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See detailMALDI-imaging guided microproteomics workflow for biomarker discovery of intra-tumor heterogeneity
Alberts, Deborah ULg; Longuespée, Rémi ULg; Smargiasso, Nicolas ULg et al

Poster (2016, June 09)

Introduction A single tumoral tissue can bear phenotypically different cell populations. This phenomenon called intra-tumor heterogeneity can lead to differential behaviors regarding metastasis seeding ... [more ▼]

Introduction A single tumoral tissue can bear phenotypically different cell populations. This phenomenon called intra-tumor heterogeneity can lead to differential behaviors regarding metastasis seeding and therapy resistance [Zardavas et al., Nature Rev. Clin. Onc. 2015]. MALDI imaging has proven its efficiency for revealing hidden molecular features offering an insight into distinct cellular regions based on their molecular content. Further, proteomics applied to these regions could allow depicting the molecular context associated to particular cells groups and enable the collection of qualitative, quantitative and spatial information for each protein. Methods Breast cancer Formalin Fixed and Paraffin Embedded tissues, from patients whose outcome had been recorded over a period of 10 years, were provided by the department of Pathology of University of Liège. After Citric Acid Antigen Retrieval and trypsin digestion, images were obtained by MALDI-TOF/TOF-MS (Bruker, Germany). From the obtained datasets, segmentation and analytical data analysis were applied using SCiLS (Bruker, Germany) and the cloud software Multimaging (ImaBiotech, France). Small tissue areas were obtained by laser microdissection (LEICA LMD 700, Germany), upon which a combination of chemical processes was applied to ensure optimal protein antigen retrieval, extraction and digestion. Finally, the tissue pieces obtained were analyzed by LC-MS/MS using UPLC Waters Nanoacquity and Thermo Q-Exactive instruments. Preliminary data Based on mathematical calculations for the MALDI imaging datasets of the breast cancer FFPE tissues, Regions Of Interest (ROIs) were detected in a single tumor, revealing intra-tumoral heterogeneity, which can be correlated to the level of aggressiveness of the affliction and to the final prognosis of the patient. We aimed to compare the proteomic profiles of each of the small ROIs. Until today, proteomics applied to tissues composed by a restricted number of cells is quite tedious due to possible tissue losses during their handling. Recently, Longuespée [Longuespée et al., Methods 2015] published a method in order to retrieve the identification of 1400 proteins from microdissected tissue pieces containing only 2700 cells. This whole procedure allowed us to identify a panel of protein that characterizes tissue heterogeneity within a single tumor. This proves the applicability of the combination of MALDI imaging for the discovery of intra-tumoral heterogeneity without a priori, on a mathematical basis, and classical proteomics applied on laser-microdissected tissue samples of very restricted areas. This method will now be applied to several MALDI datasets in order to retrieve commune ROIs and to associate their presence with the information of each patient, such as their prognosis. Those ROIs will then be microdissected and subjected to microproteomic methods that will allow us to retrieve the extensive molecular context associated to bad patient prognosis and/or therapy resistance. The possibility to identify protein/peptide markers will have the power to predict the outcome of the breast cancer patient at the beginning of their treatment, and thus, improve the clinical care for the benefit of the patients. Novel aspect The workflow combines the unique advantages of MALDI imaging for de novo molecular features characterization and LMD-based microproteomics. [less ▲]

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

Poster (2016, January 22)

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See detailEffet inflammatoire des extraits de Cupressus sempervirens planté dans l’Est de l’Algérie : du modèle murin à la stimulation des cellules BEAS-2B par l’allergène majeur Cup s 1
Bouguenoun, Imene; De Pauw-Gillet, Marie-Claire ULg; Bensakhri, Z et al

in Revue Française d'Allergologie (2016), 56

Cupressus sempervirens est l’une des espèces les plus répandues en Algérie ayant une capacité allergénique très élevée. Nous avons étudié,d’abord, comment l’instillation nasale des différentes fractions ... [more ▼]

