UV and chemical crosslinking mass spectrometry for the analysis of protein-nucleic acid interactions
von Luisa Welp
Datum der mündl. Prüfung:2024-04-03
Erschienen:2024-07-12
Betreuer:Prof. Dr. Ralf Ficner
Gutachter:Prof. Dr. Ralf Ficner
Gutachter:Prof. Dr. Melina Schuh
Zum Verlinken/Zitieren:
http://dx.doi.org/10.53846/goediss-10569
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Description:Dissertation - Main text
Lizenzbestimmungen:
Zusammenfassung
Englisch
Protein-nucleic acid interactions are a key part of essential cellular processes and their disturbance often results in the development of diseases. Crosslinking mass spectrometry (XL-MS)-based approaches have become increasingly popular for the proteome-wide discovery of nucleic acid-binding proteins. The basic principle of the methodology relies on covalent attachment of proteins and binding nucleic acids by crosslinking, which makes the heteroconjugates amenable to MS analysis. Building up on comprehensive nucleic acid-binding protein inventories, there is an increasing demand for higher resolution analyses identifying the interaction sites at the level of crosslinked amino acids. Amino acid-resolved XL-MS data is of significant value for integration in structural modelling approaches as distance restrains, and for the discovery of novel nucleic acid-binding sequence and structure motifs. At the outset of the research detailed in this doctoral thesis, amino acid-resolved XL-MS could not be applied comprehensively in native cellular environments, due to low crosslink yields and the absence of specialized software solutions. In this work, we focused on i) the optimization of crosslinking conditions comparing conventional UV-light induced crosslinking with chemical crosslinking reagents; ii) crosslinked peptide-(oligo)nucleotide enrichment; iii) identification of crosslinked (oligo)nucleotide-adducts; iv) the development of a specialized software solution for crosslinked peptide-(oligo)nucleotide database search; and v) the adaptation of a labelling-based quantitative MS approach. In the published manuscript by Stützer et al., which constitutes the first part of this thesis, an optimized UV-light based XL-MS workflow was established, leading to the identification of numerous crosslinked amino acids within eukaryotic nucleosomes and HeLa nuclei. With the aim to increase XL-MS efficiency and sensitivity, we established chemical crosslinking XL-MS, which is presented in the second, yet unpublished manuscript by Welp and colleagues. We compared the results obtained by chemical XL-MS with those from UV XL-MS, and applied it to Escherichia coli cells, which delivered a comprehensive crosslink site inventory. We further present the database search tool, NuXL, specialized on the identification of peptide-(oligo)nucleotide crosslinks and providing crosslink localisation on the level of single amino acids. Building upon these studies, we conducted a preliminary quantitative amino acid-resolved XL-MS experiment using tandem mass tag (TMT)-labelling in combination with our established workflow. This thesis provides a valuable contribution to the application toolkit for the investigation of protein-nucleic acid complexes and contains exciting protein-DNA and protein-RNA datasets from human cell nuclei and E. coli cells.
Keywords: Mass Spectrometry; Crosslinking; Protein-RNA; Protein-DNA; Escherichia coli XL MS; Chromatin
