Development of an untargeted analytical approach for the identification and quantification of proteins in complex biological and environmental matrices

Grâce aux récents progrès en terme d'instrumentation analytique, la protéomique, en tant que science qui étudie le protéome d'un organisme ou d'un milieu, a connu un véritable tournant et a permis d'étendre le champ des connaissances sur le fonctionnement du vivant dans son ensemble (structure, fonction, métabolisme, dynamisme). Néanmoins, l'étude des protéomes représente un challenge pour de nombreux biologistes, chimistes et biochimistes, en raison notamment de la complexité des échantillons étudiés. De nombreux protocoles analytiques ont d'ores et déjà été développés. Cependant, dans leur ensemble, ces stratégies sont relativement longues et multi-étapes et le plus souvent ciblées sur une protéine donnée.Dans ce contexte, ce travail de thèse a pour objectif de simplifier les protocoles expérimentaux existants afin de limiter les pertes en protéines et ainsi amplifier leurs signaux au cours d'une analyse « Bottom-up » par LC-HRMS (analyse « hors gel »). Chaque étape du processus a été décortiquée et optimisée. Dans un premier temps, les paramètres UPLC-HRMS/MS ont été optimisés afin d'améliorer la détection et la quantification des protéines présentes à des concentrations très variables dans les milieux étudiés. Ensuite, une approche de purification simplifiée qui repose sur une seule et unique étape de lavage et solubilisation des protéines a été mise au point. La démonstration de son efficacité « chimique » et « biologique » a ensuite été réalisée via une étude mécanistique au cours de laquelle les changements de conformation des protéines ont été étudiés à chacune des étapes de purification proposées. Enfin, certains paramètres influençant l'extraction des protéines à partir de ces mêmes matrices ont été étudiés afin de proposer à terme un protocole d'extraction à la carte compatible avec une analyse directe par LC-HRMS/MS.Recent advances in proteomics have been spurred by the rapid development of hybrid and/or high-resolution mass analysers (HRMS/MS). These powerful instrumentations have led to significant improvements in « Bottom-up » approach and have enabled to deepen our knowledge on the functionality of biological systems (structure, function, metabolism, dynamic, etc).Despite their high sensibilities, the potential of such instruments could be significantly lessened by an imperfect sample pre-treatment. In this context, current sample pretreatments follow multi-steps experimental workflows, which alternatively lead to low recoveries of proteins. In this line, this study aims at developing a simple and versatile strategy in order to reduce protein losses and enhance their detection in gel-free LC-MS analysis. First, an analytical method based on liquid chromatography and tandem mass spectrometry was developed to detect and quantify complex peptides mixture. Secondly, a universal, simple and fast purification approach was designed with the aim to purify protein extracts in only one-step. For this purpose, the molecular reactivity, dynamics and conformational changes of proteins at each development step were comprehensively investigated with a set of spectroscopic techniques, in order to select the best strategy. Finally, different factors influencing extraction of proteins were investigated with the goal in the long term to propose an on-demand extraction protocol for direct analysis of proteins by LC-HRMS/MS

Développement d'une approche analytique non ciblée pour l'étude des protéines dans les milieux complexes, environnementaux et biologiques

Recent advances in proteomics have been spurred by the rapid development of hybrid and/or high-resolution mass analysers (HRMS/MS). These powerful instrumentations have led to significant improvements in « Bottom-up » approach and have enabled to deepen our knowledge on the functionality of biological systems (structure, function, metabolism, dynamic, etc).Despite their high sensibilities, the potential of such instruments could be significantly lessened by an imperfect sample pre-treatment. In this context, current sample pretreatments follow multi-steps experimental workflows, which alternatively lead to low recoveries of proteins. In this line, this study aims at developing a simple and versatile strategy in order to reduce protein losses and enhance their detection in gel-free LC-MS analysis. First, an analytical method based on liquid chromatography and tandem mass spectrometry was developed to detect and quantify complex peptides mixture. Secondly, a universal, simple and fast purification approach was designed with the aim to purify protein extracts in only one-step. For this purpose, the molecular reactivity, dynamics and conformational changes of proteins at each development step were comprehensively investigated with a set of spectroscopic techniques, in order to select the best strategy. Finally, different factors influencing extraction of proteins were investigated with the goal in the long term to propose an on-demand extraction protocol for direct analysis of proteins by LC-HRMS/MS.Grâce aux récents progrès en terme d'instrumentation analytique, la protéomique, en tant que science qui étudie le protéome d'un organisme ou d'un milieu, a connu un véritable tournant et a permis d'étendre le champ des connaissances sur le fonctionnement du vivant dans son ensemble (structure, fonction, métabolisme, dynamisme). Néanmoins, l'étude des protéomes représente un challenge pour de nombreux biologistes, chimistes et biochimistes, en raison notamment de la complexité des échantillons étudiés. De nombreux protocoles analytiques ont d'ores et déjà été développés. Cependant, dans leur ensemble, ces stratégies sont relativement longues et multi-étapes et le plus souvent ciblées sur une protéine donnée.Dans ce contexte, ce travail de thèse a pour objectif de simplifier les protocoles expérimentaux existants afin de limiter les pertes en protéines et ainsi amplifier leurs signaux au cours d'une analyse « Bottom-up » par LC-HRMS (analyse « hors gel »). Chaque étape du processus a été décortiquée et optimisée. Dans un premier temps, les paramètres UPLC-HRMS/MS ont été optimisés afin d'améliorer la détection et la quantification des protéines présentes à des concentrations très variables dans les milieux étudiés. Ensuite, une approche de purification simplifiée qui repose sur une seule et unique étape de lavage et solubilisation des protéines a été mise au point. La démonstration de son efficacité « chimique » et « biologique » a ensuite été réalisée via une étude mécanistique au cours de laquelle les changements de conformation des protéines ont été étudiés à chacune des étapes de purification proposées. Enfin, certains paramètres influençant l'extraction des protéines à partir de ces mêmes matrices ont été étudiés afin de proposer à terme un protocole d'extraction à la carte compatible avec une analyse directe par LC-HRMS/MS

