References of "Smargiasso, Nicolas"
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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 detailAn Improved Molecular Histology Method for Ion Suppression Monitoring and Quantification of Phosphatidyl Cholines During MALDI MSI Lipidomics Analyses.
Jadoul, Laure ULg; Smargiasso, Nicolas ULg; Pamelard, Fabien et al

in OMICS : A Journal of Integrative Biology (2016), 20(2), 110-21

Tissue lipidomics is one of the latest omics approaches for biomarker discovery in pharmacology, pathology, and the life sciences at large. In this context, matrix-assisted laser desorption/ionization ... [more ▼]

Tissue lipidomics is one of the latest omics approaches for biomarker discovery in pharmacology, pathology, and the life sciences at large. In this context, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is the most versatile tool to map compounds within tissue sections. However, ion suppression events occurring during MALDI MSI analyses make it impossible to use this method for quantitative investigations without additional validation steps. This is especially true for lipidomics, since different lipid classes are responsible for important ion suppression events. We propose here an improved lipidomics method to assess local ion suppression of phospatidylcholines in tissues. Serial tissue sections were spiked with different amounts of PC(16:0 d31/18:1) using a nebulization device. Settings for standard nebulization were strictly controlled for a detection similar to when using spiked tissue homogenates. The sections were simultaneously analyzed by MALDI MSI using a Fourier transform ion cyclotron resonance analyzer. Such a spray-based approach allows taking into account the biochemical heterogeneity of the tissue for the detection of PC(16:0 d31/18:1). Thus, here we present the perspective to use this method for quantification purposes. The linear regression lines are considered as calibration curves and we calculate PC(16:0/18:1) quantification values for different ROIs. Although those values need to be validated by a using a different independent approach, the workflow offers an insight into new quantitative mass spectrometry imaging (q-MSI) methods. This approach of ion suppression monitoring of phosphocholines in tissues may be highly interesting for a large range of applications in MALDI MSI, particularly for pathology using translational science workflows. [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 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 detailStudy of N-linked glycosylation in different life stages of the red flour beetle (Tribolium castaneum)
Smargiasso, Nicolas ULg; Walski, Tomasz; Van Damme, Els et al

Poster (2015, June)

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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 detailCombined use of Ion Mobility and Collision-Induced Dissociation to investigate the opening of disulfide bridges by Electron-Transfer Dissociation in peptides bearing two disulfide bonds
Massonnet, Philippe ULg; Upert, Gregory; Smargiasso, Nicolas ULg et al

in Analytical Chemistry (2015)

Disulfide bonds are post-translational modifications (PTMs) often found in peptides and proteins. They increase their stability towards enzymatic degradations, and provide the structure and (consequently ... [more ▼]

Disulfide bonds are post-translational modifications (PTMs) often found in peptides and proteins. They increase their stability towards enzymatic degradations, and provide the structure and (consequently) the activity of such folded proteins. The characterization of disulfide patterns, i.e the cysteine connectivity, is crucial to achieve a global picture of the active conformation of the protein of interest. Electron Transfer Dissociation (ETD) constitutes a valuable tool to cleave the disulfide bonds in the gas phase, avoiding chemical reduction/alkylation in solution. To characterize the cysteine pairing, the present work proposes (i) to reduce by ETD one of the two disulfide bridges of model peptides , resulting in the opening of the cyclic structures, (ii) to separate the generated species by ion-mobility and, (iii) to characterize the species using CID. Results of this strategy applied to several peptides show different behaviors depending on the connectivity. The loss of SH• radical species, observed for all the peptides, confirms the cleavage of the disulfides during the ETD process. [less ▲]

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See detailIon mobility-mass spectrometry to perform structural classifications of disulfide-bridged-peptides
Massonnet, Philippe ULg; Upert, Gregory; Morsa, Denis ULg et al

Poster (2014, November)

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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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