Cupressus sempervirens est l’une des espèces les plus répandues en Algérie ayant une capacité allergénique très élevée. Nous avons étudié,d’abord, comment l’instillation nasale des différentes fractions de l’extrait pollinique de cette espèce peut moduler la réponse immunitaire chez les souris, et, ensuite, la stimulation des cellules épithéliales bronchiques humaines (BEAS-2B) par l’allergène majeur, identifié par spectrométrie de masse et purifié par chromatographie échangeuse d’ions. La sensibilisation des souris a révélé, d’une part, une diminution du nombre de neutrophiles et une augmentation des éosinophiles, et, d’autre part, une augmentation significative du total cellulaire dans le liquide du lavage bronchoalvéolaire.L’étude histologique des poumons a montré une présence d’infiltrat inflammatoire et d’oedème péri-bronchique. La spectrométrie de masse nous a permis de mettre en évidence la présence de l’allergène majeur Cup s 1 dans toutes les fractions avec une présence spécifique du Cup s 3 et Cupa 1 dans la fraction 4. Cet allergène avait le potentiel pour stimuler la libération de l’IL-8 et l’IL-6 par la ligné cellulaire, BEAS-2B, in vitro d’une manière dose-dépendante [less ▲]

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See detailComparison of serum fractionation methods by data independent label-free proteomics
Baiwir, Dominique ULg; Mazzucchelli, Gabriel ULg; Smargiasso, Nicolas ULg et al

in EuPA Open Proteomics (2015), 9

Off-line sample prefractionations applied prior to biomarker discovery proteomics are options to enable more protein identifications and detect low-abundance proteins. This work compared five commercial ... [more ▼]

Off-line sample prefractionations applied prior to biomarker discovery proteomics are options to enable more protein identifications and detect low-abundance proteins. This work compared five commercial methods efficiency to raw serum analysis using label-free proteomics. The variability of the protein quantities determined for each process was similar to the unprefractionated serum. A 49% increase in protein identifications and 12.2% of reliable quantification were obtained. A 61 times lower limit of protein quantitation was reached compared to protein concentrations observed in raw serum. The concentrations of detected proteins were confronted to estimated reference values. [less ▲]

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See detailGender differences in responses in Gammarus pulex exposed to BDE-47: a gel-free proteomic approach
Gismondi, Eric ULg; Mazzucchelli, Gabriel ULg; De Pauw, Edwin ULg et al

in Ecotoxicology & Environmental Safety (2015), 122

Very few ecotoxicological studies have considered differences in toxic effects on male and female organisms. Here, we investigated protein expression differences in caeca of Gammarus pulex males and ... [more ▼]

Very few ecotoxicological studies have considered differences in toxic effects on male and female organisms. Here, we investigated protein expression differences in caeca of Gammarus pulex males and females under control conditions (unexposed) and after 96 h exposure to BDE-47. Using gel-free proteomic analysis, we have identified 45 proteins, of which 25 were significantly differently expressed according to sex and/or BDE-47 exposure. These proteins were involved in several biological processes such as energy metabolism, chaperone proteins, or transcription/translation. In unexposed amphipods, 11 proteins were significantly over-expressed in females, and 6 proteins were over-expressed in males. Under BDE-47 stress, 7 proteins were differently impacted according to sex. For example, catalase was over-expressed in exposed females and under-expressed in exposed males, as compared to respective controls. Conversely, proteins involved in energy metabolism were up-regulated in males and down-regulated in females. Our proteomic study showed differences in responses of males and females to BDE-47 exposure, emphasizing that sex is a confounding factor in ecotoxicological assessment. However, due to the limited information existing in databases on Gammarids, it was difficult to define a BDE-47 mechanism of action. The gel-free proteomic seems to be a promising method to develop in future ecotoxicological studies and thus, to improve our understanding of the mechanism of action of xenobiotics. [less ▲]

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See detailImpact of Regiella insecticola infection on EBF production in Acyrthosiphon pisum Harris
Bauwens, Julien ULg; Mazzucchelli, Gabriel ULg; De Pauw, Edwin ULg et al

Conference (2015, November 06)

Symbionts of aphids influence their host from many points of view. We investigate the potential influence of bacterial symbionts on the production and emission of the aphid alarm pheromone, E-β-franesene ... [more ▼]

