56 research outputs found
Prediction of subplastidial localization of chloroplast proteins from spectral count data - Comparison of machine learning algorithms
To study chloroplast metabolism and functions, subplastidial localization is a prerequisite to achieve protein functional characterization. As the accurate localization of many chloroplast proteins often remains hypothetical, we set up a proteomics strategy in order to assign the accurate subplastidial localization. A comprehensive study of Arabidopsis thaliana chloroplast proteome has been carried out in our group [1], involving high performance mass spectrometry analyses of highly fractionated chloroplasts. In particular, spectral count data were acquired for the three major chloroplast sub-fractions (stroma, thylakoids and envelope) obtained by sucrose gradient purification. As the distribution of spectral counts over compartments is a fair predicator of relative abundance of proteins [2], it was justified to propose a prime statistical model [1] relating spectral counts to subplastidial localization. This predictive model was based on a logistic regression, and demonstrated an accuracy rate of 84% for chloroplast proteins. In the present work, we conducted a comparative study of various machine learning techniques to generate a predictive model of subplastidial localization of chloroplast proteins based on spectral count data
Neutral particle Mass Spectrometry with Nanomechanical Systems
Current approaches to Mass Spectrometry (MS) require ionization of the
analytes of interest. For high-mass species, the resulting charge state
distribution can be complex and difficult to interpret correctly. In this
article, using a setup comprising both conventional time-of-flight MS (TOF-MS)
and Nano-Electro-Mechanical-Systems-based MS (NEMS-MS) in situ, we show
directly that NEMS-MS analysis is insensitive to charge state: the spectrum
consists of a single peak whatever the species charge state, making it
significantly clearer than existing MS analysis. In subsequent tests, all
charged particles are electrostatically removed from the beam, and unlike
TOF-MS, NEMS-MS can still measure masses. This demonstrates the possibility to
measure mass spectra for neutral particles. Thus, it is possible to envisage
MS-based studies of analytes that are incompatible with current ionization
techniques and the way is now open for the development of cutting edge system
architectures with unique analytical capability
Compact and modular system architecture for a nano-resonator-mass spectrometer
Mass measurements in the mega-to giga-Dalton range are essential for the characterization of natural and synthetic nanoparticles, but very challenging to perform using conventional mass spectrometers. Nano-electro-mechanical system (NEMS) based MS has demonstrated unique capabilities for the analysis of ultra-high mass analytes. Yet, system designs to date included constraints transferred from conventional MS instruments, such as ion guides and high vacuum requirements. Encouraged by other reports, we investigated the influence of pressure on the performances of the NEMS sensor and the aerodynamic focusing lens that equipped our first-generation instrument. We thus realized that the NEMS spectrometer could operate at significantly higher pressures than anticipated without compromising particle focusing nor mass measurement quality. Based on these observations, we designed and constructed a new NEMS-MS prototype considerably more compact than our original system, and which features an improved aerodynamic lens alignment concept, yielding superior particle focusing. We evaluated this new prototype by performing nanoparticle deposition to characterize aerodynamic focusing, and mass measurements of calibrated gold nanoparticles samples. The particle capture efficiency showed nearly two orders of magnitude improvement compared to our previous prototype, while operating at two orders of magnitude greater pressure, and without compromising mass resolution
A dynamic ion cooling technique for FTICR mass spectrometry
AbstractA fast dynamic ion cooling technique based upon the adiabatic invariant phenomenon for Fourier transform ion cyclotron resonance mass spectrometry (FTICR) is presented. The method cools ions in the FTICR trap more efficiently, within a few hundred milliseconds without the use of a buffer gas, and results in a substantial signal enhancement. All performance aspects of the FTICR spectrum, e.g., peak intensities, mass resolution, and mass accuracy, improve significantly compared with cooling based on ion–ion interactions. The method may be useful in biological applications of FTICR, such as in proteomic studies involving extended on-line liquid chromatography (LC) separations, in which both the duty cycle and mass accuracy are crucially important
Increased proteome coverage for quantitative peptide abundance measurements based upon high performance separations and DREAMS FTICR mass spectrometry
