Descrizione del progetto
Spettrometria di massa nativa avanzata per l’analisi di miscele proteiche non covalenti
La spettrometria di massa nativa analizza le macromolecole, come le proteine intatte e i complessi proteici, e consente di caratterizzare le strutture secondarie, terziarie e quaternarie delle biomolecole. Il progetto 2D-TOPMASS, finanziato dall’UE, si propone di utilizzare la spettrometria di massa a risonanza ionica ciclotronica a trasformata di Fourier, accoppiata a tecniche di attivazione ionica laser a ultravioletti e infrarossi, per espandere l’applicazione della spettrometria di massa nativa a grandi complessi di proteine e proteine/acidi nucleici. L’applicazione della spettrometria di massa nativa in combinazione con schemi di acquisizione bidimensionali della spettrometria di massa sarà testata per l’analisi di complessi proteici non covalenti e miscele eterogenee. Il progetto farà progredire l’applicazione della spettrometria di massa nativa come efficace tecnica per lo studio dei complessi e dei gruppi proteici in un’ampia gamma di campioni.
Obiettivo
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.
Campo scientifico
- natural sciencesphysical sciencestheoretical physicsparticle physicsparticle accelerator
- natural sciencesbiological sciencesbiochemistrybiomoleculesnucleic acids
- natural sciencesbiological sciencesbiochemistrybiomoleculesproteins
- natural scienceschemical sciencesanalytical chemistrymass spectrometry
- natural sciencesbiological sciencesmolecular biologystructural biology
Programma(i)
Argomento(i)
Meccanismo di finanziamento
HORIZON-AG-UN - HORIZON Unit GrantCoordinatore
142 00 Praha 4
Cechia