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

Abstract

Protein–RNA cross-linking combined with mass spectrometry is a powerful tool to elucidate hitherto noncharacterized 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–61K–U4atac] 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 U4atac snRNA we identified His270 in the spliceosomal U4/U6 snRNP-specific protein 61K (hPrp31p) cross-linked to U44; in the U1 snRNP we show that Leu175 of the U1 snRNP-specific 70K protein is cross-linked to U30 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.

Keywords

• RNA
• protein
• cross-linking
• mass spectrometry
• hPrp31