|Urlaub, Henning Prof. Dr.
|Identification of peptide-RNA heteroconjugates by mass spectrometry
|Urlaub, Henning Prof. Dr.
|Proteins 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
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 chain.
|Bohnsack, Markus Prof. Dr.
|Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB)