Où docteurs et entreprises se rencontrent
Menu
Connexion

Vous avez déjà un compte ?

Nouvel utilisateur ?

Mass spectrometry techniques for selenium imaging in fish thin-sections: from quantitative localization to selenium species identification

ABG-91015 Sujet de Thèse
25/03/2020 < 25 K€ brut annuel
CNRS UMR 5254
pau - Nouvelle Aquitaine - France
Mass spectrometry techniques for selenium imaging in fish thin-sections: from quantitative localization to selenium species identification
  • Chimie
selenium, mass spectrometry imaging, analytical developments, rainbow trout, aquaculture

Description du sujet

I. Le contexte scientifique / Scientific Context

Metal ions affect many aspects of life as some of them are essential or beneficial while others are deleterious or even toxic [1-2]. The bioinorganic chemistry of the organism/cell affects the role each metal can play, stressing the requirement of a fine description of metal speciation. Thus visualizing metals distribution at the organism and cellular scales is a key for understanding how they impart their functions and how their functions might be altered [3]. Moreover, as metal deficiency or overload can be a proof of disease, the measurement of the concentration of metal in a specific area of a biological tissue is of paramount importance [4-6].
Due to its antioxidant properties, selenium is an essential element for humans and animals including fish [7]. It is therefore used as a supplement in plant-based diet to promote a sustainable aquaculture by replacement of fishmeal based diet [8]. The biological effect of such a selenium-supplemented diet have been the subject of numerous studies but the biological mechanisms of assimilation are unknown. The physiological role of selenium is mainly due to its incorporation to selenoproteins as selenocysteine, the 21th amino acid and catalytic site of antioxidant enzymes of the selenoproteome [9].
Given the diversity of selenized species and their low abundance, the precise and accurate quantitative localisation of distinct selenium species as well as their identification are a challenge at the forefront of analytical technology. Mass spectrometry (MS) – based imaging techniques are the best technologies available to tackle such challenge by considering three intertwined key properties: (1) sensitivity, when analytical limits of detection and quantification are low enough to reliably quantify metals at biologically relevant levels, (2) selectivity, when co-localized metals have to be discriminated and their chemical form (speciation) determined and (3) spatial resolution to localise metals within the smallest cellular structure (μm scale).
The high sensitivity (ng.g-1 LOD) and the spatial resolution (μm level) of laser ablation inductively coupled plasma mass spectrometry (LA-ICP MS) make it an attractive technique to map chemical elements at biologically relevant level [10-11]. However, one of the most challenging tasks in bioimaging studies by LA-ICP MS is the development of a reliable and validated quantification strategy [9; 11]. Indeed, to gain information of the bidimensional metal distribution, tissues samples are analyzed as thin slices (5-200 μm) without prior homogenization step. Thus the variation of the composition (heterogeneity) of biological samples, even within several thin sections, gives rise to problems, all closely related, at the level of material ablation, ablated material transport and ionization and stability of ICP MS detector over the analysis time. Additionally, the diversity of biological samples and the lack of suitable standard material make the situation even more complicated. Even if approaches have been proposed to improve quantification of trace elements in biological tissues to overcome these limitations [12], quantitative bioimaging of selenium in thin sections by LA-ICP MS, requires analytical developments to enhance its reliability and robustness.
Complementary to LA-ICP MS, Matrix assisted Laser Desorption Ionization Mass Spectrometry imaging (MALDI-MSI) is mainly used for molecules identification (proteins/peptides, metabolites, amino acids...) but very rarely for the identification of metal species because of the difficulty to ionize them in MALDI. The most advanced study reports the complementarity of LA-ICP MS and MALDI-MS to propose molecular targets of metals but without formal identification of the metal species entities [13-14]. The mapping of selenized species (selenoproteins, seleno amino acids) in thin sections of trouts under selenium-supplemented diet will bring insight into the function and regulation of selenoproteins (linked with observed effects).
II. Les objectifs / Objectives

The objective of the PhD project is to develop mass spectrometry based imaging methodologies for the quantitative mapping of selenium by LA-ICP MS and the identification by MALDI-MS of selenized species (selenoproteins, seleno amino acids) directly in thin-sections of trout.

III. Plan de travail / Work plan

Workpackage 1 : In a first step, selenium quantitative imaging will be developed by LA-ICP MS for determining:
- first the total quantity of Se in thin sections (organs, tissues) of rainbow trout by setting up an isotope dilution analysis methodology with a selenium tracer deposited either below or above the thin section,
- then the quantitative mapping of selenium associated with selenoproteins in thin sections of interest. For that purpose, a quantification strategy using immunochemistry (with available antibodies) will be developed.

Workpackage 2: The selenized species mapping by MALDI-MS be carried out in collaboration with CBMN institute in Bordeaux (France). A methodology for a direct enzymatic digestion (proteolysis) on the thin section of the trout with be developed in order to identify selenized peptides and consequently selenoproteins. The PhD student will be hosted at CBMN during several punctual stays.
Analytical methodology developments will include sample preparation (cryotomy, chemical treatment of the thin section, ...), measurement standards synthesis and characterization, imaging experiments, mass spectrometry data processing and analysis.


Teaching mission: As a E2S UPPA PhD student, a 32 hrs/year teaching task will have to be undertaken.

 

Prise de fonction :

01/10/2020

Nature du financement

Contrat doctoral

Précisions sur le financement

Présentation établissement et labo d'accueil

CNRS UMR 5254

The Institute of Analytical Sciences and Physico-Chemistry for Environment and Materials (IPREM) is a joint research unit CNRS / UPPA (UMR 5254). IPREM members are interested in the development of fundamental knowledge in physico-chemistry, analytical chemistry and microbiology, in relation to applications concerning the structure of the living, the management of the environment and the functional properties of different classes of materials.
Skills of IPREM members in physico-chemistry and analytical chemistry concern the development of methodologies for trace elements analysis and speciation (detection, identification and quantification of chemical forms of metals and metalloids) in environmental and biological samples. In fine, the research work benefits the understanding of essentiality or toxicity mechanisms of metals and metalloids thanks to interdisciplinary collaborative works. The developed methodologies include sample preparation and the coupling of separation techniques (chromatography, electrophoresis) with elemental and molecular mass spectrometry (ICP MS and ESI-FT-MS/MS). Metals imaging (direct analysis on solid samples such as biological thin sections) is being developed with laser ablation coupled to ICP MS (LA-ICP MS). IPREM has also expertise in selenium and selenoproteins analysis.

Intitulé du doctorat

Chimie

Pays d'obtention du doctorat

France

Etablissement délivrant le doctorat

Université de Pau et des Pays de l'Adour

Ecole doctorale

ED 211 SCIENCES EXACTES ET LEURS APPLICATIONS

Profil du candidat

The candidate must have a physic-chemistry and/or analytical chemistry education from a master in chemistry or engineer chemistry school. Theoretical or practical skills in mass spectrometry will be strongly appreciated.
The candidate must demonstrate:
- a good organization and autonomy,
- a good capacity to synthetize and present its research work.

Date limite de candidature

15/05/2020
Partager via
Postuler
Fermer

Vous avez déjà un compte ?

Nouvel utilisateur ?

Besoin d'informations ?

Vous souhaitez recevoir une ou plusieurs lettres d’information de l’ABG. Chaque mois des actualités, des offres, des outils, un agenda…

item1
Ils nous font confiance