A proteomic approach to the analysis of RNA degradosome composition in Escherichia coli

The RNA degradosome is a bacterial protein machine devoted to RNA degradation and processing. In Escherichia coli it is typically composed of the endoribonuclease RNase E, which also serves as a scaffold for the other components, the exoribonuclease PNPase, the RNA helicase RhlB, and enolase. The variable presence of additional proteins, however, suggests that the degradosome is a flexible machine that may vary its composition in response to different conditions. Direct analysis of large protein complexes, together with simplified purification procedures, can facilitate qualitative and quantitative identification of RNA degradosome components under different physiological and genetic conditions and can help elucidating the role in the bacterial cell and the mechanisms of action of this macromolecular assembly. Herewith we describe the application of Multidimensional Protein Identification Technology (MudPIT) to the rapid and quantitative identification of the RNA degradosome components. RNA degradosome preparations obtained from specific conditions are enzymatically digested and the peptides are first fractionated by two dimensional (ion exchange and reversed phase) chromatography and analyzed by tandem mass spectrometry. Bioinformatic analysis by means of the SEQUEST algorithm, which correlates the experimental mass spectra with those predicted by peptide sequences in proteomic and translated genomic databases, allows for the identification of the corresponding proteins that compose the complex. The output protein lists of two or more degradosome samples is then compared so as to obtain a rapid evaluation of qualitative and quantitative differences in protein composition. Quantitative analysis is based on the observation that changes of relative protein abundance in different samples are reflected by the score values assigned to each protein component of the RNA degradosome identified by the MudPIT approach. This correlation can be validated by means of orthogonal independent methods. This general fully automated procedure may be applied to the characterization of any complex protein mixture.