown roles in the bone metastatic process as well as proteins notably involved in cancer cell invasiveness and in bone metabolism. The comparison of these clinical data with those previously obtained using a human osteotropic breast cancer cell line highlighted an overlapping group of proteins. Certain differentially expressed proteins are validated in the present study using immunohistochemistry on a retrospective collection of breast tumors and matched bone metastases. Our exclusive set of selected proteins supports the set-up of further investigations on both clinical samples and experimental bone metastasis models that will help to reveal the finely coordinated expression of proteins that favor the development of metastases in the bone microenvironment. [less ▲]

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See detailOverexpression of CD9 in human breast cancer cells promotes the development of bone metastases.
Kischel, Philippe; Bellahcene, Akeila ULg; Deux, Blandine et al

in Anticancer Research (2012), 32(12), 5211-20

BACKGROUND: Bone is a preferred target for circulating metastatic breast cancer cells. We found that the CD9 protein was up-regulated in the B02 osteotropic cell line, derived from the aggressive parental ... [more ▼]

BACKGROUND: Bone is a preferred target for circulating metastatic breast cancer cells. We found that the CD9 protein was up-regulated in the B02 osteotropic cell line, derived from the aggressive parental MDA-MB-231 breast cancer cell line. Here, we investigated the putative relationship between CD9 expression and the osteotropic phenotype. MATERIALS AND METHODS: Overexpression of CD9 was analyzed by immunoblotting in different cell lines. Immunohistochemistry was used to assess CD9 expression in primary tumors and metastatic lesions. In vivo experiments were conducted in mice using a monoclonal antibody against CD9. RESULTS: CD9 overexpression was confirmed in osteotropic cells. CD9 was significantly overexpressed in bone metastases versus primary tumors and visceral metastatic lesions. Finally, in vivo experiments showed that an antibody against CD9 delays homing of B02 cells in bone marrow, slowing down bone destruction. CONCLUSION: Our study reveals a potential implication of CD9 in the formation of bony metastases from breast cancer cells. [less ▲]

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See detailMALDI In-Source Decay for High Throughput sequencing of peptide animal toxins
Quinton, Loïc ULg; Degueldre, Michel ULg; Gilles, Nicolas et al

Poster (2012)

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See detailMass spectrometry as a tool to search specific ligands for G protein coupled receptors.
Cologna, Camila Takeno; Echterbille, Julien ULg; De Pauw, Edwin ULg et al

Conference (2012)

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See detailAdvances in proteomics for the FP7 Venomics project
Degueldre, Michel ULg; Quinton, Loïc ULg; De Pauw, Edwin ULg

Scientific conference (2012)

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See detailRaman spectroscopy and laser desorption mass spectrometry for minimal destructive forensic analysis of black and color inkjet printed documents
Heudt, Laetitia ULg; Debois, Delphine ULg; Zimmerman, Tyler ULg et al

in Forensic Science International (2012), 219

Inkjet ink analysis is the best way to discriminate between printed documents, or even though more difficult, to connect an inkjet printed document with a brand or model of printers. Raman spectroscopy ... [more ▼]

Inkjet ink analysis is the best way to discriminate between printed documents, or even though more difficult, to connect an inkjet printed document with a brand or model of printers. Raman spectroscopy and laser desorption mass spectrometry (LDMS) have been demonstrated as powerful tools for dyes and pigments analysis, which are ink components. The aim of this work is to evaluate the aforementioned techniques for inkjet inks analysis in terms of discriminating power, information quality, and nondestructive capability. So, we investigated 10 different inkjet ink cartridges (primary colors and black), 7 from the HP manufacturer and one each from Epson, Canon and Lexmark. This paper demonstrates the capabilities of three methods: Raman spectroscopy, LDMS and MALDI-MS. Raman spectroscopy, as it is preferable to try the nondestructive approach first, is successfully adapted to the analysis of color printed documents in most cases. For analysis of color inkjet inks by LDMS, we show that a MALDI matrix (9- aminoacridine, 9AA) is needed to desorb and to ionize dyes from most inkjet inks (except Epson inks). Therefore, a method was developed to apply the 9AA MALDI matrix directly onto the piece of paper while avoiding analyte spreading. The obtained mass spectra are very discriminating and lead to information about ink additives and paper compositions. Discrimination of black inkjet printed documents is more difficult because of the common use of carbon black as the principal pigment. We show for the first time the possibility to discriminate between two black-printed documents coming from different, as well as from the same, manufacturers. Mass spectra recorded from black inks in positive ion mode LDMS detect polyethylene glycol polymers which have characteristic mass distributions and end groups. Moreover, software has been developed for rapid and objective comparison of the low mass range of these positive mode LDMS spectra which have characteristic unknown peaks. [less ▲]

