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  Photo-cross-linking and high-resolution mass spectrometry for assignment of RNA-binding sites in RNA-binding proteins.

Kramer, K., Sachsenberg, T., Beckmann, B. M., Qamar, S., Boon, K. L., Hentze, M. W., et al. (2014). Photo-cross-linking and high-resolution mass spectrometry for assignment of RNA-binding sites in RNA-binding proteins. Nature Methods, 11(10), 1064-1070. doi:10.1038/nmeth.3092.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0024-265D-3 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0028-4828-7
Genre: Journal Article

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 Creators:
Kramer, K.1, Author              
Sachsenberg, T., Author
Beckmann, B. M., Author
Qamar, S.1, Author              
Boon, K. L.2, Author              
Hentze, M. W., Author
Kohlbacher, O., Author
Urlaub, H.1, Author              
Affiliations:
1Research Group of Bioanalytical Mass Spectrometry, MPI for biophysical chemistry, Max Planck Society, ou_578613              
2Department of Cellular Biochemistry, MPI for Biophysical Chemistry, Max Planck Society, ou_578576              

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 Abstract: RNA-protein complexes play pivotal roles in many central biological processes. Although methods based on high-throughput sequencing have advanced our ability to identify the specific RNAs bound by a particular protein, there is a need for precise and systematic ways to identify RNA interaction sites on proteins. We have developed an experimental and computational workflow combining photo-induced cross-linking, high-resolution mass spectrometry and automated analysis of the resulting mass spectra for the identification of cross-linked peptides, cross-linking sites and the cross-linked RNA oligonucleotide moieties of such RNA-binding proteins. The workflow can be applied to any RNA-protein complex of interest or to whole proteomes. We applied the approach to human and yeast mRNA-protein complexes in vitro and in vivo, demonstrating its powerful utility by identifying 257 cross-linking sites on 124 distinct RNA-binding proteins. The open-source software pipeline developed for this purpose, RNPxl, is available as part of the OpenMS project.

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Language(s): eng - English
 Dates: 2014-08-312014-10
 Publication Status: Published in print
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 Rev. Method: Peer
 Identifiers: DOI: 10.1038/nmeth.3092
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Title: Nature Methods
Source Genre: Journal
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Pages: - Volume / Issue: 11 (10) Sequence Number: - Start / End Page: 1064 - 1070 Identifier: -