Description du sujet de thèse

Development of improved cross-linking mass spectrometry workflows to decipher protein-nucleic acid interactions.

Objectives of the thesis: The objective of this PhD will be to develop structural mass spectrometry methods, mostly based on native mass spectrometry (nMS) and cross-inking (XL-MS) for the characterization of protein/RNA and protein/DNA complexes.

Structural mass spectrometry (MS) approaches have emerged as key techniques for the in vitro characterization of protein/DNA or RNA complexes. Native MS affords mass measurement of intact non-covalent assemblies, allowing fast and accurate determination of stoichiometries. Hydrogen/deuterium exchange MS (HDX-MS) reveals protein regions affected upon NA binding, but lacks in providing information on the DNA/RNA side. Finally, cross-linking MS approaches (XL-MS) appear as the most potent methods to gain information on both protein and nucleic acid sides. However, while XL-MS has gained maturity and popularity for protein-protein interactions studies, it is still scarcely used for providing information on protein/DNA or RNA complexes, mostly because of the complexity of both analytical and data processing workflows.
The NAProtXLMS project aims at :
1) synthesize new chemical XL reagents based on the repurposing of drugs used in therapy cross-linking multifunctional probes and their benchmarking against the reference UV-XL method.
2) develop sensitive, robust analytical MS-based proteomic workflows for chemical protein/DNA and protein/RNA cross-linking with new proteomic LC-MS/MS methods for improved enrichment, separation and identification of Peptide-Oligo conjugates both in vitro and in vivo.
3) evaluate the new XL-MS workflows not only on reference commercial samples but also on two well-characterized systems for proof-of-concept (nuclear receptor/DNA and ribonucleoproteins/RNA complexes), for a more exhaustive and comprehensive characterization of their role in biological processes.
The objective of this PhD work will thus be to develop new XL-MS approach for the characterization of protein/nucleic acids. In collaboration with chemists, new chemical cross-linking reagents for protein/nucleic acid will be developed and evaluated. All steps of the XL-MS workflow, including the chemical cross-linking reaction, the enzymatic digestions, MS methods and data treatment for peptide/nucleic acid cross-links identification will have to be optimized. This PhD work will allow the fellow to use latest generation MS instruments (Eclipse Tribrid Orbitrap, TimsTOF). The methodology will be applied to a series of collaborations in the framework of the ANR project.

Contexte de travail

The Institut Pluridisciplinaire Hubert CURIEN (IPHC), a joint research unit under the joint supervision of CNRS and the University of Strasbourg (UMR7178), is a multidisciplinary laboratory where research teams from different scientific cultures (ecology, physiology and ethology, chemistry and subatomic physics) develop very high level programs based on scientific instrumentation. The IPHC is structured into 4 departments and has a total staff of 393 staff including 257 permanent staff (ie 119 researchers and teachers / researchers and 138 engineers and technicians), 46 staff on fixed-term contracts and 102 doctoral students.

The PhD student will join the "Laboratoire de Spectrométrie de Masse Bio-Organique" (LSMBO) team of the IPHC comprised 9 researchers and teacher-researchers (M/F), 11 engineers (M/F), post-docs and PhD students and will be able to benefit from all the material conditions (LC-MS / MS instrumentation), human support (sharing of expertise) and IT (access to all the software tools for the processing of LC-MS / MS data) necessary for the successful completion of this thesis project.

The expected skills are :
- hold a master's degree in analytical science
- solid knowledge of protein biochemistry of proteins, nucleic acids, mass spectrometry and chomatography
- experience in mass spectrometry and chomatography. Experience in biomolecular MS (peptide and protein analysis) or XL-MS will be a strong advantage.
- fluency in English in writing and orally (at level B2 according to the Common European Framework of Reference for Languages) in order to be able to write results reports and present them at meetings and conferences.
- The multidisciplinary and collaborative context of this project requires that the candidate has a strong motivation, curiosity to expand his area of expertise, autonomy and ability to work in a team with strong constraints to meet deadlines.
The candidate will benefit from access to an administrative restaurant, partial reimbursement of travel expenses, a works council, the possibility of teleworking and training to adapt to the job.

Le poste se situe dans un secteur relevant de la protection du potentiel scientifique et technique (PPST), et nécessite donc, conformément à la réglementation, que votre arrivée soit autorisée par l'autorité compétente du MESR.

Contraintes et risques

-Casual work in staggered schedule
-Work in an ISO 9001 certified environment