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See detailFundamental Studies on Poly(2-oxazoline) Side Chain Isomers Using Tandem Mass Spectrometry and Ion Mobility-Mass Spectrometry
Haler, Jean ULiege; de la Rosa, Victor R.; Massonnet, Philippe ULiege et al

in Journal of the American Society for Mass Spectrometry (in press)

When polymer mixtures become increasingly complex, the conventional analysis techniques become insufficient for complete characterization. Mass spectrometric techniques can satisfy this increasing demand ... [more ▼]

When polymer mixtures become increasingly complex, the conventional analysis techniques become insufficient for complete characterization. Mass spectrometric techniques can satisfy this increasing demand for detailed sample characterization. Even though isobaric polymers are indistinguishable using simple mass spectrometry (MS) analyses, more advanced techniques such as tandem MS (MS/MS) or ion mobility (IM) can be used. Here, we report proof of concept for characterizing isomeric polymers, namely poly(2-n-propyl-2-oxazoline) (Pn-PrOx) and poly(2-isopropyl-2-oxazoline) (Pi-PrOx), using MS/MS and IM-MS. Pi-PrOx ions lose in intensity at higher accelerating voltages than Pn-PrOx ions during collision-induced dissociation (CID) MS/MS experiments. A Pn/i-PrOx mixture could also be titrated using survival yield calculations of either precursor ions or cation ejection species. IM-MS yielded shape differences in the degree of polymerization (DP) regions showing the structural rearrangements. Combined MS techniques are thus able to identify and deconvolute the molar mass distributions of the two isomers in a mixture. Finally, the MS/MS and IM-MS behaviors are compared for interpretation. [less ▲]

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See detailDevelopment of bimetallic nanoparticles for Surface-Assisted Laser Desorption/Ionisation Mass Spectrometry imaging of small molecules
Verdin, Alexandre ULiege; Malherbe, Cédric ULiege; Bertrand, Virginie ULiege et al

Poster (2019, June 04)

Mass Spectrometry Imaging (MSI) allows the simultaneous visualisation of hundreds to thousands of molecules in biological tissue slides enabling the comprehension of complex biochemical processes ... [more ▼]

Mass Spectrometry Imaging (MSI) allows the simultaneous visualisation of hundreds to thousands of molecules in biological tissue slides enabling the comprehension of complex biochemical processes. Additionally, the analysis of untargeted MSI data with advanced statistical tools has the potential to reveal pertinent features of the sample such as the progression or outcome of a disease or the response to a treatment. Recently, metallic nanoparticles (NPs) have received increasing attention as desorption/ionization substrate for Surface-Assisted Laser Desorption/Ionization-MS (SALDI-MS). Metallic NPs provide several advantages over organic MALDI matrices for the analysis of small molecules: high absorption coefficients of the ionization laser (in the UV-Visible range), the ability to load small molecules on their surface by physical or chemical adsorption, little interferences in the low m/z range and a simple sample preparation (no crystallization required). SALDI-MSI is adapted to study the variations of the molecular composition associated with various diseases and to visualize the localization of pertinent small molecules in complex biological tissues. Innovative Au@Ag (core@shell) bimetallic NPs are interesting for SALDI applications, especially because controlling the thickness of the Ag shell around the Au core allows the tuning of the absorption in near-UV. These Au@Ag NPs permits to maximize the energy absorption for an efficient ionization and desorption of analytes adsorbed to the NPs. Moreover Au@Ag NPs can also be used for Surface-Enhanced Raman Spectroscopy (SERS) imaging which is a highly complementary technique. We report here a study of ovarian cancer tissue using Au@Ag NPs to characterize the overexpression of membrane receptor in the tumor by SERS imaging and to gain molecular insights by SALDI-MSI. [less ▲]

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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 detailGas-Phase Dynamics of Collision Induced Unfolding, Collision Induced Dissociation, and Electron Transfer Dissociation-Activated Polymer Ions
Haler, Jean ULiege; Massonnet, Philippe ULiege; Far, Johann ULiege et al

in Journal of the American Society for Mass Spectrometry (2019), 30(4), 563572

Polymer characterizations are often performed using mass spectrometry (MS). Aside from MS and different tandem MS (MS/MS) tech- niques, ion mobility–mass spectrometry (IM-MS) has been recently added to ... [more ▼]

