References of "De Pauw, Edwin"
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See detailMolecular imaging through in combinaison with quantitative proteomic approaches unraveling the molecular players of breast cancer adaptation to anti-angiogenic therapy.
Cimino, Jonathan ULg; Sounni, Nor Eddine ULg; Calligaris, David ULg et al

Poster (2012, June 22)

Breast carcinoma is the most common and second leading cause of cancer mortality in women. The recognition of the “angiogenic switch” as a rate-limiting secondary step in tumorigenesis led to extensive ... [more ▼]

Breast carcinoma is the most common and second leading cause of cancer mortality in women. The recognition of the “angiogenic switch” as a rate-limiting secondary step in tumorigenesis led to extensive pre-clinical researches on angiogenesis and finally the approval of VEGF-neutralizing antibodies (bevacizumab) and VEGF receptor tyrosine kinase inhibitors (RTKs:Sunitinib). The Sunitinib has been used clinically in patients with breast cancer refractory to other therapeutic agents. Unfortunately, like the cytotoxic therapies, these drugs do not produce lasting effects and resistance to treatment appeared clinically. Questions have emerged about the failure of anti-angiogenic therapy in clinic and the limitations of predictive preclinical models, and also about the molecular assessment of all stages of tumor adaptation and metastatic disease. To this end, we applied quantitative proteomics and imaging mass spectrometry tools to visualize and study the profiles of proteins and small molecules associated with tumor treated or not with Sunitinib using a novel preclinical model of breast carcinoma cells. In this project, we first developed a reproducible model of resistance to Sunitinib of human triple negative breast cancer MDA-MB-231 cells expressing luciferase gene. Cells were subcutaneously injected into mice RAG1-/- and divided into four experimental groups including, control mice treated with vehicle or Sunitinib for 30 days and sacrificed 1 days after treatment withdrawal or when tumor reached a volume of 300 mm3. In the second step. Tumors were analyzed using a nanoAcquity UPLC Synapt TM HDMS TM G1 (Waters, Manchester,UK) and Mass Spectrometry Imaging. For quantitative proteomic analyses of tumors, a bioinformatics analysis was used with the Protein lynx global server 2.2.5 software. Imaging mass spectrometry was performed on tissue sections of tumors and organs subsequently colonized by metastases. Matrix sublimation was used to coat tumor sections (14 µm-tick) with 1.5 Diaminonaphthalene for lipids analysis and Sinapinic acid for entire proteins analysis. Ion cartographies were recorded with a Solarix 9.4T FTMS instrument for lipids and with an Ultraflex II TOF-TOF instrument for entire proteins (Bruker Daltonics, Germany) with a spatial resolution of 100 µm. Global protemic revealed different protein profiles between tumor treated or not with Sunitinib. The Mass Spectrometry Imaging detected differences in intensity and location of some proteins and lipids are also associated with some histological features including inflammatory, necrotic and angiogenic areas. Bioinformatics analysis will be applied to ensure the integration of all data in order to provide the basis for identifying molecular pathways activated during the acquisition of refractoriness to drug treatments. [less ▲]

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See detailSelection and cultivation of hydrolytic microorganisms extracted from the digestive tract of the termite Reticulitermes santonensis (3DV.1.55)
Tarayre, Cédric ULg; Bauwens, Julien ULg; Matteotti, Christel ULg et al

Poster (2012, June 21)

Biofuel production can be based on the use of fermentable substrates issued from the hydrolysis of lignocellulosic biomass stemming from agricultural residues and by-products. However, such substrates are ... [more ▼]

Biofuel production can be based on the use of fermentable substrates issued from the hydrolysis of lignocellulosic biomass stemming from agricultural residues and by-products. However, such substrates are not easy to degrade. Enzymes (cellulases, xylanases, etc.) can be used for this purpose and pre-treatments can increase their action by providing more available extremities. The digestive tract of the termite Reticulitermes santonensis contains various microorganisms (bacteria, molds, protists) able to degrade the wood components. These microorganisms act as consortia, leading to a better hydrolysis than in the cow rumen. Our purpose is the isolation of microorganisms from termite guts in order to evaluate their potential for hydrolysis of lignocellulosic materials. This approach led us to isolate and to study a bacteria (Bacillus sp.) displaying a xylanase activity, a mold (Aspergillus sp.) displaying a cellulase activity and a chrysophyte (protist) displaying an amylase activity. [less ▲]