TCA precipitation and ethanol/HCl single-step purification evaluation: One-dimensional gel electrophoresis, bradford assays, spectrofluorometry and Raman spectroscopy data on HSA, Rnase, lysozyme - Mascots and Skyline data

The data presented here are related to the research paper entitled “Study of a Novel Agent for TCA Precipitated Proteins Washing - Comprehensive Insights into the Role of Ethanol/HCl on Molten Globule State by Multi-Spectroscopic Analyses” (Eddhif et al., submitted for publication) [1]. The suitability of ethanol/HCl for the washing of TCA-precipitated proteins was first investigated on standard solution of HSA, cellulase, ribonuclease and lysozyme. Recoveries were assessed by one-dimensional gel electrophoresis, Bradford assays and UPLC-HRMS. The mechanistic that triggers protein conformational changes at each purification stage was then investigated by Raman spectroscopy and spectrofluorometry. Finally, the efficiency of the method was evaluated on three different complex samples (mouse liver, river biofilm, loamy soil surface). Proteins profiling was assessed by gel electrophoresis and by UPLC-HRMS

The Challenging Detection of Nucleobases from Pre-accretional Astrophysical Ice Analogs

International audienceAmino acids, sugars, and nucleobases are considered as the so-called molecular bricks of life, the major subunits of proteins and genetic materials. All three chemical families have been previously detected in meteorites. In dense molecular cloud ice analogs, the formation of a large set of amino acids and sugars (+derivatives) has been observed. In this contribution, we demonstrate that similar ices (H2O:13CH3OH:NH3 ices, 2:1:1) can also lead to the formation of nucleobases. Using combined UPLC-Orbitrap mass spectrometric and UPLC-SRM-triple quadrupole mass spectrometric analyses, we have unambiguously detected cytosine in these primitive, realistic astrophysical ice analogs. Additionally, a huge variety of nucleobase isomers was observed. These results indicate that all central subunits of biochemical materials may have already been present at early stages of chemical evolution of the protosolar nebula, before accretion toward planetesimals. Consequently, the formation of amino acids, sugars, and nucleobases does not necessarily require secondary alteration processes inside meteoritic parent bodies. They might have been supplied from dense molecular cloud ices toward post-accretional objects, such as nonaqueously modified comets, and subsequently delivered onto the early Earth's surface, potentially triggering the emergence of prebiotic chemistry leading to the first living systems

Primary Step Towards In Situ Detection of Chemical Biomarkers in the UNIVERSE via Liquid-Based Analytical System: Development of an Automated Online Trapping/Liquid Chromatography System

The search for biomarkers in our solar system is a fundamental challenge for the space research community. It encompasses major difficulties linked to their very low concentration levels, their ambiguous origins (biotic or abiotic), as well as their diversity and complexity. Even if, in 40 years’ time, great improvements in sample pre-treatment, chromatographic separation and mass spectrometry detection have been achieved, there is still a need for new in situ scientific instrumentation. This work presents an original liquid chromatographic system with a trapping unit dedicated to the one-pot detection of a large set of non-volatile extra-terrestrial compounds. It is composed of two units, monitored by a single pump. The first unit is an online trapping unit able to trap polar, apolar, monomeric and polymeric organics. The second unit is an online analytical unit with a high-resolution Q-Orbitrap mass spectrometer. The designed single pump system was as efficient as a laboratory dual-trap LC system for the analysis of amino acids, nucleobases and oligopeptides. The overall setup significantly improves sensitivity, providing limits of detection ranging from ppb to ppt levels, thus meeting with in situ enquiries

Study of a novel agent for TCA precipitated proteins washing - comprehensive insights into the role of ethanol/HCl on molten globule state by multi-spectroscopic analyses

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Controlled Release of a Micelle Payload via Sequential Enzymatic and Bioorthogonal Reactions in Living Systems

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Analyse de peptides dans des échantillons extraterrestre

International audienceMethod development is part of the objectives of astrophysical community for characterizing the organic matter in objects of the Solar System. In this context, we report on the development of an enzyme-catalyzed stereoselective hydrolysis, inspired from the proteomics discipline, which has enabled the indirect detection of peptide sequences in extraterrestrial samples. A proof of concept has been performed on a Murchison extract. We showed that our approach can successfully highlight L- and D-amino acids involved in peptide bonds. While we showed that some D-amino acids must have been involved in peptide bonds, we cannot at this stage conclude on the indigenous or exogenous nature of these biopolymers. However, our strategy constitutes the first step towards the direct UPLC-MS evidence of peptide sequences in extraterrestrial samples. It should thus participate to deepen knowledge on the molecules available in the Solar System, hence providing new clues on their chemical history, especially on Earth

Development of liquid chromatography high resolution mass spectrometry strategies for the screening of complex organic matter: Application to astrophysical simulated materials

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TCA precipitation and ethanol/HCl single-step purification evaluation: One-dimensional gel electrophoresis, bradford assays, spectrofluorometry and Raman spectroscopy data on HSA, Rnase, lysozyme - Mascots and Skyline data

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