Description du projet
Spectrométrie de masse native avancée pour l’analyse des complexes protéiques non-covalents
La spectrométrie de masse native (SMN) analyse les macromolécules, telles que les protéines intactes et les complexes protéiques, et permet de caractériser les structures secondaires, tertiaires et quaternaires des biomolécules. Le projet 2D-TOPMASS, financé par l’UE, entend utiliser la spectrométrie de masse (SM) par résonance cyclotronique ionique à transformée de Fourier couplée à des techniques d’activation laser des ions par ultraviolet et par infrarouge pour élargir l’application de la SMN aux grands complexes de protéines et de protéines/acides nucléiques. L’application de la SMN en combinaison avec des schémas d’acquisition de SM bidimensionnels sera testée aux fins de l’analyse de complexes protéiques non-covalents et de mélanges hétérogènes. Le projet développera l’application de la SMN en tant que technique efficace d’étude des complexes et des assemblages protéiques dans un large éventail d’échantillons.
Objectif
Native mass spectrometry (MS) is a powerful structural biology tool to study protein complexes, their dynamics, assembly and function. In its so-called top-down approach, it aims to link specific proteoforms to higher assemblies such as non-covalent complexes, in which they function naturally and/or in a disease. The proposed project will leverage the power and versatility offered by state-of-the-art Fourier transform ion cyclotron resonance (FTICR) mass spectrometry coupled to ultraviolet and infrared laser ion activation techniques to advance native top-down MS of large protein- and protein / nucleic acid complexes. Studying a range of protein complexes up to small viral capsids, the project will push the boundaries of FTICR MS in the analysis of high-mass protein complex samples. Utilizing both standard commercially available protein complexes for method setup and benchmarking as well as biologically-relevant FOXO, TEAD and nucleosome protein / DNA complexes from the host laboratory, advanced laser- and electron-induced dissociation and fragmentation techniques and their combinations will be tested to obtain the best possible information on multiple levels of the complexes’ structure. Finally, the hitherto untried combination of native MS of non-covalent protein complexes with two-dimensional MS acquisition schemes will be explored to probe all species in a heterogeneous mixture at the same time. Linking the obtained sequential information on proteoforms to their complex and subcomplex stoichiometry, this approach will help increase the utility of native MS as a powerful technique for the study of proteoforms and protein assemblies applicable to a broad range of biologically / medically relevant samples promoting and reinforcing the position of native MS in the modern integrative structural biology portfolio as a technique providing information complementary to (and difficult to access by) classical high-resolution approaches.
Champ scientifique
- natural sciencesphysical sciencestheoretical physicsparticle physicsparticle accelerator
- natural sciencesbiological sciencesbiochemistrybiomoleculesnucleic acids
- natural sciencesbiological sciencesbiochemistrybiomoleculesproteins
- natural scienceschemical sciencesanalytical chemistrymass spectrometry
- natural sciencesbiological sciencesmolecular biologystructural biology
Programme(s)
Régime de financement
HORIZON-AG-UN - HORIZON Unit GrantCoordinateur
142 00 Praha 4
Tchéquie