References of "Mc Cann, Andréa"
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See detailComputational chemistry and ion mobility – mass spectrometry at high resolving power suggest prototropism of cyclic lipopeptides
Mc Cann, Andréa ULiege; Kune, Christopher ULiege; Far, Johann ULiege et al

Poster (2019, June)

Introduction Cyclic lipopeptides (CLPs) are cyclic hydrophilic peptides with a lipid ramification using a β-hydroxy fatty acid that are produced by bacteria in a ribosome independent manner. Despite CLPs ... [more ▼]

Introduction Cyclic lipopeptides (CLPs) are cyclic hydrophilic peptides with a lipid ramification using a β-hydroxy fatty acid that are produced by bacteria in a ribosome independent manner. Despite CLPs have relatively low molecular weight between 800 and 2,000 Da, the analysis of lipopeptides remains challenging due to the wide variety of synthetized isoforms differing in fatty acid chain length, in methyl group branching position, and in the nature of the amino-acids residues. These isoforms are suspected to have different biological activities requiring development of reliable methods for CLPs characterization. We present here an original approach combining UPLC and ion mobility - mass spectrometry at high resolving powers to separate the different species. Experimentally determined CCS will be compared with theoretical ones. Methods Lipopeptides were separated by UPLC (I-class, Waters, U.K.) on a C18 BEH column and identified by CID MS/MS mass spectrometry. Ion mobility – mass spectrometry (IM-MS) measurements were performed on a traveling wave ion mobility mass spectrometer (Synapt G2 HDMS from Waters, U.K.) and on a trapped Ion Mobility Mass Spectrometer (timsTOF, Bruker Daltonics, U.S.A.) to investigate the 3D structures of the ionized lipopeptides. Accurate Collison Cross Section (CCS) were obtained in both positive and negative mode and compared with theoretical CCS. Density Functional Theory (Gaussian) was used for structure optimizations at the CAM-B3LYP level of theory and 3-21G as basis set. Theoretical CCS have been computed from optimized structures using the trajectory method from IMoS V2. Preliminary data Separation of lipopeptides such as surfactins was successfully performed by reverse phase liquid chromatography. Lipopeptides were separated according to lipidic chain length and the branching position of the methyl group (iso/anteiso/linear). In positive ionization mode, the infusion of each isolated isoforms in the Synapt G2 showed a broad IMS distribution. These ion mobility profiles suggested the presence of different conformers. The higher IMS resolving power of the TIMS allowed the detection of at least three near-resolved peaks for a single isomer. In negative ionization mode however, only one peak was observed in the IM-MS profile on both Synapt G2 and TIMS, corresponding to one CCS. We included prototropic hypotheses where all the potential protonation and deprotonation sites on each lipopeptide had been determined by theoretical calculation. The abundances of the species in the CCS distributions of the resulting structures were obtained based on the Boltzmann distribution. Regarding the surfactin family, preliminary calculations by DFT shows that several protonation sites are energetically favorable and that the proton localization has a significant effect on the resulting CCS (∆CCS = 10Ų). These results are in good agreement with the experimental IMS profiles, obtained in both positive and negative ionization mode. Lipopeptides are then not related to a unique CCS value but a set of IM-MS profile that probably contains additional structural and physicochemical information. Novel aspect Experimental and theoretical approaches for lipopeptides IMS profiles analysis: protonation site determination, peaks intensity prediction and structural information extraction. [less ▲]

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See detailRapid visualization of chemically related compounds using Kendrick mass defect as a filter in mass spectrometry imaging
Kune, Christopher ULiege; Mc Cann, Andréa ULiege; La Rocca, Raphaël ULiege et al

E-print/Working paper (2019)

Kendrick mass defect (KMD) analysis is widely used for helping the detection and identification of chemically related compounds based on exact mass measurements. We report here the use of KMD as a ... [more ▼]

Kendrick mass defect (KMD) analysis is widely used for helping the detection and identification of chemically related compounds based on exact mass measurements. We report here the use of KMD as a criterion for filtering complex mass spectrometry data. The method enables an automated, faster and efficient data processing, enabling the reconstruction of 2D distributions of family of homologous compound in MSI images. We show that the KMD filtering, based on a homemade software, is suitable for low resolution and high resolution MSI data. This method has been successfully applied to two different types of samples, bacteria co-cultures and brain tissue section. [less ▲]

