Improved identification of enriched peptide RNA cross-links from ribonucleoprotein particles (RNPs) by mass spectrometry.
Direct UV cross-linking combined with mass spectrometry (MS) is a powerful tool to identify hitherto non-characterized protein-RNA contact sites in native ribonucleoprotein particles (RNPs) such as the spliceosome. Identification of contact sites after cross-linking is restricted by: (i) the relatively low cross-linking yield and (ii) the amount of starting material available for cross-linking studies. Therefore, the most critical step in such analyses is the extensive purification of the cross-linked peptide-RNA heteroconjugates from the excess of non-crosslinked material before MS analysis. Here, we describe a strategy that combines small-scale reversed-phase liquid chromatography (RP-HPLC) of UV-irradiated and hydrolyzed RNPs, immobilized metal-ion affinity chromatography (IMAC) to enrich cross-linked species and their analysis by matrix-assisted laser desorption/ionisation (MALDI) MS(/MS). In cases where no MS/MS analysis can be performed, treatment of the enriched fractions with alkaline phosphatase leads to unambiguous identification of the cross-linked species. We demonstrate the feasibility of this strategy by MS analysis of enriched peptide-RNA cross-links from UV-irradiated reconstituted [15.5K-61K-U4atac snRNA] snRNPs and native U1 snRNPs. Applying our approach to a partial complex of U2 snRNP allowed us to identify the contact site between the U2 snRNP-specific protein p14/SF3b14a and the branch-site interacting region (BSiR) of U2 snRNA.
Kühn-Hölsken
Eva
E
Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Dybkov
Olexandr
O
Sander
Björn
B
Lührmann
Reinhard
R
Urlaub
Henning
H
D000469:Alkaline Phosphatase; D000595:Amino Acid Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid / Q000379:methods; D019295:Computational Biology; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification; D012342:RNA, Small Nuclear / Q000737:chemistry* / Q000302:isolation & purification; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry / Q000528:radiation effects; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000737:chemistry / Q000528:radiation effects; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry* / Q000528:radiation effects; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014466:Ultraviolet Rays
D010455:Peptides; D012342:RNA, Small Nuclear; D017412:Ribonucleoprotein, U1 Small Nuclear; D017413:Ribonucleoprotein, U2 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; D000469:Alkaline Phosphatase