Symbionts of aphids influence their host from many points of view. We investigate the potential influence of bacterial symbionts on the production and emission of the aphid alarm pheromone, E-β-franesene. Some trends could be observed in the total EβF production. Particularly, aphid strains infected by Buchnera only seemed to produce less alarm pheromone. By contrast, the presence of Regiella insecticola seemed to increase EβF production. Mevalonate pathway was investigated by RT-qPCR. This analysis showed a slightly lower transcription level o mIPPS in Regiella-infected strains. This enzyme is involved in the last step of EβF production. By contrast, two enzymes involved respectively in the linkage and release of farnesyl moeities on proteins c-terminal ends. Escape tests were conducted to assay if these results were traduced by differential behavior in front of a predator. Preliminary results showed significantly higher dropping behavior for Regiella-infected strains. [less ▲]

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

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

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

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

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

Poster (2015, May 06)

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

in Acta Gastroenterologica (2015, February 27)

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

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

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

in Acta gastroenterologica (2015, February 26)

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

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

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See detailProteomic analysis of Arabidopsis thaliana (L.) Heynh responses to a generalist sucking pest (Myzus persicae Sulzer)
Truong, Thi Dieu; Bauwens, Julien ULg; Delaplace, Pierre ULg et al

in Plant Biology (2015), 17(6), 1210-1217

Herbivorous insects can cause deep cellular changes to plant foliage following infestations depending on feeding 41 behavior. Here, a proteomic study was conducted to investigate green peach aphid (Myzus ... [more ▼]

Herbivorous insects can cause deep cellular changes to plant foliage following infestations depending on feeding 41 behavior. Here, a proteomic study was conducted to investigate green peach aphid (Myzus persicae Sulzer) 42 influence as a polyphagous pest on the defense response of Arabidopsis thaliana (L.) Heynh after aphid colony 43 set up on host plant (3 days). Analysis of about 574 protein spots on 2-DE gel revealed 31 differentially 44 expressed protein spots. Twenty out of 31 differential proteins were selected to be analyzed by mass 45 spectrometry. From 12 out of the 20 analyzed spots, we identified 7 and 9 proteins by MALDI-TOF-MS and LC-46 ESI-MS/MS, respectively. Twenty five percents of the analyzed spots contain a couple of proteins. Different 47 metabolic pathways were modulated in Arabidopsis leaves according to aphid feeding: most of them 48 corresponded to carbohydrate, amino acid and energy metabolism, photosynthesis, defense response and 49 translation. This paper has established a survey of early alterations induced in the proteome of Arabidopsis plants 50 by the M. persicae aphids. It provides valuable insights to uncover the complex response of plants to biological 51 stress, particularly with herbivorous insects with sucking feeding behavior. 52 53 [less ▲]

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See detailPotential Proteomic Biomarkers Associated To Mucosal Healing In Crohn’s Disease
MEUWIS, Marie-Alice ULg; Baiwir, Dominique ULg; Mazzucchelli, Gabriel ULg et al

Poster (2014, October 06)

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

Introduction and objectives: In Crohn's disease (CD), there is a discrepancy between clinical activity of the disease (symptoms) and intestinal healing. However absence of tissue healing is associated with the risk of relapse and tissue damage progression. Endoscopy is costly and invasive. Hence biomarkers correlating with intestinal healing could improve disease management. We aimed to identify potential biomarkers associated to CD mucosal healing by a shotgun proteomics label free study. Methods: We used the STORI clinical trial cohort (n=103) aiming at identifying markers associated to relapse prediction after Infliximab treatment withdrawals. We used serum samples of patients in clinical remission, grouped according to the degree of intestinal healing seen at endoscopy. We performed depletion of the 20 most abundant plasma proteins on each serum pools and ran a proteomics label free differential analysis using 2D-nanoUPLC-MSE HDMS Synapt G1 for data acquisition and Protein Lynks Global Server vs 2.4 for data analysis (Waters, Corp., Milford, USA). Results and Discussion: We obtained potential biomarkers and designed a multiplexed -selected reaction monitoring (SRM) method for validation of these candidates in each individual patient. The method may also be tested in an independent set of IBD patients with and without mucosal healing. Conclusions: This research strategy and results of SRM validation of potential biomarkers associated to mucosal healing in this cohort of CD patients as well as the tests done on other CD patients, may provide new opportunities for CD follow-up tests development. [less ▲]

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