Poster abstracts

Poster number 32 submitted by Kady Krivos

A comparison of two methods used to investigate peptide:RNA heteroconjugates by mass spectrometry

Kady L. Krivos (Chemistry, University of Cincinnati), Larry Sallans (Chemistry, University of Cincinnati), Patrick A. Limbach (Chemistry, University of Cincinnati)

Abstract:
Protein:RNA complexes participate in transcription, translation (ribosomes), chaperoning (signal recognition particles), and enzymatic cleavages (ribozyme and splicesome) to name a few. To date, the biochemical methods for obtaining sequence and structural information on these complexes have been arduous tasks often involving hazardous radioactive labeling and some the methods lack the ability to identify peptide or RNA modifications. Mass spectrometry has previously been developed as a method to investigate peptide:oligonucleotide complexes, however it has been shown to result in the preferential loss of the modifications. A recently developed method of fragmentation called electron capture dissociation (ECD) has been employed in recent years in mass spectrometry as a fragmentation technique of peptides because it can provide more extensive sequence coverage and improved identification of the location of labile post translational modifications when compared to CID. The focus of the present work is a comparison of the two fragmentation techniques, CID and ECD, to see if more sequence information can be elucidated for peptide:oligonucleotide heteroconjugates in mass spectrometry. In particular, we seek to learn if ECD will provide a more effective approach for identifying amino acids containing the site of RNA cross-linking. Synthetic heteroconjugates containing a 2-5 oligonucleotides covalently linked through amide chemistry to peptide sequences less than 15 amino acids in length have served as the model systems for this investigation. CID of these complexes has revealed the propensity for sequencing the RNA portion of the heteroconjugate and has not allowed for the identification of the point of crosslinking. Initial ECD studies have shown the fragmentation and sequencing of the peptide moiety to be favored. On going investigation of ECD as well as the heteroconjugate properties are being investigated to optimize this method.

Keywords: RNAProtein, mass spectrometry