Improved Identification of Enriched Peptide Rna Cross-links from Ribonucleoprotein Particles (Rnps) by Mass Spectrometry.
From: Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Nucleic acids research
- Publish Date: 2007
- ISSN: 1362-4962
- Volume: 35
- Issue: 15
- Pages: e95
- Medium: Internet
- Language: English
- Citation (JAMA): Kühn-Hölsken Eva, Dybkov Olexandr, Sander Björn, et al. Improved Identification of Enriched Peptide Rna Cross-links from Ribonucleoprotein Particles (Rnps) by Mass Spectrometry.. Nucleic Acids Res. 2007;35:e95
Abstract
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.
Mesh Headings (Keywords): Alkaline Phosphatase, Amino Acid Sequence, Binding Sites, Chromatography, Affinity, Chromatography, Liquid, Computational Biology, Molecular Sequence Data, Peptides, RNA, Small Nuclear, Ribonucleoprotein, U1 Small Nuclear, Ribonucleoprotein, U2 Small Nuclear, Ribonucleoproteins, Small Nuclear, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Ultraviolet Rays
Check for Full Text / PubMed Unique Identifier (PMID): 17652325
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