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Identification of peptide-RNA heteroconjugates by mass spectrometry

dc.contributor.advisorUrlaub, Henning Prof. Dr.
dc.contributor.authorChernev, Aleksandar
dc.titleIdentification of peptide-RNA heteroconjugates by mass spectrometryde
dc.contributor.refereeUrlaub, Henning Prof. Dr.
dc.description.abstractengProteins and nucleic acids are two of the major constituents of life and their interplay is at the center of most biological processes. A deep understanding of the interaction between these biomolecules is crucial for structural and functional elucidation of numerous cellular mechanisms. Cross-linking of proteins to nucleic acids by UV irradiation or chemical reagents enables the preservation of association information in covalent bonds that can be examined by a variety of analytical methods. Mass spectrometry (MS) is particularly useful in detecting the proteins associated with RNA. In recent years, significant progress was made in identifying cross-linked peptide-RNA heteroconjugates, which provide direct evidence for the contact sites between proteins and RNA. However, the analysis of protein-RNA cross-links by MS remains a very challenging task due to the low yield of the cross-linking reactions and the laborious manual annotation of mass spectra that is required to validate the results. In this work, a strategy for fully automated annotation was developed that substantially speeds up the manual analysis of cross-links. The different elements of peptide-RNA heteroconjugate fragment spectra were identified and categorized, allowing the development of comprehensive and fully descriptive scoring functions. The created scores are a prerequisite for the employment of a false discovery rate estimation and pave the path towards full automation of the cross-link analysis. Furthermore, the cross-linking behavior of the four canonical ribonucleotides was examined in controlled experiments with model RNA-binding proteins Hsh49 and GAPDH. In addition to the previously described cross-links to uracil, UV-induced heteroconjugates formed with cytosine, guanine and adenine could be detected by mass spectrometry. All identified spectra were formed by generating a covalent bond between the nucleobase and various amino acids. The mass characteristics of the observed precursors and their fragmentation products were investigated in detail and are summarized for future utility in mass spectrometric analyses. As an alternative to UV irradiation, sulfite-mediated cross-linking was demonstrated to be useful in the identification of cytosine contacts with lysines. The toolkit for cross-link enrichment was complemented with two novel workflows for purification of oligoribonucleotide heteroconjugates, based on silica-based purification and strong anion exchange chromatography. Both methods result in significant depletion of interfering non-cross-link species. The workflows could be successfully employed in the study of in vivo UV-generated cross-links of E. coli, as well as in investigation of the protein-RNA interactions in HeLa cytoplasmic extract. A large number of cross-link sites 2   could be detected, providing contact information for known RNA-binding proteins and identifying novel RNA interaction partners. Finally, the ability of nanoelectrospray mass spectrometry to identify heteroconjugates with large RNA moieties was explored. A synthetic peptide-RNA standard was generated by click chemistry and used to determine appropriate chromatographic separation and electrospray ionization conditions. The RNA moiety could be successfully fragmented by collision-induced dissociation, providing comprehensive sequencing information. Thus, demonstrating the capability of nanoelectrospray mass spectrometry to acquire additional structural information from cross-linked samples by revealing the identity of the interacting RNA and the localization of the cross-link site on the nucleotide
dc.contributor.coRefereeBohnsack, Markus Prof. Dr.
dc.subject.engMass spectrometryde
dc.subject.engPeptide-RNA heteroconjugatesde
dc.subject.engProtein-RNA interactionsde
dc.affiliation.instituteGöttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB)de
dc.subject.gokfullBiologie (PPN619462639)de

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