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See detailInvestigation of data independent acquisition for small molecules structure elucidation in image by FT-ICR MS
La Rocca, Raphaël ULiege; Mc Cann, Andréa ULiege; Far, Johann ULiege et al

Poster (2019, April 14)

Mass Spectrometry Imaging (MSI) is an analytical method allowing the mapping of molecules from thin sections of samples after their ionization/desorption usually obtained by MALDI. However, in the case of ... [more ▼]

Mass Spectrometry Imaging (MSI) is an analytical method allowing the mapping of molecules from thin sections of samples after their ionization/desorption usually obtained by MALDI. However, in the case of biological samples, the study of localized metabolic pathways is hampered by the lack of structural identification of the detected molecules even with high mass resolution. In this context, we are developing an independent data acquisition method for MSI by fragmenting all the precursors without prior m/z selection. The obtained MS/MS spectra are then attempted to be deconvoluted with the aim of attributing the fragments to their parents by the help of their spatial distributions.The chosen model for acquiring MSI data is the chemical communication observed between bacteria and fungi colonies, grown on agar- based medium. On this sample, 2 images have been acquired using MALDI-FT-ICR (SolariX XR, 9.4T, fitted with the ParaCell®). The first one, called precursors’ image (PI), is obtained at low collision energy and contains parent ions. The second one, called fragments’ image (FI), has been acquired at greater collision energy, and thus contains mostly their fragment ions. The fragmentation is made in the hexapole. A deconvoluted MS/MS spectrum for each ion in PI is calculated with the help of non-negative least square regression. In a few words, the algorithm tries to recreate the spatial distribution of an ion from FI by a weighted sum of the ions in PI. A deconvoluted MS/MS spectrum of an ion in PI corresponds thus to its weights for each associated ion in FI. A first stage of quality control for evaluating the deconvolution process is made by comparing the reconstructed MS/MS spectrum of a reference ion, to its experimentally acquired MS/MS spectrum. In the present study, the reference molecule is a lipopeptide called viscosin added as reference spots before the image acquisition.The reconstructed MS/MS spectrum for all the peaks in PI are compared to the experimental MS/MS spectrum of viscosin based on two parameters: cosine similarity and the proportion of intensity in the reconstructed spectrum belonging to a true fragment. The obtained results shows a good correlation between the experimentally acquired MS/MS and the reconstructed MS/MS of viscosin, however the algorithm is not yet able to distinguish variables with corresponding spatial distributions. In the future, new algorithms will be developed and tested to overcome the issue of correlated variables to implement the recording of data independent MS/MS images. [less ▲]

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See detailFrom noise to signal: Kendrick mass filtering for high-resolution mass spectrometry imaging analysis
Mc Cann, Andréa ULiege; Kune, Christopher ULiege; Arguelles Arias, Anthony ULiege et al

Poster (2019, April 14)

Introduction Over the last years, lots of progress have been done in the development of mass spectrometry imaging, making the technique more and more accessible for various applications, such as ... [more ▼]

Introduction Over the last years, lots of progress have been done in the development of mass spectrometry imaging, making the technique more and more accessible for various applications, such as biomarkers discovery or bioactive compounds identification. However, the progresses made in terms of spatial and instrumental resolution has for consequences the dramatic increase of dataset size, shifting the burden from data production to data analysis. We propose here to use a semi-targeted method based on Kendrick mass defect (KMD) analysis to immediately identify the chemistry-related compounds in mass spectrometry imaging applied to microbiology samples. Thanks to an in-house software, we are now able to better understand the bacteria-bacteria interactions. Materials and methods Bacillus velezensis GA1 and Pseudomonas sp. CMR12a were inoculated on a semi-solid agar-based medium and incubated at 30°C. Region of interest was cut directly from the petri dish and transferred to the target ITO plate, previously covered with double sided conductive carbon tape. This assembly was then put in a vacuum desiccator until complete drying (overnight), and covered with HCCA matrix. Mass spectrometry images were obtained using a FT-ICR mass spectrometer (9.4T SolariX, Bruker Daltonics, Bremen, Germany). Data analysis was performed on an in-house software. Results & Discussion Thanks to the KMD analysis, we were able to directly compare and identify the nature of the compounds detected in MSI such as lipids (1) or lipopeptides (2a), without the need of an extensive database search. It was also possible to identify some lipopeptides degradation occurring nearby Pseudomonas (2b). Thanks to our in-house software, the compounds with a similar chemistry can now be filtrated and the image can be reconstructed, removing thus the noise and focusing only on the signal. [less ▲]