Polymer characterizations are often performed using mass spectrometry (MS). Aside from MS and different tandem MS (MS/MS) tech- niques, ion mobility–mass spectrometry (IM-MS) has been recently added to the inventory of char- acterization technique. However, only few studies have focused on the reproducibility and robust- ness of polymer IM-MS analyses. Here, we per- form collisional and electron-mediated activation of polymer ions before measuring IM drift times, collision cross-sections (CCS), or reduced ion mobilities (K0). The resulting IM behavior of different activated product ions is then compared to non-activated native intact polymer ions. First, we analyzed collision induced unfolding (CIU) of precursor ions to test the robustness of polymer ion shapes. Then, we focused on fragmen- tation product ions to test for shape retentions from the precursor ions: cation ejection species (CES) and product ions with m/z and charge state values identical to native intact polymer ions. The CES species are formed using both collision induced dissociation (CID) and electron transfer dissociation (ETD, formally ETnoD) experiments. Only small drift time, CCS, or K0 deviations between the activated/formed ions are observed compared to the native intact polymer ions. The polymer ion shapes seem to depend solely on their mass and charge state. The experiments were performed on three synthetic homopolymers: poly(ethoxy phosphate) (PEtP), poly(2-n-propyl- 2-oxazoline) (Pn-PrOx), and poly(ethylene oxide) (PEO). These results confirm the robustness of polymer ion CCSs for IM calibration, especially singly charged polymer ions. The results are also discussed in the context of polymer analyses, CCS predictions, and probing ion–drift gas interaction potentials. [less ▲]

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See detailUse of IMMS for the investigation of the effects of shapes, densities and dipole moments on the CCS of small metal complexes
Kune, Christopher ULiege; Haler, Jean ULiege; Rappe, Sophie ULiege et al

Conference (2019, March 31)

Ion mobility mass spectrometry provides the collision cross section (CCS) of ions in the gas phase. The CCS growth of ions sharing similar physicochemical properties such as charge state, geometry, shape ... [more ▼]

Ion mobility mass spectrometry provides the collision cross section (CCS) of ions in the gas phase. The CCS growth of ions sharing similar physicochemical properties such as charge state, geometry, shape, apparent density and dipole moments correlates well with their masses evolution. In this presentation, we study the effect on the CCS/mass correlations after variation of these physicochemical properties using a set of negatively charged iron center complexes with halogens (chloride, bromide and iodide) and carboxylates (linear or branched mono carboxylate) as ligands. Due to the halogen ligands are more compact and denser than the carboxylate ligands, all combinations of these ligands lead to a set of iron center complexes (theoretically superior to 250 complexes) having different CCS growth. Indeed, the number of ligands imposes the geometry (linear, trigonal plan, tetrahedral) while the halogen/carboxylate ligands ratio influences the apparent densities and the shape (spherical or near spherical) of the complex ions. Moreover, theses complex ions also share different dipole moment. These properties have been estimated by computational chemistry (Molecular Mechanism or Density Functional Theory levels) for each complex whereas the CCS and mass values have been measured by traveling wave ion mobility (Water’s Synapt G2). Comparing all the experimental CCS/mass trends in function of the physicochemical properties shows that the apparent density is the main factor influencing these correlations following by the shape and geometry factors. The variations on the dipole moment of theses complexes seems to be insufficient to significantly affect the CCS/mass correlation. This work also highlights the capability of CCS prediction of a given ion based on its mass, apparent density, shape, geometry, and the dipole moment. [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 detailAutomatic exploration of images using ultra high resolution MS
La Rocca, Raphaël ULiege; Tiquet, Mathieu ULiege; Far, Johann ULiege et al

Poster (2019)

Mass Spectrometry Imaging (MSI) is an analytical method allowing the mapping of molecules in thin sections of samples after their ionization/desorption thanks to an appropriate method. In the case of ... [more ▼]