AbstractA primary challenge in proteome measurements is to be able to detect, identify, and quantify the extremely complex mixtures of proteins. The relative abundances of interest span at least six orders of magnitude for mammalian proteomes, and this constitutes an intractable challenge for high throughput proteome studies. We have recently described a new approach, Dynamic Range Enhancement Applied to Mass Spectrometry (DREAMS), which is based upon the selective ejection of the most abundant species to expand the dynamic range of Fourier transform ion cyclotron resonanace (FTICR) measurements. The basis of our approach is on-the-fly data-dependent selective ejection of highly abundant species, followed by prolonged accumulation of remaining low-abundance species in a quadrupole external to the FTICR ion trap. Here we report the initial implementation of this approach with high efficiency capillary reverse phase LC separations and high magnetic field electrospray ionization FTICR mass spectrometry for obtaining enhanced coverage in quantitative measurements for mammalian proteomes. We describe the analysis of a sample derived from a tryptic digest of proteins from mouse B16 cells cultured in both natural isotopic abundance and 15N-labeled media. The FTICR mass spectrometric analysis allows the assignment of peptide pairs (corresponding to the two distinctive versions of each peptide), and thus provides the basis for quantiative measurements when one of the two proteomes in the mixture is perturbed or altered in some fashion. We show that implementation of the DREAMS approach allows assignment of approximately 80% more peptide pairs, thus providing quantitative information for approximately 18,000 peptide pairs in a single analysis
Screen-printed digital microfluidics combined with surface acoustic wave nebulization for hydrogen-deuterium exchange measurements
International audienc
The emerging landscape of single-molecule protein sequencing technologies
Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.This Perspective describes new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell proteomics.</p
Etude des mécanismes d'ablation-ionisation laser de pesticides adsorbés sur membranes par spectrométrie de masse à transformée de Fourier
Laser desorption/ionisation coupled with Fourier transform mass spectrometry is an ever growing technique with promising applications in the field of surface analysis. However, this method is very sensitive to the experimental parameters. Through this work, after having defined the physical phenomena invoked in the process, we have put the stress on the practical implications of the microscopic processes which have to be taken into account during the analysis. Several examples illustrate this demonstration. The feasability of the method for the analysis of pesticides adsorbed on solid phase extraction membranes has been evaluated for the first time. Five compounds with very different structures and various applications in agriculture have been analysed by classical mass spectrometry and their fragmentation products have been compared to those obtained by laser desorption at 248 nm wavelength. The analysis of the membrane by laser desorption has allowed us to evaluate the possible interferences and the ablation depth at various irradiances. The experiments concerning atrazine and its metabolite have allowed us to propose two distinct ionisation mecanisms : gas phase ionisation versus "true" desorption. Moreover, at the 248 nm wevelength, the various compounds adsorbed on membranes have shown very different behaviors which are better explained by their respective octanol/water partition coefficient than by their absorbance at the laser wavelength. The use of a tripled Nd-YAG laser has allowed the detection of the compounds which couldn't be detected at 248 nm and to confirm the hypothesis of a deeper adsorption of these compounds into the membraneLa désorption/ionisation laser couplée à la spectrométrie de masse à transformée de Fourier est une technique d'analyse en plein développement dont les applications en analyse de surfaces sont particulièrement prometteuses. Il s'agit cependant d'une méthode extrêmement sensible aux conditions opératoires. Après avoir défini les phénomènes physiques sous-jacents, nous avons, à travers des exemples d'applications, mis l'accent sur les implications pratiques des phénomènes microscopiques dont il faut tenir compte lors d'une analyse. La faisabilité de cette méthode pour l'analyse de pesticides adsorbes sur supports d'extraction a été évaluée pour la première fois. Cinq composés de structures très différentes et d'applications variées en agriculture ont été analyses sous forme de standard par spectrométrie de masse classique et les modes de fragmentation comparés à ceux obtenus par désorption laser à 248 nm. L'analyse des membranes utilisées a permis de déterminer les interférences possibles et d'évaluer la profondeur d'ablation à différentes irradiances. Les essais concernant l'atrazine et son métabolite hydroxyle adsorbes sur membranes d'extraction ont conduit à proposer deux mécanismes distincts d'ionisation : un processus d'ionisation en phase gazeuse compare à une désorption vraie. De plus, à 248 nm, les différents composes analyses sur membranes ont montré des comportements très différents qui sont mieux expliqués par leurs coefficients de partages octanol/eau respectifs que par leur absorption a la longueur d'onde utilisée. L'utilisation d'un laser nd-yag triple a permis de détecter les composés qui posaient des problèmes à 248 nm et de confirmer l'hypothèse d'une adsorption plus profonde dans la membran
Multiplexed and data-independent tandem mass spectrometry for global proteome profiling
International audienc
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