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See detailNanobodies as structural probes to investigate the mechanism of fibril formation by the amyloidogenic variants of human lysozyme
Dumont, Janice ULg; pardon, Els; Aumont-Nicaise, Magali et al

Poster (2012, June)

Six variants of human lysozyme (single-point mutatants I56T, F57I, W64R, D67H and double mutants F57I/T70N, W112R/T70N) are associated with a hereditary non-neuropathic systemic amyloidosis. These ... [more ▼]

Six variants of human lysozyme (single-point mutatants I56T, F57I, W64R, D67H and double mutants F57I/T70N, W112R/T70N) are associated with a hereditary non-neuropathic systemic amyloidosis. These proteins form extracellular amyloid fibrils that deposit in a wide range of tissues and organs such as liver, spleen and kidneys where they cause damages [1]. It was shown that the D67H and I56T mutations cause a loss in stability and more particularly a loss of global cooperativity of protein [1]. Consequently, under physiologically relevant conditions, these variants can transiently populate a partially unfolded state in which the beta-domain and the C-helix are cooperatively unfolded while the rest of the protein remains native like [1]. The formation of intermolecular interactions between the regions that are unfolded in this intermediate state is likely to be a fundamental trigger of the aggregation process that ultimately leads to the formation and deposition of fibrils in tissues. We have also shown that the binding of three variable domain of camelid antibodies (VHHs) - raised against the wild type human lysozyme inhibit in vitro the formation of amyloid fibrils by the lysozyme variants. These three VHHs bind on different regions of lysozyme and act as amyloid fibril inhibitor through different mechanisms [2, 3, and unpublished results]. In the present work, sixteen new VHHs specific of human lysozyme have been generated. Competition experiments have shown that they bind to five non-overlapping epitopes. We have demonstrated that five of these VHHs are able to bind lysozyme in conditions used for amyloid fibril formation, and interestingly two of them recognize two epitopes that are different from those of the three VHHs previously characterized [2, 3, and unpublished results]. The effects of these new VHHs on the properties of lysozyme variants such as stability, cooperativity and aggregation will be discussed. [1] Dumoulin, M., J.R. Kumita, and C.M. Dobson, Normal and aberrant biological self-assembly: Insights from studies of human lysozyme and its amyloidogenic variants. Acc Chem Res, 2006, 39(9), 603-610. [2] Dumoulin, M., et al., A camelid antibody fragment inhibits the formation of amyloid fibrils by human lysozyme. Nature, 2003, 424, 783-788. [3] Chan, P.H., et al., Engineering a camelid antibody fragment that binds to the active site of human lysozyme and inhibits its conversion into amyloid fibrils. Biochemistry, 2008, 47, 11041-11054. [less ▲]

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See detailA Promising Perspective for Pathologies Diagnosis by MALDI In-Source Decay Imaging with a FTMS System.
Calligaris, David ULg; Debois, Delphine ULg; Turtoi, Andrei ULg et al

Poster (2012, May 23)

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

Introduction MALDI imaging mass spectrometry has proven to be effective for the discovery and the monitoring of disease-related proteins. With this technique a molecular diagnosis could be done directly on tissue sections in the environment of the diseased area. The use of in-source decay (ISD), that does allow fast and reliable sequences assignments of proteins termini, is a crucial tool for the identification of known biomarkers during MALDI imaging experiments. Combined with ultra-high mass resolution and high mass measurement accuracy of Fourier transform ion-cyclotron (FTICR) mass spectrometry, it is possible to unambiguously assign sequences of proteins present in tissue slices. In this study, we have shown that FTICR mass spectrometry could be a powerful tool to diagnose pathologies by MALDI-ISD imaging. Methods All measurements were carried out on a SolariX FTMS (9.4 tesla) equipped with a Dual Source including smartbeamTMII laser which includes a robust solid state 1 kHz laser with advanced optics for molecular imaging (Bruker Daltonics). Lysozyme (14.3-kDa) or Human Serum Albumin (66.3-kDa) solution (1 mg/ml in 0.1 % TFA) was mixed with 1,5-diaminonaphthalene (DAN) and analyzed by MALDI-ISD and MALDI-ISD imaging. Mouse brain and rabbit eye tissue slices were washed (fixed) to obtain optimal sensitivity and high-quality ion. Before DAN application with an ImagePrep (Bruker Daltonics) and MALDI-ISD imaging analyzes, spots of myelin and crystalline were deposited near mouse brain or rabbit eye tissues, respectively. Results were interpreted using BioToolsTM 3.2 in combination with MascotTM (Matrix Science) for ISD spectra and FlexImagingTM 2.1 for MALDI-ISD imaging experiments. α Preliminary data The studies were carried out by MALDI-ISD and MALDI-ISD imaging analyses to evidence the 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 lysozyme and HSA in presence of DAN. Supplementary an-, bn-, xn- and yn-series ions can also be observed. 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), whether from pure solutions or included in tissue slices. Moreover, spots of pure proteins solution (myelin or crystalline) near tissue slices allows to unambiguously validate the proteins identification during MALDI ISD imaging experiments. 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 detailImaging Guided Proteomics Unveils Heterogeniety in Colorectal Carcinoma Liver Metastases – Implications for Targeted Therapies.
blomme, Arnaud; Turtoi, Andrei ULg; Delvaux, David ULg et al

