UV crosslinking is an appropriate method to identify proteins that directly contact nucleic acid, e.g., RNA. In combination with modern mass spectrometric (MS) analysis such an approach provides the opportunity to reveal not only the nature of the crosslinked proteins but also to identify the actual crosslinking sites between the protein and the nucleic acid. However, the relatively low yield in UV-induced crosslinking makes it difficult to identify in particular those species by MS that represent peptide-nucleic acid conjugates, as the great excess of noncrosslinked material interferes with their detection in MS. Here, we present an automated enrichment strategy of crosslinked peptide-RNA oligonucleotides derived from crude mixtures of UV-irradiated ribonucleoprotein (RNP) particles that uses TiO(2) columns integrated within a two-dimensional (2D) nanoliquid chromatography (LC) system. The setup combines two C18 precolumns, a TiO(2) enrichment column and a nanoanalytical column. It allows the removal of the noncrosslinked RNA and protein moiety and the specific enrichment of crosslinked peptide-RNA conjugates so that UV-irradiated and subsequently completely hydrolyzed RNP complexes can directly be loaded and analyzed by MS. In this feasibility study, we demonstrate the specific enrichment of peptide-RNA oligonucleotides derived from UV-irradiated native spliceosomal U1 snRNPs and spliceosomal [15.5K-61K-U4atac snRNA] complex reconstituted in vitro.
2008 Wiley Periodicals, Inc