Mass Spectrometry Imaging (MSI) is an analytical method allowing the mapping of molecules in thin sections of samples after their ionization/desorption thanks to an appropriate method. In the case of biological samples, the attribution of molecular formula, and if possible structures, related to the m/z values with their localization can be useful for improving our understanding of biological metabolic pathways. Unfortunately, bioinformatics tools dedicated to the analysis of such data are not open source and are still limited in terms of features. These lacks constrained scientists to build their own tools. In this context, we have developed a tool from existing methods to process images acquired by MALDI FT-ICR-MS, aiming the unambiguous identification of lipids. The image is processed automatically by taking into account the spatial distribution of each m/z value (ionic image). The goal of this approach is to discriminate unstructured ionic images from informative ionic images which revealed regions of interest. This classification is made by an automated evaluation of the level of structure within each image. Briefly, the level of structure of an ionic image is determined by counting the number of clusters of pixels displaying similar levels of intensity, in contrast to ubiquitous ions images in which the levels of intensity in the pixels are randomly spread. The MS image is analysed as follows: m/z values corresponding to lipids are selected based on their exact masses provided by FT-ICR MS (similarly to a peak list). These selected m/z are then curated by evaluating their level of structure. The used of a false discovery rate (FDR) to control this latter step is investigated. Finally, the different validated m/z values are exploited to discover regions of interest in the sample by the means of unsupervised learning algorithms. We describe in this work how to compute the FDR and the spatial distribution for each m/z value. The identifications of lipids were performed using the LIPID MAPS database and different informatics tools were developed in the programming language R. [less ▲]

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See detailBenchmarking IM-MS gas-phase data based on cross-linking strategies
Hanozin, Emeline ULiege; Grifnée, Elodie ULiege; Gattuso, Hugo ULiege et al

Conference (2019)

Since the apparition of conservative soft-ionization sources in the late 80s, i.e. electrospray (ESI) and matrix-assisted laser desorption-ionization, mass spectrometry (MS) has been extensively used to ... [more ▼]

Since the apparition of conservative soft-ionization sources in the late 80s, i.e. electrospray (ESI) and matrix-assisted laser desorption-ionization, mass spectrometry (MS) has been extensively used to identify, quantify and characterize wide types of biomolecules. Especially, ESI sources make feasible the transfer of intact biomolecules and macromolecular complexes in the mass spectrometer, opening the so-called field of “native” MS. However, major questioning and inherent skepticism yet surround the native MS field concerning the ability to preserve non-bonded biological interactions during and after the molecular transfer from the physiological aqueous media to the gas-phase. Indeed, the introduction of biological structures into the gas-phase may eventually result in a reshape of their tridimensional fold, therefore leading to a misinterpretation of further derived structural data. In an effort to circumvent gas-phase unfolding scenarios, we here suggest a structural strategy relying on intra-molecular covalent cross-linkers acting as molecular scaffolds. We focused on small 10 kDa to 20 kDa proteins, such as cytochrome c, myoglobin and β-lactoglobulin, which are yet often used as model systems to gauge wide range of structural, kinetic and thermodynamics methodologies. We studied the products of “type 0” and “type 1” cross-linking reactions achieved using bissulfosuccinimidyl suberate (BS3) as a linker reagent and compared them with their non-linked counter-parts regarding their respective structural properties. The model systems were structurally investigated both in solution, through circular dichroism and thermal denaturation measurements, and in the gas-phase following ESI injection, through IM-MS and collision induced unfolding (CIU) measurements. The gas-phase data were acquired from different solvent conditions and compared with “native-compliant” benchmarks furnished by NMR spectroscopy. In general, the content in secondary structures within the proteins was found barely unaffected by the presence of the linkers when operating in physiological solution, therefore witnessing a preservation of the native fold upon linking reaction. However, after ionization and transfer into the gas-phase, we monitored significant reshapes in the conformational landscapes materialized through a shift of the collision cross-sections distribution towards lower values when linkers are present. The magnitude of the compaction effect is directly dependent on the protein charge and linker content. Altogether, the comprehension of the fate of electro-sprayed cross-linked conformers in the gas-phase and their respective correlation with the physiological fold achieved in solution constitute major steps in the validation of this technique as part of native MS workflows. [less ▲]