in Proceedings Giga Day 2012 (2012, May 04)

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See detailIn situ protein identification in imaging mass spectrometry
Calligaris, David ULg; Debois, Delphine ULg; De Pauw, Edwin ULg

Scientific conference (2012, May 04)

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is an emerging tool for clinical research. MALDI MSI can be used to elucidate the relative abundance and spatial ... [more ▼]

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is an emerging tool for clinical research. MALDI MSI can be used to elucidate the relative abundance and spatial localization of peptides and proteins throughout a tissue section. For this, a matrix is applied on the tissue in either a spotted array or a homogenous coating. Acquisition of mass spectra is then carried out by performing a raster with a laser across the tissue section in a defined pattern. The spectra acquired from each position on the tissue section contain molecular weight and intensity information representative of the biomolecules at that position. One can plot the intensity of any measured ion as a function of individual pixel locations to generate m/z specific images. But, if protein desorption/ionization and subsequent MS analyses provides a measurement of molecular weight, no protein identification is performed. To achieve this, several methods have been developed. In this talk, I will first present the methods inspired by classical proteomics techniques that are regularly used to identify proteins. Bottom-up and top-down approaches have been used directly from a tissue slice, leading to the identification of some of the most abundant proteins present within the tissue slice. Then, I will present the new developments led in our lab for imaging and especially for in situ protein identification. The first example will deal with the exceptional features of FT-ICR mass spectrometry for in-source decay (ISD)-based protein identification. The benefit of mass accuracy and high mass resolution allow unequivocal assignment of ISD fragments of proteins, in the low mass range (m/z between 400 and 900), whether from pure solutions or from tissue slices. The next example is the use of a matrix “cleaning” software that reduce/remove matrix peaks thus facilitating ISD spectra analyses. Finally, proteins identification by localization and MALDI-ISD profile matching will also be a really simplistic and interesting method that will complement the immunohistological techniques commonly used to validate expression of known biomarkers within diseased tissues. [less ▲]

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See detailApplication of molecular imaging in combination with quantitative proteomic approaches to determine the molecular players of adaptation to anti-angiogenic therapy in breast cancer.
Cimino, Jonathan ULg; Sounni, Nor Eddine ULg; Calligaris, David ULg et al

Poster (2012, May 04)

The recognition of the “angiogenic switch” as a rate-limiting secondary step in tumorigenesis led to extensive pre-clinical researches on angiogenesis and finally the approval of VEGF-neutralizing ... [more ▼]

The recognition of the “angiogenic switch” as a rate-limiting secondary step in tumorigenesis led to extensive pre-clinical researches on angiogenesis and finally the approval of VEGF-neutralizing antibodies (bevacizumab) and VEGF receptor tyrosine kinase inhibitors (RTKs:Sunitinib). The Sunitinib has been used clinically in patients with breast cancer refractory to other therapeutic agents. Unfortunately, like the cytotoxic therapies, these drugs do not produce lasting effects and resistance to treatment appeared clinically. Questions have emerged about the failure of anti-angiogenic therapy in clinic and the limitations of predictive preclinical models, and also about the molecular assessment of all stages of tumor adaptation and metastatic disease. To this end, we applied a quantitative proteomics and imaging mass spectrometry tools to visualize and study the profiles of proteins and small molecules associated with tumor treated or not with Sunitinib using a novel preclinical model of breast carcinoma cells. [less ▲]

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See detailIdentification and quantification of concentration-dependent biomarkers in MCF-7/BOS cells exposed to 17β-estradiol by 2-D DIGE and label-free proteomics
Collodoro, Mike ULg; Lemaire, Pascale ULg; Eppe, Gauthier ULg et al

in Journal of Proteomics (2012), in press

This paper reports the identification of biomarkers resulting from the exposure of MCF-7/BOS cells to 17β-estradiol (E2). The biomarkers were identified using 2 independent and complementary techniques, 2 ... [more ▼]

This paper reports the identification of biomarkers resulting from the exposure of MCF-7/BOS cells to 17β-estradiol (E2). The biomarkers were identified using 2 independent and complementary techniques, 2-D DIGE / MALDI-TOF peptide mass fingerprint, and 2-D UPLC-ESI MS/MS. These markers form a preliminary molecular signature that can be used when testing the estrogenic activity of xenobiotics, either pure or in mixtures. [less ▲]

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See detailINTRA-TUMORAL HETEROGENEITY AND RATIONAL SELECTION OF ANTIGENS FOR TARGETED THERAPY OF LIVER METASTASES
Turtoi, Andrei ULg; Blomme, Arnaud ULg; Delvaux, David ULg et al

in Acta Chirurgica Belgica (2012, May), 112(3), 8953

Objectives: Targeted therapies of liver metastases are gaining a major stake in current and future treatment options. However, the malignant lesions are heterogeneous in nature offering niches for cancer ... [more ▼]

Objectives: Targeted therapies of liver metastases are gaining a major stake in current and future treatment options. However, the malignant lesions are heterogeneous in nature offering niches for cancer cells causing treatment resistance and relapse. Therefore, a rational strategy is needed to select targetable antigens that would overcome this intra-tumoral heterogeneity. Methods: After ethical committee approval, 48 fresh liver metastases of colorectal origin were prospectively collected from patients undergoing liver resection. Here we macroscopically divided the lesion in different zones and generated a unique quantitative picture of the proteome heterogeneity in colorectal carcinoma liver metastases. Particular focus was laid on accessible proteins, a protein subclass comprising cell membrane associated and extracellular proteins. Accordingly, the tissues were ex-vivo biotinylated, affinity purified and analyzed for each zone separately using nano-UPLC-MSe proteomics technique. In total over 1500 unique proteins were statistically divided into different patterns of expression. Results: We have generated a quantitative picture of the proteome heterogeneity in colorectal carcinoma liver metastases. The study offers insight into novel targets but also antigens against which the antibodies are already involved in clinical trials or treatment of liver metastases. Extensive clustering and validation experiments highlight novel markers that offer the potential to homogeneously cover the metastatic lesion and become better targets. Conclusions: Two such antigens, LTBP2 and TGFBI were selected for functional analysis in colorectal carcinoma cells. In vitro and in vivo experiments showed that in particular TGFBI is relevant for migration and proliferation capacity of colorectal cancer cells. The suppression of this protein led to significant inhibition of tumor growth, crystalizing it as bona fide target for the development of anti-metastases therapies. [less ▲]

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See detailUV Spectroscopy of DNA Duplex and Quadruplex Structures in the Gas Phase
Rosu, Frédéric ULg; Gabelica, Valérie ULg; De Pauw, Edwin ULg et al

in Journal of Physical Chemistry A (2012), 116

UV absorption spectroscopy is one of the most widely used methods to monitor nucleic acid folding in solution, but the absorption readout is the weighted average contribution of all species present in ... [more ▼]

UV absorption spectroscopy is one of the most widely used methods to monitor nucleic acid folding in solution, but the absorption readout is the weighted average contribution of all species present in solution. Mass spectrometry, on the other hand, is able to separate constituents of the solution based on their mass, but methods to probe the structure of each constituent are needed. Here, we explored whether gas-phase UV spectroscopy can give an indication of DNA folding in ions isolated by electrospray mass spectrometry. Model DNA single strands, duplexes, and G-quadruplexes were extracted from solution by electrospray; the anions were stored in a quadrupole ion trap and irradiated by a tunable laser to obtain the UV action spectra of each complex. We found that the duplex and quadruplex spectra are significantly different from the spectra of single strands, thereby suggesting that electronic spectroscopy can be used to probe the DNA gas-phase structure and obtain information about the intrinsic properties of high-order DNA structure. [less ▲]

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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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