Characterization of Ribonucleoproteins by Cross-linking and Mass Spectrometry
Nucleic acid and protein complexes play crucial roles in cell functions such as DNA replication, packaging, repair and transcription and RNA maturation, transport and translation. Biophysical methods such as X-ray crystallography, nuclear magnetic resonance (NMR) and cryo-electron microscopy are typically used to gain insight into the structure and function of these complexes. While X-ray crystallography and NMR can provide high resolution information, not all nucleic acid-protein complexes are readily characterized by these methods. Cross-linking combined with mass spectrometry (MS) is one alternative approach for structurally characterizing nucleic acid-protein complexes, although sample preparation, analysis and data interpretation are not trivial.
Chromatographic and mass spectrometric methods for analyzing RNA-Protein cross-links are presented here and encountered difficulties and shortcomings are discussed. A new approach based on fractional mass for the identification of oligonucleotide-peptide conjugates in the mass spectrum is introduced, which eliminates the need for isotope labeling. Developed methods were applied to three different studies: identification of ribosomal proteins involved in recognition of leaderless mRNA, identification of cross-linked ribosomal proteins in a cross-linking study of E. coli 30S risosomal subunit cross-linked with 2-iminothiolane and investigation of nucleocapsid protein cross-linked to the s4U incorporated RNA packaging signal of the Murine leukemia virus.
A mobile phase containing ammonium acetate could provide conditions needed for the chromatographic separation and mass spectrometric detection of the oligonucleotide-peptide conjugates. In the cross-linking studies of leaderless mRNA, seven ribosomal proteins were identified. Data suggests that these proteins, positioned at or near the mRNA binding tunnel, are involved in the recognition of leaderless mRNA by 30S ribosomal subunit. Cross-linked ribosomal proteins in the 30S ribosomal subunit and cross-links in the nuclecapsid protein cross-linking were identified. However, oligonucleotide-peptide heteroconjugates were not identified on the fractional mass plots. These projects show that cross-linking in combination with mass spectrometry is a valuable tool for the analysis of ribonucleoproteins.
School:University of Cincinnati
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:mass spectrometry ribonucleoprotein cross linking fractional
Date of Publication:01/01/2008