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Protein-RNA cross-linking combined with mass spectrometry is a powerful tool to elucidate hitherto non-characterized protein-RNA contacts in ribonucleoprotein particles, as, for example, within spliceosomes. Here, we describe an improved methodology for the sequence analysis of purified peptide-RNA oligonucleotide cross-links that is based solely on MALDI-ToF mass spectrometry. The utility of this methodology is demonstrated on cross-links isolated from UV-irradiated spliceosomal particles; these were (1) [15.5K-61 K-U4 atac] small nuclear ribonucleoprotein (snRNP) particles prepared by reconstitution in vitro, and (2) U1 snRNP particles purified from HeLa cells. We show that the use of 2',4',6'-trihydroxyacetophenone (THAP) as MALDI matrix allows analysis of cross-linked peptide-RNA oligonucleotides in the reflectron mode at high resolution, enabling sufficient accuracy to assign unambiguously cross-linked RNA sequences. Most important, post-source decay (PSD) analysis under these conditions was successfully applied to obtain sequence information about the cross-linked peptide and RNA moieties within a single spectrum, including the identification of the actual cross-linking site. Thus, in U4 atac snRNA we identified His 270 in the spliceosomal U4/U6 snRNP-specific protein 61 K (hPrp31p) cross-linked to U 44; in the U1 snRNP we show that Leu175 of the U1 snRNP-specific 70K protein is cross-linked to U 30 of U1 snRNA. This type of analysis is applicable to any type of RNP complex and may be expected to pave the way for the further analysis of protein-RNA complexes in much lower abundance and/or of cross-links that are obtained in low yield.

Complete MALDI-ToF MS analysis of cross-linked peptide–RNA oligonucleotides derived from nonlabeled UV-irradiated ribonucleoprotein particles