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See detailSparc-like protein 1 is a new marker of human glioma progression.
Turtoi, Andrei ULg; Musmeci, Davide; Naccarato, Antonio Giuseppe et al

in Journal of Proteome Research (2012), 11(10), 5011-21

High-grade gliomas (glioblastomas) are the most common and deadly brain tumors in adults, currently with no satisfactory treatment available. Apart from de novo glioblastoma, it is currently accepted that ... [more ▼]

High-grade gliomas (glioblastomas) are the most common and deadly brain tumors in adults, currently with no satisfactory treatment available. Apart from de novo glioblastoma, it is currently accepted that these malignancies mainly progress from lower grade glial tumors. However, the molecular entities governing the progression of gliomas are poorly understood. Extracellular and membrane proteins are key biomolecules found at the cell-to-cell communication interface and hence are a promising proteome subpopulation that could help understand the development of glioma. Accordingly, the current study aims at identifying new protein markers of human glioma progression. For this purpose, we used glial tumors generated orthotopically with T98G and U373 human glioma cells in nude mice. This setup allowed also to discriminate the protein origin, namely, human (tumor) or mouse (host). Extracellular and membrane proteins were selectively purified using biotinylation followed by streptavidin affinity chromatography. Isolated proteins were digested and then identified and quantified employing 2D-nano-HPLC-MS/MS analysis. A total of 23 and 27 up-regulated extracellular and membrane proteins were identified in the T98G and U373 models, respectively. Approximately two-thirds of these were predominantly produced by the tumor, whereas the remaining proteins appeared to be mainly overexpressed by the host tissue. Following extensive validation, we have focused our attention on sparc-like protein 1. This protein was further investigated using immunohistochemistry in a large collection of human glioma samples of different grades. The results showed that sparc-like protein 1 expression correlates with glioma grade, suggesting the possible role for this protein in the progression of this malignancy. [less ▲]

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See detailThe angiogenesis suppressor gene AKAP12 is under the epigenetic control of HDAC7 in endothelial cells.
Turtoi, Andrei ULg; Mottet, Denis ULg; Matheus, Nicolas ULg et al

in Angiogenesis (2012)

Histone deacetylases (HDACs) are a family of 18 enzymes that deacetylate lysine residues of both histone and nonhistone proteins and to a large extent govern the process of angiogenesis. Previous studies ... [more ▼]

Histone deacetylases (HDACs) are a family of 18 enzymes that deacetylate lysine residues of both histone and nonhistone proteins and to a large extent govern the process of angiogenesis. Previous studies have shown that specific inhibition of HDAC7 blocks angiogenesis both in vitro and in vivo. However, the underlying molecular mechanisms are not fully understood and hence preclude any meaningful development of suitable therapeutic modalities. The goal of the present study was to further the understanding of HDAC7 epigenetic control of angiogenesis in human endothelial cells using the proteomic approach. The underlying problem was approached through siRNA-mediated gene-expression silencing of HDAC7 in human umbilical vein endothelial cells (HUVECs). To this end, HUVEC proteins were extracted and proteomically analyzed. The emphasis was placed on up-regulated proteins, as these may represent potential direct epigenetic targets of HDAC7. Among several proteins, A-kinase anchor protein 12 (AKAP12) was the most reproducibly up-regulated protein following HDAC7 depletion. This overexpression of AKAP12 was responsible for the inhibition of migration and tube formation in HDAC7-depleted HUVEC. Mechanistically, H3 histones associated with AKAP12 promoter were acetylated following the removal of HDAC7, leading to an increase in its mRNA and protein levels. AKAP12 is responsible for protein kinase C mediated phosphorylation of signal transducer and activator of transcription 3 (STAT3). Phosphorylated STAT3 increasingly binds to the chromatin and AKAP12 promoter and is necessary for maintaining the elevated levels of AKAP12 following HDAC7 knockdown. We demonstrated for the first time that AKAP12 tumor/angiogenesis suppressor gene is an epigenetic target of HDAC7, whose elevated levels lead to a negative regulation of HUVEC migration and inhibit formation of tube-like structures. [less ▲]

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