Integrating 'top-down' and 'bottom-up' mass spectrometric approaches for proteomic analysis of Shewanella oneidensis

Abstract

Here we present a comprehensive method for proteome analysis that integrates both intact protein measurement ('top-down') and proteolytic fragment characterization ('bottom-up') mass spectrometric approaches, capitalizing on the unique capabilities of each method. This integrated approach was applied in a preliminary proteomic analysis of Shewanella oneidensis, a metal-reducing microbe of potential importance to the field of bioremediation. Cellular lysates were examined directly by the 'bottom-up' approach as well as fractionated via anion-exchange liquid chromatography for integrated studies. A portion of each fraction was proteolytically digested, with the resulting peptides characterized by on-line liquid chromatography/tandem mass spectrometry. The remaining portion of each fraction containing the intact proteins was examined by high-resolution Fourier transform mass spectrometry. This 'top-down' technique provided direct measurement of the molecular masses for the intact proteins and thereby enabled confirmation of post-translational modifications, signal peptides, and gene start sites of proteins detected in the 'bottom-up' experiments. A total of 868 proteins from virtually every functional class, including hypotheticals, were identified from this organism