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

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-U4atacsnRNA] snRNPs and native U1snRNPs. Applying our approach to a partial complex of U2snRNP allowed us to identify the contact site between the U2snRNP-specific protein p14/SF3b14a and the branch-site interacting region (BSiR) of U2snRNA.