2D-nanoLC approach using TiO_(2) columns for the enrichment of protein-RNA cross-links and phosphopeptides derived from ribonucleoprotein particles for MS-based identification

摘要

Ribonucleoprotein particles (RNPs) play essential roles in a number of fundamental cellular processes, including pre-mRNA splicing. A detailed knowledge of contact sites between proteins and RNA within the RNP is crucial for a full understanding of their functions. UV cross-linking at 254 nm combined with mass spectrometry (MS) is a powerful and straightforward strategy to identify such contact sites in purified native RNPs [1]. A bottleneck in this is the low yield (about 1percent) of protein-RNA cross-linking, when non-modified RNA, as present in native particles, is used. Therefore, we exploit the feature of phosphate groups, common to phosphopeptides and cross-linked species, to purify the latter selectively over the excess of non-cross-linked peptide and RNA. Here, we report an enrichment strategy based on titanium dioxide (TiO_(2)) [2, 3] columns integrated within a 2D-nano LC system. Enriched fractions are either spotted onto MALDI targets and analyzed by MALDI-ToF/ToF [1] or analyzed by directly connecting the LC output to an ESI mass spectrometer [4]. The strategy also proves useful for the enrichment and subsequent analysis of phosphopeptides derived from non-cross-linked RNPs.