Poster abstracts

Poster number 32 submitted by Romel Dator

Mass spectrometry-based quantitation of proteins in ribonucleoprotein complexes

Romel P. Dator (Department of Chemistry, University of Cincinnati), Patrick A. Limbach (Department of Chemistry, University of Cincinnati)

Abstract:
Ribonucleoprotein (RNP) complexes perform crucial roles in diverse cellular processes including DNA replication, repair, RNA transcription and maturation, and protein synthesis. The ability to accurately determine the absolute quantity of proteins within such complexes provides useful information relating to complex stoichiometry and biogenesis. Here, mass spectrometry based-approaches are being used to characterize and obtain absolute quantitative information of proteins associated with RNPs. By virtue of parallel data acquisition, alternating scans of low energy collision-induced dissociation (CID) and high-energy CID during liquid chromatography-mass spectrometry analyses reveal both protein identification and quantitation in a single experiment. The low energy CID mode is used to obtain accurate mass measurements of precursor ions and intensity data for quantitation, while the high-energy CID mode generates peptide fragmentation of all precursor ions for database searching and subsequent protein identification. Because the information obtained in these experiments is related to the number of peptides identified per protein (within the complex), optimization of experimental conditions is necessary to provide an unbiased analysis of all low and high molecular weight components. The approach was developed using E. coli wild-type ribosomes and then applied to ribosome assembly particles that accumulate as a result of perturbation. Ribosomal proteins and extra-ribosomal protein factors associated with these particles were identified. In addition, absolute quantitative information of proteins provided insights on the degree of heterogeneity of these ribonucleoprotein particles. This MS-based platform enables the rapid characterization of RNA-protein complexes including ribosome assembly particles resulting from perturbations, stress conditions and deletion strains of proteins or assembly factors.

Keywords: ribosomal proteins, ribonucleoprotein complexes, mass spectrometry