Analyzing RNA-protein crosslinking sites in unlabeled ribonucleoprotein complexes by mass spectrometry.
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 the databases, it has proven to be the most straightforward and sensitive technique for the sequence analysis and hence the identification of protein components in the cells, their (post)translational modifications, and their relative and absolute abundance. In addition, mass spectrometric methods are successfully applied for the structural analysis of biomolecules (i.e., deciphering molecule-ligand interactions and spatial quartenary arrangements of molecule complexes). We describe a methodology for the mass spectrometric analysis of protein-RNA contact sites in purified ribonucleoprotein (RNP) particles. The method comprises ultraviolet (UV) crosslinking of proteins to RNA, hydrolysis of the protein and RNA moieties, isolation of cross-linked peptide-RNA oligonucleotides, MALDI (matrix-assisted laser desorption/ionization) mass spectrometry of the isolated conjugates to determine the sequence of the crosslinked peptide and RNA part. The utility of this methodology is demonstrated on crosslinks isolated from UV-irradiated spliceosomal particles; these were [15.5 K-61 K-U4atac] small nuclear ribonucleoprotein (snRNP) particles prepared by reconstitution in vitro and U1 snRNP particles purified from HeLa cells.
Urlaub
Henning
H
Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany.
Kühn-Hölsken
Eva
E
Lührmann
Reinhard
R
D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008954:Models, Biological; D011485:Protein Binding / Q000528:radiation effects; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry / Q000378:metabolism; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D011506:Proteins; D017411:Ribonucleoproteins, Small Nuclear; D012313:RNA