Integrating 'top-down' and 'bottom-up' mass spectrometric approaches for proteomic analysis of Shewanella oneidensis
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
VerBerkmoes, NC., Bundy, J., Hauser, L., Asano, KG., Razumovskaya, J., Larimer, F., ... Stephenson, J. (2002). Integrating 'top-down' and 'bottom-up' mass spectrometric approaches for proteomic analysis of Shewanella oneidensis. Journal of Proteome Research, 1(3), 239-252.