Henning Urlaub

RNA/DNA-protein crosslinking mass spectrometry – Applications of UV and chemical crosslinking and data analysis with NuXL

Luisa M. Welp1,2, Alexander Chernev1, Alexander Wulf1, Fanni Bazso1, Timo Sachsenberg3,4, Yehor Horokhovskyi5, Monika Raabe1, Arslan Siraj3, Aditi Sharma6, Yi He7, Bernard Delanghe6, Rosa Viner7, Juliane Liepe5, Oliver Kohlbacher3,4,8, Henning Urlaub1,2

1 Bioanalytical Mass Spectrometry Group, Max Planck Institute for Multidisciplinary Sciences, 37077 Göttingen, Germany
2 Bioanalytics Group, Institute of Clinical Chemistry, University Medical Center Göttingen, 37075 Göttingen, Germany
3 Institute of Bioinformatics and Medical Informatics, University of Tübingen, 72076 Tübingen, Germany
4 Applied Bioinformatics, Dept. of Computer Science, University of Tübingen, 72076 Tübingen, Germany
5 Quantitative and Systems Biology; Max Planck Institute for Multidisciplinary Sciences, 37077 Göttingen, Germany
6 Thermo Fisher Scientific (Bremen) GmbH, 28199 Bremen, Germany
7 Thermo Fisher Scientific, San Jose, USA 8 Institute of Translational Bioinformatics, University Hospital Tübingen, 72076 Tübingen, Germany

Introduction: Crosslinking mass spectrometry (XL-MS) can resolve sites of interaction in protein-RNA and DNA complexes at single amino acid and nucleic acid resolution. Processing of (large) XL-MS data, reliable assignment, and visualization of crosslinking results – including annotation of spectra – remain key challenges in the database search of crosslinked species. We have developed NuXL, a database search engine, which robustly identifies UV and chemically induced crosslink sites within peptide sequences from MS2 spectra, and significantly improves data-processing and identification of protein–RNA/DNA crosslinks, irrespective of source (i.e., isolated complexes or cellular entities). NuXL not only supports all types of UV crosslinking with non-substituted and substituted nucleotides but also the use of various chemical crosslinking reagents including formaldehyde.

Methods: We used UV and chemical XL-MS to investigate protein-RNA/DNA interaction sites/regions in E. coli and analyzed the data with NuXL. For this, crosslinked E. coli cells were processed by nuclease and endoproteinase digestions and crosslinked peptides were enriched using TiO2 or SILICA-based chromatography. Finally, enriched crosslinked peptide-RNA/DNA (oligo)nucleotides were separated by off-line chromatography followed by LC-MS analysis. MS data were analyzed with NuXL, using different presets for the applied crosslinkers. Crosslink spectrum matches (CSMs) were rescored by percolator and filtered for 1% FDR. Resulting crosslink sites were mapped to available protein-DNA/RNA complex 3D structures and located to conserved domains within the crosslinked proteins.

Results: We developed NuXL, a dedicated software package for the analysis of XL-MS data obtained from UV and chemically crosslinked protein–RNA/DNA samples. NuXL is available in the OpenMS platform and as a node in Proteome Discoverer (PD). It allows for reliable, FDR-controlled assignment of protein–nucleic acid crosslinking sites from samples treated with UV light or chemical crosslinkers and offers user-friendly matched spectra visualization including ion annotations. We demonstrate the robust and sensitive performance of NuXL on MS data acquired from UV and chemically crosslinked cells. With respect to the different crosslinking approaches, i.e. UV and chemical crosslinking, we show that C, G, F, H, K, Y and C, K, H, M are the most prominent crosslinked amino acids, respectively, and that chemical XL-MS almost exclusively identifies crosslinks to purines, whereas UV XL-MS identifies pyrimidine crosslinks in both RNA and DNA-protein crosslinking experiments. In E. coli cells, we identified in total more than 4800 unique crosslinked peptides of more than 1490 proteins crosslinked to RNA. Proteins found in all crosslinking approaches are enriched for RNA-binding proteins, while proteins that are specifically identified through chemical crosslinking are enriched for proteins with oxidoreductase activity, small molecule binding proteins, and proteins that have been mainly categorized to participate in protein-protein interactions. Importantly, the crosslinked peptides/amino acids that we identified through NuXL are located within or in close neighborhood to the conserved domains/sites of these proteins. In summary, NuXL provides rapid, robust, sensitive, and reliable data annotation of protein-RNA and DNA crosslinking sites irrespectively of sample complexity.

Novel aspect: NuXL search tool for XL-MS data annotation form protein-RNA and DNA crosslinking with UV and chemicals applicable on in vivo crosslinking.

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