3 research outputs found
Detection of protein-RNA crosslinks by nanoLC-ESI-MS/MS using precursor ion scanning and multiple reaction monitoring (MRM) experiments
ProteināRNA interactions within ribonucleoprotein particles (RNPs) can be investigated by UV-induced crosslinking of proteins to their cognate RNAs and subsequent isolation and mass-spectrometric analysis of crosslinked peptideāRNA oligonucleotides. Because of the low crosslinking yield, a major challenge in proteināRNA UV crosslinking is the detection of the crosslinked species over the excess of non-crosslinked material, especially when complex systems (native RNPs) are investigated. Here, we applied a novel approach that uses on-line nanoLC-ESI-MS/MS to detect and subsequently sequence peptideāRNA oligonucleotide crosslinks from crude mixtures. To detect the crosslinks we made use of features shared by crosslinks and phosphopeptides, that is, the phosphate groups that both carry. A precursor ion scan for m/z 79 (negative-ion mode, āve) is applied to selectively detect analytes bearing the phosphate-containing species (i.e., residual non-crosslinked RNA and peptideāRNA crosslinks) from crude mixtures and to determine their exact m/z values. On this basis, a multiple reaction monitoring (MRM) experiment monitors the expected decomposition from the different precursor charge states of the putative crosslinks to one of the four possible RNA nucleobases [m/z 112, 113, 136, 152 (positive-ion mode, +ve)]. On detection, a high-quality MS/MS is triggered to establish the structure of the crosslink. In a feasibility study, we detected and subsequently sequenced peptideāRNA crosslinks obtained by UV-irradiation of (1) native U1 snRNPs and (2) [15.5K-61K-U4atac] snRNPs prepared by reconstitution in vitro. MRM-triggered collision-induced dissociation (CID) MS/MS enabled us to obtain sequence information about the crosslinked peptide and RNA moiety