Automatic differential analysis of NMR experiments in complex samples

Liquid state NMR is a powerful tool for the analysis of complex mixtures of unknown molecules. This capacity has been used in many analytical approaches: metabolomics, identification of active compounds in natural extracts, characterization of species, and such studies require the acquisition of many diverse NMR measurements on series of samples. While acquisition can easily be performed automatically, the number of NMR experiments involved in these studies increases very rapidly and this data avalanche requires to resort to automatic processing and analysis. We present here a program that allows the autonomous, unsupervised processing of a large corpus of 1D, 2D and DOSY experiments from a series of samples acquired in different conditions. The program provides all the signal processing steps, as well as peak-picking and bucketing of 1D and 2D spectra, the program and its components are fully available. In an experiment mimicking the search of an active species in natural extract, we use it for the automatic detection of small amounts of artemisin added to a series of plant extracts, and for the generation of the spectral fingerprint of this molecules. This program called Plasmodesma is a novel tool which should be useful to decipher complex mixtures, particularly in the discovery of biologically active natural products from plants extracts, but can also in drug discovery or metabolomics studies.Comment: 35 pages, 36 figures, 26 reference

Application of tandem two-dimensional mass spectrometry for top-down deep sequencing of calmodulin

Two-dimensional mass spectrometry (2DMS) involves simultaneous acquisition of the fragmentation patterns of all the analytes in a mixture by correlating their precursor and fragment ions by modulating precursor ions systematically through a fragmentation zone. Tandem two-dimensional mass spectrometry (MS/2DMS) unites the ultra-high accuracy of Fourier transform ion cyclotron resonance (FT-ICR) MS/MS and the simultaneous data-independent fragmentation of 2DMS to achieve extensive inter-residue fragmentation of entire proteins. 2DMS was recently developed for top-down proteomics (TDP), and applied to the analysis of calmodulin (CaM), reporting a cleavage coverage of about ~23% using infrared multiphoton dissociation (IRMPD) as fragmentation technique. The goal of this work is to expand the utility of top-down protein analysis using MS/2DMS in order to extend the cleavage coverage in top-down proteomics further into the interior regions of the protein. In this case, using MS/2DMS, the cleavage coverage of CaM increased from ~23% to ~42%

2D FT-ICR MS of Calmodulin : a top-down and bottom-up approach

Two-dimensional Fourier transform ion cyclotron resonance mass spectrometry (2D FT-ICR MS) allows data-independent fragmentation of all ions in a sample and correlation of fragment ions to their precursors through the modulation of precursor ion cyclotron radii prior to fragmentation. Previous results show that implementation of 2D FT-ICR MS with infrared multi-photon dissociation (IRMPD) and electron capture dissociation (ECD) has turned this method into a useful analytical tool. In this work, IRMPD tandem mass spectrometry of calmodulin (CaM) has been performed both in one-dimensional and two-dimensional FT-ICR MS using a top-down and bottom-up approach. 2D IRMPD FT-ICR MS is used to achieve extensive inter-residue bond cleavage and assignment for CaM, using its unique features for fragment identification in a less time- and sample-consuming experiment than doing the same thing using sequential MS/MS experiments

Couplage optimal d'une antenne IRM supraconductrice avec un transistor HEMT refroidi

En IRM la sensibilité du détecteur joue un rôle déterminant sur la résolution spatiale et temporelle accessible. Le circuit inductif du détecteur doit avoir un facteur de qualité (Q) élevé comme en IRM bas champ ou en microscopie RMN sur de petites régions anatomiques. L'adaptation du détecteur à un préamplificateur est alors difficile à réaliser sur la bande de fréquence nécessaire à la transmission du signal d'imagerie. Le chapitre 1 traite du couplage antenne-préamplificateur dans la littérature. Il existe une limite théorique en bande passante, dite limite de Fano. Il est montré que pour une dégradation constante du rapport signal-surbruit (RSB), la largeur de bande atteignable est inversement proportionnelle à la température de bruit Tn du préamplificateur. Le chapitre II est consacré à la recherche et à la caractérisation d'un amplificateur refroidi dépassant les limites de l'état de l'art en électronique RMN faible bruit. Les HEMTs sont les meilleurs candidats aux fréquences IRM mais leurs caractéristiques ne sont pas bien connues à ces fréquences et à basse température. Une méthode de caractérisation a été appliquée en combinant la mesure de paramètres S et le modèle de transistor bruyant de Pospieszalski. La caractérisation a été menée à 293 K et à 100 K. Le chapitre III traite de l'optimisation du couplage antenne-préamplificateur. L'étude est détaillée dans le cas d'un couplage inductif du second ordre qui offre le meilleur compromis efficacité/simplicité. Dans le chapitre IV les résultats précédents sont appliqués à la conception d'un prototype de préamplification intégré à un détecteur supraconducteur dédié à la microscopie localisée in-vivo à 1,5T.In MRI the sensitivity of the detector plays a determining part on the spatial and temporal resolution available. The detector inductive circuit must have a high quality factor (Q) as in low field MRI or in NMR microscopy on small anatomical areas. The adaptation of the detector to a preamplifier is then difficult to realize on the frequency band necessary to the transmission of the signal. The chapter 1 deals with antenna-preamplifier coupling in the literature. There is a theoretical limit in band-width, known as limit of Fano is shown that for a constant degradation of the signal to noise ratio (SNR) the bandwidth achievable is inversely proportional to the noise temperature Tn noise of the preamplifier. Chapter II is devoted to the search and the characterization of a cooled amplifier exceeding the limits of l'état de l'art in weak noise NMR electronics. HEMTs are the best candidates at MRI frequencies but their characteristics are not well-known at these frequencies and low temperature. A method of characterization was applied by combining measurement of parameters S and the model of Pospieszalski nois transistor. The characterization was carried out to 293 K and 100 K. The chapter III deals with the optimisation of antenna-preamplifier coupling. The study is detailed in the case of an inductive coupling of the second order which offers the best tradeoff efficiency/simplicity. In chapter IV the preceding results are applied to the prototype preamplification design of a superconductive detector dedicated to microscopy located in-vivo with 1,5T.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Nonuniform Sampling Acquisition of Two-Dimensional Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Increased Mass Resolution of Tandem Mass Spectrometry Precursor Ions