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See detailMulti-Enzymatic Limited Digestion: The Next-Generation Sequencing for Proteomics?
Morsa, Denis ULiege; Baiwir, Dominique ULiege; La Rocca, Raphaël ULiege et al

in Journal of proteome research (2019)

Over the past 40 years, proteomics, generically defined as the field dedicated to the identification and analysis of proteins, has tremendously gained in popularity and potency through advancements in ... [more ▼]

Over the past 40 years, proteomics, generically defined as the field dedicated to the identification and analysis of proteins, has tremendously gained in popularity and potency through advancements in genome sequencing, separative techniques, mass spectrometry, and bioinformatics algorithms. As a consequence, its scope of application has gradually enlarged and diversified to meet specialized topical biomedical subjects. Although the tryptic bottom-up approach is widely regarded as the gold standard for rapid screening of complex samples, its application for precise and confident mapping of protein modifications is often hindered due to partial sequence coverage, poor redundancy in indicative peptides, and lack of method flexibility. We here show how the synergic and time-limited action of a properly diluted mix of multiple enzymes can be exploited in a versatile yet straightforward protocol to alleviate present-day drawbacks. Merging bottom-up and middle-down ideologies, our results highlight broad assemblies of overlapping peptides that enable refined and reliable characterizations of proteins, including variant identification, and their carried modifications, including post-translational modifications, truncations, and cleavages. Beyond this boost in performance, our methodology also offers efficient de novo sequencing capabilities, in view of which we here present a dedicated custom assembly algorithm. [less ▲]

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See detailDevelopment of bimetallic nanoparticles for Surface-Assisted Laser Desorption/Ionisation Mass Spectrometry imaging of small molecules
Verdin, Alexandre ULiege; Malherbe, Cédric ULiege; Bertrand, Virginie ULiege et al

Poster (2019)

Metallic nanoparticles (NPs) receive increasing attention as desorption/ionization substrate for Surface-Assisted Laser Desorption Ionization-MS (SALDI-MS) [1]. Metallic NPs offer high extinction ... [more ▼]

Metallic nanoparticles (NPs) receive increasing attention as desorption/ionization substrate for Surface-Assisted Laser Desorption Ionization-MS (SALDI-MS) [1]. Metallic NPs offer high extinction coefficients of the ionization laser (in the UV-Visible range of wavelengths) and are able to ionize small molecules with little interferences in the low m/z range with a simple sample preparation [2]. SALDI-MSI is adapted to study the variations of the molecular composition associated with various diseases and to visualize the localization of pertinent small molecules in complex biological tissues. Innovative Au@Ag (core@shell) bimetallic NPs are interesting for SALDI applications, especially because controlling the thickness of the Ag shell around the Au core allows the tuning of the absorption in near-UV. The Au@Ag NPs permits to vary the energy transfer for an efficient ionization and desorption of analytes adsorbed to the NPs. Moreover Au@Ag NPs can also also be used for Surface-Enhanced Raman Spectroscopy (SERS) imaging. We report here a study of ovarian cancer tissue using Au@Ag NPs to localize the tumor by SERS imaging and to gain molecular insight of tumors by SALDI-MSI. [less ▲]

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See detailMicroproteomic Profiling of High-Grade Squamous Intraepithelial Lesion of the Cervix: Insight into Biological Mechanisms of Dysplasia and New Potential Diagnostic Markers
Pottier, Charles ULiege; Kriegsmann, M.; Alberts, D. et al

in Proteomics. Clinical Applications (2019), 13(1)

Purpose: High-grade squamous intraepithelial lesion (HSIL) is a known precursor for squamous cell carcinoma of uterine cervix. Although it is known that SILs are associated to infection by human ... [more ▼]

Purpose: High-grade squamous intraepithelial lesion (HSIL) is a known precursor for squamous cell carcinoma of uterine cervix. Although it is known that SILs are associated to infection by human papillomavirus, downstream biological mechanisms are still poorly described. In this study, we compared the microproteomic profile of HSIL to normal tissues: ectocervix (ectoC) and endocervix (endoC). Experimental design: Tissue regions of endoC, ectoC, and HSlL were collected by laser microdissection (3500 cells each) from five patients. Samples were processed and analyzed using our recently developed laser microdissection-based microproteomic method. Tissues were compared in order to retrieve HSIL's proteomic profile. Potentially interesting proteins for pathology were stained by immunohistochemistry. Results: We identified 3072 proteins among the fifteen samples and 2386 were quantified in at least four out of the five biological replicates of at least one tissue type. We found 236 proteins more abundant in HSIL. Gene ontology enrichments revealed mechanisms of DNA replication and RNA splicing. Despite the squamous nature of HSIL, a common signature between HSIL and endoC could be found. Finally, potential new markers could support diagnosis of dysplasia in SILs. Conclusion and clinical relevance: This microproteomic investigation of HSIL gives insights into the biology of cervical precancerous lesions. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim [less ▲]

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See detailIdentification of new bioactive peptides from Kefir milk through proteopeptidomics: Bioprospection of antihypertensive molecules
Amorim, F. G.; Coitinho, L. B.; Dias, A. T. et al

in Food Chemistry (2019), 282

Kefir, a probiotic beverage prepared from fermented milk, has been associated with antihypertensive activity. However, the bioactive molecules responsible for this activity still remain unclear. Therefore ... [more ▼]

Kefir, a probiotic beverage prepared from fermented milk, has been associated with antihypertensive activity. However, the bioactive molecules responsible for this activity still remain unclear. Therefore, in this study we aim to evaluate the mechanisms of the antihypertensive effects of Kefir in the two-kidney one-clip hypertension model, and to bioprospect for bioactive peptides identified by proteomic methodologies. Treatment with Kefir was performed via gavage, and resulted in a 37 mmHg reduction in systolic arterial pressure and 19% inhibition of angiotensin converting enzyme (ACE) activity. For the proteopeptidomic study, the protein extract of Kefir beverage and non-fermented bovine milk were analysed by MALDI-TOF mass spectrometry, and their tryptic digestion products sequenced via Shotgun proteomics (Q-Exactive mass spectrometer). A list of 35 peptides with potential hypertensive activity due to ACE inhibition were identified. These results demonstrate the benefits of Kefir products, and may guide the design of new antihypertensive drugs. © 2019 Elsevier Ltd [less ▲]

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See detailTowards the use of ion mobility mass spectrometry-derived collision cross section as a screening approach for unambiguous identification of targeted pesticides in food
Goscinny, Severine; McCullagh, Michael; Far, Johann ULiege et al

in Rapid Communications in Mass Spectrometry (2019)

Rationale Mass Spectrometry (MS) is the reference method for the screening of ultra‐trace residues of pesticides in food because MS offers the required selectivity/sensitivity to gather information and ... [more ▼]

Rationale Mass Spectrometry (MS) is the reference method for the screening of ultra‐trace residues of pesticides in food because MS offers the required selectivity/sensitivity to gather information and enable the analyst to make informed decisions during the identification process. Here we present and discuss the use of collision cross section (CCS) values in addition to mass accuracy and retention times in a pesticide screening method that integrates all the features offered by coupling UPLC with IMS‐MS. Methods All experiments were carried out using UHPLC coupled to a travelling wave ion mobility mass spectrometer equipped with an ESI source working in positive mode. An in‐house library containing 200 pesticides was built using standard solutions and used as reference for a TWCCS calibration study. Matrix extracts were analysed to evaluate the performance of different screening workflows based on TWCCS, mass accuracy and retention times. Results The results proved that TWCCS values are very consistent, as the measured values do not differ more than 1% from the in‐house reference data library and emphasized the importance of the first low m/z mobility calibration point to guarantee full independence from instrument parameters and calibrant. The screening procedure was simplified to a single step by fully exploiting the content of ion mobility without generating any false detections, either positive or negative from spiked samples and a previous proficiency test. Conclusions The screening approach proposed in this study is unconventional and based on large mass accuracy (20 ppm) and retention time windows (0.5 min) to capture; in a first step, a maximum of detected compounds. Compounds of interest are then identified by comparing measured collision cross sections with the measured reference library collision cross sections (with relative error tolerance lower than 2%). [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)

Detailed reference viewed: 54 (19 ULiège)