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Nucleic Acids Research Advance Access published online on July 25, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm540
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© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Improved identification of enriched peptide–RNA cross-links from ribonucleoprotein particles (RNPs) by mass spectrometry

Eva Kühn-Hölsken1, Olexandr Dybkov2, Björn Sander2, Reinhard Lührmann2 and Henning Urlaub1,*

1Bioanalytical Mass Spectrometry Group and 2Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany

*To whom correspondence should be addressed. Tel: +49 551 2011060; Fax: +49 551 2011197; Email: henning.urlaub{at}mpi-bpc.mpg.de

Received May 9, 2007. Revised June 21, 2007. Accepted June 28, 2007.

Direct UV cross-linking combined with mass spectrometry (MS) is a powerful tool to identify hitherto non-characterized protein–RNA contact sites in native ribonucleoprotein particles (RNPs) such as the spliceosome. Identification of contact sites after cross-linking is restricted by: (i) the relatively low cross-linking yield and (ii) the amount of starting material available for cross-linking studies. Therefore, the most critical step in such analyses is the extensive purification of the cross-linked peptide–RNA heteroconjugates from the excess of non-crosslinked material before MS analysis. Here, we describe a strategy that combines small-scale reversed-phase liquid chromatography (RP-HPLC) of UV-irradiated and hydrolyzed RNPs, immobilized metal-ion affinity chromatography (IMAC) to enrich cross-linked species and their analysis by matrix-assisted laser desorption/ionisation (MALDI) MS(/MS). In cases where no MS/MS analysis can be performed, treatment of the enriched fractions with alkaline phosphatase leads to unambiguous identification of the cross-linked species.

We demonstrate the feasibility of this strategy by MS analysis of enriched peptide–RNA cross-links from UV-irradiated reconstituted [15.5K-61K-U4atac snRNA] snRNPs and native U1 snRNPs. Applying our approach to a partial complex of U2 snRNP allowed us to identify the contact site between the U2 snRNP-specific protein p14/SF3b14a and the branch-site interacting region (BSiR) of U2 snRNA.


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Identification of protein binding sites on U3 snoRNA and pre-rRNA by UV cross-linking and high-throughput analysis of cDNAs
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[Abstract] [Full Text] [PDF]


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