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See detailIon mobility mass spectrometry for lipopeptides analysis
Mc Cann, Andréa ULiege; Kune, Christopher ULiege; Far, Johann ULiege et al

Poster (2019, March 31)

Cyclic lipopeptides (CLPs) are produced by bacteria in a ribosome independent manner and consists in a hydrophilic peptide linked to a B-hydroxy fatty acid chain. Despite CLPs have relatively low ... [more ▼]

Cyclic lipopeptides (CLPs) are produced by bacteria in a ribosome independent manner and consists in a hydrophilic peptide linked to a B-hydroxy fatty acid chain. Despite CLPs have relatively low molecular weight (between 800 and 2,000 Da), the analysis of lipopeptides remains challenging due to the wide variety of synthetized isoforms differing in fatty acid chain length, in methyl group branching position, and in the nature of the amino-acids residues. These isoforms are suspected to have different biological activities requiring development of reliable methods for CLPs identification and characterization. In this work, we present an original approach combining UPLC and ion mobility - mass spectrometry at high resolving powers to separate and identify the different species. Lipopeptides were separated by UPLC (I-class, Waters, U.K.) on a C18 BEH column and identified by CID MS/MS mass spectrometry. Ion mobility – mass spectrometry (IM-MS) measurements were performed on a trapped Ion Mobility Mass Spectrometer (TimsTOF, Bruker Daltonics, U.S.A.). Accurate Collison Cross Section (CCS) were obtained in both positive and negative mode and compared with theoretical CCS obtained by the trajectory method from IMoS V2. Separation of lipopeptides such as surfactins, iturins, or mycosubtilins was successfully performed by reverse phase liquid chromatography. Lipopeptides were separated according to lipid chain length and according to the branching position of the methyl group (iso/anteiso/linear). Ion mobility analysis with computational chemistry support gave additional information on the lipopeptides structure and family. Structural information are obtained by experimental and theoretical CCS comparison while the lipopeptides family could be determined with CCS/mass trends. These results pave the way to a new strategy for fast lipopeptides identification and could potentially contribute to lipopeptides structure elucidation. [less ▲]

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See detailAnalytical tool for lipopeptide identification
Mc Cann, Andréa ULiege; Kune, Christopher ULiege; Far, Johann ULiege et al

Conference (2018, November 30)

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See detailApplication of a curated genome-scale metabolic model of CHO DG44 to an industrial fed-batch process
Calmels, Cyrielle; Mc Cann, Andréa ULiege; Malphettes, Laetitia et al

in Metabolic Engineering (2018)

CHO cells have become the favorite expression system for large scale production of complex biopharmaceuticals. However, industrial strategies for upstream process development are based on empirical ... [more ▼]

CHO cells have become the favorite expression system for large scale production of complex biopharmaceuticals. However, industrial strategies for upstream process development are based on empirical results, due to a lack of fundamental understanding of intracellular activities. Genome scale models of CHO cells have been reconstructed to provide an economical way of analyzing and interpreting large-omics datasets, since they add cellular context to the data. Here the most recently available CHO-DG44 genome-scale specific model was manually curated and tailored to the metabolic profile of cell lines used for industrial protein production, by modifying 601 reactions. Generic changes were applied to simplify the model and cope with missing constraints related to regulatory effects as well as thermodynamic and osmotic forces. Cell line specific changes were related to the metabolism of high yielding production cell lines. The model was semi-constrained with 24 metabolites measured on a daily basis in n=4 independent industrial 2 L fed batch cell culture processes for a therapeutic antibody production. This study is the first adaptation of a genome scale model for CHO cells to an industrial process, that successfully predicted cell phenotype. The tailored model predicted accurately both the exometabolomics data (r²≥ 0.8 for 96% of the considered metabolites) and growth rate (r²=0.91) of the industrial cell line. Flux distributions at different days of the process were analyzed for validation and suggestion of strategies for medium optimization. This study shows how to adapt a genome scale model to an industrial process and sheds light on the metabolic specificities of a high production process. The curated genome scale model is a great tool to gain insights into intracellular fluxes and to identify possible bottlenecks impacting cell performances during production process. The general use of genome scale models for modeling industrial recombinant cell lines is a long-term investment that will highly benefit process development and speed up time to market. [less ▲]

