Implementation of chemical protein-nucleic acid cross-linking into mass spectrometric workflows and mass spectrometric database searches
by Fanni Laura Bazsó
Date of Examination:2021-10-27
Date of issue:2021-11-22
Advisor:Prof. Dr. Henning Urlaub
Referee:Prof. Dr. Henning Urlaub
Referee:Dr. Vladimir Pena
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EnglishProtein nucleic acid interactions play a pivotal role in cells, from transcription to translation. Functions of proteins in protein nucleic acid interactions are diverse: histones are responsible for the packaging of the genomic DNA; ribosomal proteins are part of the ribosome, which is responsible for protein synthesis. Crosslinking mass spectrometry proved to be a useful tool to identify protein-nucleic acid interactions and their dynamics in the cell. A vast amount of effort has been spent to elucidate protein-nucleic acid interactions with UV crosslinking mass spectrometry, whereas chemical crosslinking techniques were left untouched. Therefore, the current study was aiming for the implementation of chemical crosslinking into available mass spectrometric workflows. Two chemical crosslinkers were investigated with crosslinking mass spectrometry for the elucidation of protein-nucleic acid complexes: formaldehyde and 1,2:3,4-diepoxybutane (DEB). DEB-crosslinking was applied in the study of DNA-containing complexes such as linker histone double stranded DNA complexes and nucleosome-based complexes. DEB crosslinking could provide information about the crosslinking site localization in the protein sequences, often to single amino acid resolution. Formaldehyde-crosslinking was applied on in vitro reconstituted and native nucleosome complexes, the bacterial 70S ribosome and in vivo crosslinked Escherichia coli and HeLa cells. The simple fragmentation pattern of formaldehyde-crosslinking and the developed sequential RNA digestion technique made it possible to identify hundreds of proteins crosslinked to RNA in vivo. The established MS based crosslinking strategy can give an extensive picture of the RNA interactome in well studied and less investigated organisms.
Keywords: Chemical crosslinking; Mass Spectrometry; Protein-nucleic acid complexes