International audienc

Top-Down Deep Sequencing of Ubiquitin Using Two-Dimensional Mass Spectrometry

International audienc

CyberSco.Py an open-source software for event-based, conditional microscopy

International audienceAbstract Timelapse fluorescence microscopy imaging is routinely used in quantitative cell biology. However, microscopes could become much more powerful investigation systems if they were endowed with simple unsupervised decision-making algorithms to transform them into fully responsive and automated measurement devices. Here, we report CyberSco.Py, Python software for advanced automated timelapse experiments. We provide proof-of-principle of a user-friendly framework that increases the tunability and flexibility when setting up and running fluorescence timelapse microscopy experiments. Importantly, CyberSco.Py combines real-time image analysis with automation capability, which allows users to create conditional, event-based experiments in which the imaging acquisition parameters and the status of various devices can be changed automatically based on the image analysis. We exemplify the relevance of CyberSco.Py to cell biology using several use case experiments with budding yeast. We anticipate that CyberSco.Py could be used to address the growing need for smart microscopy systems to implement more informative quantitative cell biology experiments

Two-Dimensional ECD FT-ICR Mass Spectrometry of Peptides and Glycopeptides

2D FT-ICR MS allows the correlation between precursor and fragment ions by modulating ion cyclotron radii for fragmentation modes with radius-dependent efficiency in the ICR cell without the need for prior ion isolation. This technique has been successfully applied to ion–molecule reactions, Collision-induced dissociation and infrared multiphoton dissociation. In this study, we used electron capture dissociation for 2D FT-ICR MS for the first time, and we recorded two-dimensional mass spectra of peptides and a mixture of glycopeptides that showed fragments that are characteristic of ECD for each of the precursor ions in the sample. We compare the sequence coverage obtained with 2D ECD FT-ICR MS with the sequence coverage obtained with ECD MS/MS and compare the sensitivities of both techniques. We demonstrate how 2D ECD FT-ICR MS can be implemented to identify peptides and glycopeptides for proteomics analysis

Polymer Analysis in the Second Dimension: Preliminary Studies for the Characterization of Polymers with 2D MS

Two-dimensional Fourier transform ion cyclotron resonance mass spectrometry (2D FTICR MS or 2D MS) allows direct correlation between precursor and fragment ions without isolation prior to fragmentation. The method has been optimized for the analysis of complex mixtures and used so far for the analysis of small molecules and peptides obtained by tryptic digestion of proteins and entire proteins. In this work, a 2D MS method is developed to characterize complex mixtures of polymers using infrared multiphoton decay (IRMPD) and electron capture dissociation (ECD) as fragmentation techniques, and D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), Polysorbate 80, and poly­(methyl methacrylate) (PMMA) as analytes. The use of 2D MS allowed generation of fragment <i>m</i>/<i>z</i> values for all the compounds in the mixture at once and allowed tandem mass spectrometry of species very close in <i>m</i>/<i>z</i> that would have been difficult to isolate with a quadrupole for standard MS/MS. Furthermore, the use of unique features of 2D MS such as the extraction of neutral-loss lines allowed the successful assignment of peaks from low abundant species that would have been more difficult with standard MS/MS. For all the samples, the amount of information obtained with 2D MS was comparable with what obtained with multiple 1D MS/MS experiments targeted on each individual component within each mixture but required a single experiment of about 20–40 min