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See detailMass spectrometry based techniques for lipopeptides analysis
Mc Cann, Andréa ULiege; Delvaux, Cédric ULiege; Far, Johann ULiege et al

Conference (2018, July 05)

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See detailIon mobility mass spectrometry for lipopeptides analysis
Mc Cann, Andréa ULiege; Delvaux, Cédric ULiege; Far, Johann ULiege et al

Scientific conference (2018, July 04)

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See detailEvaluation of capillary electrophoresis separation of cyclic lipopeptides
Mc Cann, Andréa ULiege; Far, Johann ULiege; De Pauw, Edwin ULiege et al

Poster (2018, March 29)

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See detailEmerging mass spectrometry techniques for lipopeptide understanding
Mc Cann, Andréa ULiege; Far, Johann ULiege; Delvaux, Cédric ULiege et al

Scientific conference (2018, March 05)

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See detailLipidomics for robust high performance process development
Mc Cann, Andréa ULiege; Mathy, Grégory; Malphettes, Laetitia

Conference (2017, May 14)

Background and novelty: CHO cell lines are common hosts for the production of biopharmaceuticals proteins. So far, considerable progress has been made increasing productivity of cell culture to meet the ... [more ▼]

Background and novelty: CHO cell lines are common hosts for the production of biopharmaceuticals proteins. So far, considerable progress has been made increasing productivity of cell culture to meet the rapidly growing demand for antibody biopharmaceuticals through increased cell densities and longer cultured time. These major improvements have nevertheless lead to an important drawback : the increase of the process related impurities, bringing new challenges for process and harvest development. Among the process related impurities such as HCPs or DNA the potential impact of lipids production and release during a cell culture is still poorly understood due to the complex nature and diversity of this class of molecules. Thanks to recent advances in analytical tools especially mass spectrometry, the advent of lipidomics offers now the feasibility to study several thousands of lipid species, unravelling the possibility to understand and potentially control the interactions between high performance bioreactor processes, harvest conditions and purification. Experimental approach: In order to analyze and quantify lipids, we developed a three steps method. In a first step, lipids were extracted with Methyl tert-butyl ether (MTBE) according to Matyash method. Lipids were then separated by liquid chromatography using either HILIC of reverse phase column prior to detection and quantification by mass spectrometry. All lipid classes were detected by ESI-MS/MS excepted cholesterol (APCI-MS/MS). Finally we applied this method to analyze the lipid content of four different cell lines each expressing a different recombinant protein, during a 14 days fed batch process. Results and discussion: Lipid from CHO cells were successfully extracted with a yield between 80% and 95% depending on the different lipid classes. Stable isotope labelled lipids were used as internal standard in order to have comparable results between batches. The lipid profiles were not only different for the 4 tested cell lines, it was also different for a given cell line cultivated under various experimental conditions. Interestingly, in some cell lines/experimental conditions, we highlighted an overproduction of triglycerides and cholesterol leading to the accumulation of lipid droplets known as energy storage sink. At the metabolic level, these finding suggest a relative overflow of the carbon metabolism. From a process development perspective these findings can be considered on the one hand as a resource waste since the stored energy is not used for protein/biomass biosynthesis and on second hand to additional process problems during the harvest and the first capture steps given the hydrophobic nature of these molecules. Additional analysis will required to fully understand the link existing between lipids metabolism, cell line, and process development conditions. We believe that these investigation will open the door to many applications such as clone selection, process, and harvest development. [less ▲]

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