Analyzing RNA-protein crosslinking sites in unlabeled ribonucleoprotein complexes by mass spectrometry.

摘要: Mass spectrometry is a powerful tool for the analysis of biomolecules, proteins, nucleic acids, carbohydrates, lipids. In combination with genome sequences that are available in databases, it has proven to be most straightforward and sensitive technique sequence hence identification protein components cells, their (post)translational modifications, relative absolute abundance. addition, mass spectrometric methods successfully applied structural biomolecules (i.e., deciphering molecule-ligand interactions spatial quartenary arrangements molecule complexes). We describe methodology protein-RNA contact sites purified ribonucleoprotein (RNP) particles. The method comprises ultraviolet (UV) crosslinking proteins RNA, hydrolysis RNA moieties, isolation cross-linked peptide-RNA oligonucleotides, MALDI (matrix-assisted laser desorption/ionization) isolated conjugates determine crosslinked peptide part. utility this demonstrated on crosslinks from UV-irradiated spliceosomal particles; these were [15.5 K-61 K-U4atac] small nuclear (snRNP) particles prepared by reconstitution vitro U1 snRNP HeLa cells.